General Information

GENERAL INFORMATION

injection device— Equilibration temperature A constant temperature of G1 Physics and Chemistry for inside vial about 809C Equilibration time for 60 minutes inside vial Guideline for Residual Solvents Transfer-line temperature A constant temperature of about 859C and Models for the Residual Carrier gas Nitrogen Solvents Test Pressurisation time 30 seconds Injection volume of sample 1.0 mL 1. Guideline for Residual Solvents (ii) Operating conditions (2) for the head-space sample Refer to the Guideline for Residual Solvents in Phar- injection device— maceuticals (PAB/ELD NotificationNo.307;datedMarch Equilibration temperature A constant temperature of 30, 1998). for inside vial about 1059C Since the acceptable limits of residual solvents recom- Equilibration time for 45 minutes mended in the Guideline were estimated to keep the safety of inside vial patients, the levels of residual solvents in pharmaceuticals Transfer-line temperature A constant temperature of must not exceed the limits, except for in a special case. Phar- about 1109C maceutical manufacturers should assure the quality of their Carrier gas Nitrogen products by establishing their own specification limits or Pressurisation time 30 seconds manufacturing process control limits for residual solvents Injection volume of sample 1.0 mL present in their products in consideration of the limits (iii) Operating conditions (3) for the head-space sample recommended in the Guideline and the observed values in injection device— their products, and by performing the test with the products Equilibration temperature A constant temperature of according to the Residual Solvents Test. for inside vial about 809C Equilibration time for 45 minutes 2. Residual Solvents Test inside vial Generally, the test is performed by using gas chromato- Transfer-line temperature A constant temperature of graphy <2.02> as directed in the Residual Solvents Test about 1059C 2.46 < >. If only the class 3 solvents with low toxic potential to Carrier gas Nitrogen man are present in the products, Loss on Drying Test <2.41> Pressurisation time 30 seconds can be applied in place of gas chromatography, in which case Injection volume of sample 1.0 mL the limit value of residual solvents is not more than 0.5z. The test may also be performed according to the EP 3.2. Models for operating conditions and system suitability method (2.4.24 Identification and control of residual sol- of gas chromatography vents) or the USP method (<467> Residual Solvents). Even in (i) Test conditions (1) (A model described under Proce- this case, description should be in the JP style and the system dure A in the EP and the USP) suitability test should be performed according to the JP rule. Operating conditions— Detector: Hydrogen flame-ionization detector. 3. Models for the operating conditions and system suita- Column: Coat the inside wall of a fused silica tube, 0.32 bility of gas chromatography for residual solvents test mm (or 0.53 mm) in inside diameter and 30 m in length, to The following are typical examples of the operating condi- 1.8 mm(or3mm) thickness with 6z cyanopropylphenyl- tions of gas chromatography for residual solvents test, de- methyl silicon polymer for gas chromatography. Use a guard scribed in the EP and the USP, but these do not necessarily column if necessary. imply that other suitable operating conditions can not be Column temperature: Maintain at 409C for 20 minutes, used. In the operating conditions, generally, items required then increase to 2409Cat109C per minute if necessary, and for the test such as detector, column, column temperature, keep at 2409C for 20 minutes. injection port temperature, detector temperature, carrier Injection port temperature: A constant temperature of gas, flow rate, and time span of measurement should be spe- about 1409C. cified, and in the system suitability, items such as test for re- Detector temperature: A constant temperature of about quired detectability, system performance, and system repeat- 2509C. ability should be specified. The following are several models Carrier gas: Helium. for the operating conditions and the system suitability: Flow rate: 35 cm/second. 3.1. Models for operating conditions for a head-space Split ratio: 1:5. sample injection device (Models described in the EP and the System suitability— USP) System performance: When the procedure is run with the (i) Operating conditions (1) for the head-space sample standard solution under the above operating conditions, the

2135 2136 Physics and Chemistry / General Information JP XVI resolution between the peaks is not less than 1.0. (Note: In System repeatability: When the test is repeated 6 times the case that the number of substances to be tested is two or with the standard solution under the above operating condi- more.) tions, the relative standard deviation of the peak areas of the System repeatability: When the test is repeated 3 times substance to be tested is not more than 15z. with the standard solution under the above operating conditions, the relative standard deviation of the peak areas of the substance to be tested is not more than 15z. (ii) Test conditions (2) (A model described under Proce- Inductively Coupled Plasma Atomic dure B in the EP and the USP) Emission Spectrometry Operating conditions— Detector: Hydrogen flame-ionization detector. Inductively coupled plasma (ICP) atomic emission spec- Column: Coat the inside wall of a fused silica tube, 0.32 trometry is a method for qualitative and quantitative analy- mm (or 0.53 mm) in inside diameter and 30 m in length, to sis for the element in the sample specimen, which is nebu- 0.25 mm thickness with polyethylene glycol 20M for gas lized into argon plasma induced by radio frequency power, chromatography. Use a guard column if necessary. by measuring the wavelength and the intensity of the emis- Column temperature: Maintain at 509C for 20 minutes, sion spectra generated by the target element, atomized and then increase to 1659Cat69C per minute if necessary, and excited in the plasma. Argon gas plasma used in this method keep at 1659C for 20 minutes. is characteristic of higher temperature of 6000 ¿ 8000 K and Injection port temperature: A constant temperature of electronic density of 1015 cm－3. about 1409C. When the high energy is externally given to an atom, the Detector temperature: A constant temperature of about orbit transition of the most exterior electron is occurred and 2509C. attained to the excited state. When the excited state of atom Carrier gas: Helium. returns to the basic state, the energy obtained by the excita- Flow rate: 35 cm/second. tion is radiated as a light. The emitted light has an intrinsic Split ratio: 1:5. wave number n and wave length l, attributed to the individ- System suitability— ual element. Assuming the Plank constant h and the light System performance: When the procedure is run with the velocity c, the emitted light energy DE is expressed by the standard solution under the above operating conditions, the following formula. resolution between the peaks is not less than 1.0. (Note: In DE ＝ hn ＝ hc/l the case that the number of substances to be tested is two or more.) Since there are many of combinations of the orbit transi- System repeatability: When the test is repeated 3 times tion energy level of the most exterior electron and the emit- with the standard solution under the above operating condi- ted light energy, usually, there are plenty of spectral lines tions, the relative standard deviation of the peak areas of the emitted from an element, summing up every strong and substance to be tested is not more than 15z. weak emitted lights. However, the number of emitted lights (iii) Test conditions (3) (A model described in Method I of being in the ultra-violet and visible region and having suit- under Other Analytical Procedures in the USP) able detection sensitivity for qualitative and quantitative Operating conditions— analysis are limited to a certain number. Since the atomic Detector: Hydrogen flame-ionization detector. emission spectra give intrinsic wave number or wave length Column: Coat the inside wall of a fused silica tube, 0.53 for the respective element, many of elements contained in a mm in inside diameter and 30 m in length, to 5 mm thickness sample specimen can be identified by the spectroscopic with 5z phenyl-methyl silicon polymer for gas chromatog- analysis of the emitted lights. Furthermore, the quantitative raphy. If necessary use a guard column prepared by coating analysis for an element can also be done by measuring the the inside wall of a fused silica tube, 0.53 mm in inside diam- spectral intensity of the emitted light. eter and 5 m in length, to 5 mm thickness with 5z phenyl- The characteristics of ICP emission spectrometry are sum- methyl silicon polymer for gas chromatography. marized as follows; Column temperature: Maintain at 359C for 5 minutes, ( i ) Microanalysis for plenty of elements is possible. then increase to 1759Cat89C per minute, further increase to ( ii ) Analytical precision is high, since the stable plasma 2609Cat359C per minute if necessary, and keep at 2609C generation can be kept. for 16 minutes. (iii) Linearity of the calibration curve is ensured in the Injection port temperature: A constant temperature of wide range of 4 ¿ 5digit. about 709C. (iv) Multi-element simultaneous analysis is possible. Detector temperature: A constant temperature of about ( v ) Chemical interferences are almost negligible. 2609C. Carrier gas: Helium. The most serious problem in the ICP emission spectro- Flow rate: 35 cm/second. metry is that the spectral interferences are inevitable to this Split ratio: Splitless. analysis, and disturb the analysis for the target element. It System suitability— results from many of emitted spectral lines due to coexisting System performance: When the procedure is run with the elements, because the atomization and excitation of elements standard solution under the above operating conditions, the are done in very high temperature of plasma. Thus it is the resolution between the peaks is not less than 1.0. (Note: In key technique to obtain an accurate analytical result how to the case that the number of substances to be tested is two or reduce the spectral interferences and how to correct the in- more.) JP XVI General Information / Physics and Chemistry 2137 terfered spectra. 1.2. Attachments The present method is superior to analyze inorganic im- 1.2.1. Ultrasonic nebulizer purities and/or coexisting elements in drug substances and It is an instrument for atomizing the sample solution with preparations specifically, and also to analyze metal residues an ultrasonic vibrator, dehydrating it by heating and cool- in crude drugs and preparations. The qualitative and quan- ing, and introducing it into the emission unit by the carrier titative analysis of alkali, alkali earth, and heavy metals are gas. By using this attachment, the sample introducing possible, and also of many other elements which are required efficiency is remarkably increased. for suitable control for the safety assurance of drug medi- 1.2.2. Hydride generation apparatus cines. Furthermore, since the simultaneous analysis for lots It is an instrument that, after reducing the compounds of elements are possible, this method can be used for the such as arsenic, selenium, and antimony in the sample solu- profile analysis of inorganic impurities such as metal ele- tion to the volatile hydrides with sodium tetrahydroborate ments for the quality control of drug substances. etc., the vapor/liquid separation is carried out and the gas In this General Information, a spectroscopic detection components only introduced into the emission unit with the method of atomic emission spectra (AES) due to metal ele- carrier gas. In comparison with the usual nebulizer, the ments introduced into inductively coupled plasma (ICP), is sample introducing efficiency is remarkably increased. described as ICP-AES. Separately, since ICP is not only a 2. Pretreatment of sample good excitation source, but also a good ionizing source, Usually, organic samples such as drug substances are inductively coupled plasma-mass spectrometry (ICP-MS) is ignited or digested by the dry ashing method or the acid also possible by using ICP as an ionizing source. digestion method, respectively. Then the sample solution is 1. Apparatus prepared by dissolving the residues with small amount of 1.1. Composition of the apparatus nitric acid or hydrochloric acid. Separately, as for the diges- The apparatus is composed of the excitation source unit, tion resistant samples such as crude drugs, the microwave sample introduction unit, the emission unit, the spectropho- digestion apparatus is available to the digestion of those tometry unit, the detection unit, and the data processing samples in tightly closed vessels. unit. Where the sample can be dissolved in water or an ap- The excitation source unit is composed of the electric propriate solvent, the sample solution can be simply pre- power source circuit, the control circuit to supply and to pared by dilution or dissolution, provided that a correct control electric energy, and the gas supplying part. The sam- analysis is assured previously, either by the dry ashing ple introduction unit is the part of introducing the sample method or the acid digestion method. into the emission unit and composed of a nebulizer and a 2.1. Dilution and/or dissolution method spray chamber, etc. Take a specified amount of sample in the respective mono- The emission unit is a part where the target element in the graph, then dilute by water or an appropriate solvent, or dis- sample is excited and emitted, and it is composed of the solve in water or a specified solvent to prepare the sample so- torch and the radio frequency induction coil. The torch usu- lution. ally consists of a threefold tube, in which the sample is intro- 2.2. Dry ashing method duced by the central tube. Argon is used for the gas to form Applying the ``Residue on Ignition Test'', take a specified plasmaandalsotocarrythesample.Therearetwotypesof amount of sample in the crucible, moisten it with a small observation way of the light emitted, one is the radial view- amount of sulfuric acid, then heat gently to carbonize it ing, and the other the axial viewing. thoroughly. After cooling, moisten the charred residue with The spectroscope unit is a part separating the emitted light a small amount of sulfuric acid, heat gently again, then into the individual spectral line and composed of convergent ignite the residue to ash in the range of 400 ¿ 6009C. lens system and optical elements such as the diffraction grat- Dissolve the ash by adding a small amount of nitric acid or ing, etc. There are two types of spectroscopes, one is the hydrochloric acid to prepare the sample solution. wavelength scanning type of monochrometer, the other the Separately, applying the 3rd Method in the ``Heavy Metals fixed wavelength type of polychrometer. For measuring Limit Test'', ignite the sample to ash without moistening it spectral lines in the vacuum UV region, in which the wave- with sulfuric acid. In this case, since it is digested and incin- length is not more than 190 nm, the evacuation of the in- erated in the open system, be careful to the vaporization loss terior of the spectroscope or the substitution of air for argon of low boiling point elements such as mercury. or nitrogen gas are necessary. 2.3. Acid digestion method Detection unit is a part converting the incident light inten- Take an amount of sample into the beaker or the flask as sity to the corresponding strength of the electric signal and specified in the monograph, add nitric acid, sulfuric acid, or composed of the detector and the signal processing system. the mixture of these acids, then digest by heating. If it is nec- Regarding the detector, either the photomultiplier or the essary, an auxiliary oxidant such as hydrogen peroxide may charge transfer device (CTD) such as the charge coupled be available. After the digestion is completed, add a small device, the charge injection device, and the photo diode ar- amount of nitric acid or hydrochloric acid to the residues, ray etc.,isused. dissolve them by heating to prepare the sample solution. Data processing unit is a part of processing the detected Acid digestion can be done either in the open or in the signal and the measurement result and of giving the calibra- closed system. Regarding the closed system, the heat digestion curve etc., which are given by the displaying apparatus tion at high temperature under high pressure conditions or the printer, etc. might be applicable by using the pressure tolerable digestion vessel of polytetrafluoroethylene fitted with the outer tube of stainless steel. Where the heat digestion method is used in the 2138 Physics and Chemistry / General Information JP XVI closed system, be careful enough to accidents such as the taining the target element of high purity (not less than expansion and the liquid leak. 99.99z) can be used for preparing the calibration standards, 2.4. Microwave digestion method in which one or more target elements are included. In case of Take an amount of sample into the pressure tolerable preparing multi-component standard solution, the reagent closed vessel as specified in the monograph, add an ap- solutions and the element combinations are required not to propriate amount of nitric acid, then digest under heating form precipitates, and not to give spectral interferences with conditions by using the microwave digestion apparatus. the analytical line of the target element. Since it is a digestion procedure under high temperature/ 3.3. Optimization of the operating conditions high pressure conditions, the temperature and the pressure in The operating conditions are usually as follows. the closed vessel should be appropriately controlled, in con- After the plasma is attained to the stable generation by the sideration of amounts of the acid and the sample. warm-up driving for 15 ¿ 30 minutes, the optimum operat- The pretreatment method should be selected appropriate- ing conditions are adjusted for the intended use. Radio fre- ly, corresponding to the attribute of the sample and the tar- quency power output of 0.8 ¿ 1.4 kW, argon gas flow rate get element. Where the ignition or the digestion is done in for the cooling gas of 10 ¿ 18 L/min, for auxiliary gas of the open system, be careful to the vaporization loss of the 0 ¿ 2 L/min, and for carrier gas of 0.5 ¿ 2 L/min, sample target element or the contamination from the operating en- introduction of 0.5 ¿ 2 mL/min are usual operating condi- vironment. Furthermore, where the residues obtained by the tions. Where the radial viewing method is applied, the obser- ignition or the digestion are dissolved in acid solution and vation height is adjusted to 10 ¿ 25 mm above the induction prepared to the sample solution, if necessary, the filtration coil, and the axial viewing is applied, the optical axis is with membrane filter (pore size: 1 mm) may be useful. adjusted to obtain the greatest emission intensity. The integration time is set appropriately between 1 and several de- 3. Procedure cades of second, in consideration of the stability of the emis- 3.1. Performance test of the spectroscope sion intensity. 3.1.1. Wavelength calibration As the analytical line of the individual target element, the The wavelength calibration should be suitably performed representative emission lines are shown in Table 1. However, according to the indicated methods and procedures of the where the concentration of the target element is markedly apparatus, since there is specific calibration method of the high, it is recommended either to dilute the sample solution individual instrument. For example, some of calibration or to select an appropriate another spectral line, based on the methods are available, such as a method of using standard expected concentration of the element. Furthermore, when solution containing several elements, spectral lines of mer- the spectral interferences due to coexisting elements are oc- cury lamp, and emission lines of argon gas, etc. curred, another spectral line should be selected so as to be 3.1.2. Wavelength resolution free from the interference. The wavelength resolution is usually specified by the half Where this test method is specified in the respective mono- wavelength (nm) of the spectral lines for some representative graph, the necessary operating conditions such as the ana- elements. During the lower and the higher wavelength, fol- lytical line (nm), the radio frequency power unit (kW), argon lowing spectral lines of As (193.696 nm), Mn (257.610 nm), gas flow rate (L/min) etc., should be specified for the in- Cu (324.754 nm), and Ba (455.403 nm) are usually selected. tended use. However, all the specified conditions except for The specification of the half wavelength for the individual the analytical line are the referring ones, and the optimiza- element is responsible to the user, because the characteristics tion of those conditions are expected to be found according of the instrument and the spectroscope are intrinsic and to the individual instrument and the observation way etc. different each other. 3.4. Water and reagents However, in the case of the simultaneous analysis type of Water and reagents for this test are as follows. instrument fitted with semiconductor detector, this specifica- (i) As for water, use water for ICP analysis described tion is not always required. below, 3.2. Preparation of sample solution and standard solution Water for ICP analysis: The electric conductivity should Sample of drug substances, preparations, and crude drugs be below 1 mS･cm－1 (259C). Further it should be con- are pretreated by using anyone of the method described in firmed that the contaminated impurities do not inter- the above section (2. Pretreatment of sample) and prepared fere with the emission of the target element. to the sample solution. Usually, the sample solution is pre- (ii) The quality of reagents should be suitable, as it does pared to as a dilute nitric acid solution, while hydrochloric not contain the interfering substances of the test. acid is also available in place of nitric acid. (iii) As for argon gas, use that specified below, Where the reference standard solutions are specified in the Argon gas: The purity should be not less than 99.99 Japanese Pharmacopoeia (JP) and/or the Japanese Industri- volz, as specified in JIS K 1105. Either the liquefied al Standard (JIS), the standard solutions are prepared by argon or pressurized argon gas can be used. diluting those official standards to the definite concentra- 3.5. Procedure tions with the water for ICP analysis etc. When those refer- After confirmation of the normal action of the instrumen- ence standard solutions are not specified both in JP and JIS, tal parts usually switched on electrically, the electric source the reference substances certified and provided by the offi- of the instrument body and the surrounding equipments cial organization or the academic association etc.,areused should be switched on. After setting argon gas flow rate to for the preparation of the standard solutions. Where the ref- the decided value, switch on the radio frequency source, and erence standard solutions or the reference substances are not generate argon plasma. After confirming the stable plasma is given by the above mentioned official or semi-official or- given, the sample solution and the standard solution pre- ganizations, either the pure metals or the compounds con- pared as specified in the individual monograph, are intro- JP XVI General Information / Physics and Chemistry 2139

Table 1 The representative analytical line (nm) of various In case of the assay test, one of the standard solutions for metal elements the calibration curve, which are specified in the monograph, can be selected as a test solution of the system repeatability. Al 396.153 Fe 259.940 Os 225.585 Sn 189.980 As 193.759 Hg 184.950 Pb 220.351 Sr 407.771 5. Interferences and their reduction or correction B 249.773 In 230.606 Pd 340.458 Tl 276.787 Interferences accompanied to ICP-AES are a general ex- Ba 455.404 Ir 224.268 Pt 214.423 V 309.311 pression of the negative effects caused by the coexisting sub- Be 313.042 Li 670.784 Rb 780.023 W 207.911 stances or the matrix containing the target element. Various Cd 214.438 Mg 279.553 Rh 233.477 Zn 213.856 interferences are classified to the spectral interference and Co 228.616 Mn 257.610 Ru 240.272 the non-spectral one, and in the latter there are interferences Cr 205.552 Mo 202.030 Sb 206.833 such as the physical interference and the ionization interfer- Cu 324.754 Ni 221.647 Se 196.090 ence etc. Those interferences can be removed or reduced by the application of a suitable reduction or correction methods. 5.1. Physical interference duced into the plasma, and the emission intensity is meas- When there are differences of physical properties such as ured at the indicated analytical line. Where the qualitative the viscosity, the density, and the surface tension etc. be- test for the confirmation or the identification is performed, tween the sample solution and the standard solution, the the measurement wavelength range of the emission spectra analytical result might be affected by the differences, due to should be sufficiently wide, in which all the specified analyti- the induction of the difference in the sample nebulization cal lines are included. efficiency. It is called the physical interference. In order to Where the emission lines in vacuum UV region are meas- remove or reduce this kind of interference, dilute the sample ured by using the vacuum type spectroscope, the air around solution until the interference cannot occur, or conform the the optical axis between the emission part and the spectro- liquid characteristics of the sample solution to those of the scope part should be substituted for argon or nitrogen gas. standard solution (Matrix matching method ). Separately, it 4. System suitability is a useful correction method to adopt the internal standard When this test is applied to the limit test or the quantita- method (Intensity ratio method) or the standard addition tive test of metal elements etc., it is required to confirm pre- method as an assay method. viously whether the performance ability of the apparatus is 5.2. Ionization interference adequate or not by the system suitability test specified The ionization interference indicates the effect that the below. However, in the assay test, the test for 4.1. Confir- marked increase of electronic density in plasma may affect mation of detection and evaluation of linearity, is not the ionization ratio of the target element, where the coexist- required. ing element highly contained in the sample solution are easily 4.1. Confirmation of detection and evaluation of linearity ionized and release a great number of electrons in the plas- In the limit test of metal elements etc., where the pretreat- ma. The reduction or the correction method against the ioni- ment method of the sample specimen is specified in the re- zation interference effect is essentially the same as that spective monograph, it can be speculated what is the concen- described in the section 5.1 Physical interference. Further- tration of the specified limit in the sample solution (mg/mL). more, the ionization interference is moderately reduced by Assuming that the concentration of the target element in the the selection of the observation way and the adjustments of standard solution is 10 ¿ 20timesofthatofthesampleso- the observation height, the radio frequency power output, lution, the standard solution of the target element can be and the flow rate of carrier gas, etc. prepared, as mentioned in the section 3.2. Next, diluting this 5.3. Spectral interference standard solution to 1/10 times of the concentration, this is The spectral interference indicates that analytical results the system suitability test solution. are affected by the overlapping of various emission lines or Under the optimum conditions for an individual instru- continuous spectra to the analytical line of the target element, ICP emission spectra of the standard solution and the ment. The causes and the correction methods to this interfer- system suitability test solution of the target element are ence are shown below. measured and the followings should be confirmed. Regard- 5.3.1. Interference by the coexisting elements ing the system suitability test solution, it should be con- This interference occurs when the emission line of coexist- firmed that the spectra of the target element are clearly ob- ing element in the sample solution come close to the analyti- served at the specified wavelength, and the emission intensity cal line of the target element. The degree of the interference is within a specified range (for example, 80 ¿ 120z)com- depends on the resolution ability of the spectroscope, the pared to the theoretical emission intensity, which is calcu- wavelength difference between two emission lines, and the lated by multiplying the concentration of the standard solu- intensity ratio. To avoid this interference another analytical tion by the dilution ratio. line can also be available. Where an appropriate analytical 4.2. System repeatability line cannot be found, it is necessary to do the spectral inter- As for the standard solution of the target element ference correction. described in the section 4.1, when the emission intensity The inter-element correction is one of the correction measurement is repeated 6 times under the optimum condi- methods for the spectral interference. If the effect of coexist- tions for an individual instrument, the relative standard de- ing element on the analytical line of the target element are viation of the emission intensity for the target element measured in advance as a function of the emission intensity should be below a certain specified value (for example, not or the concentration by using the known concentration of more than 3.0z). two-component or multi-component standard solution, the 2140 Physics and Chemistry / General Information JP XVI degree of interference on the emission intensity of the ana- suitable concentration, in consideration of a permitted limit lytical line or the concentration will be speculated by measur- of the respective element, specified separately. However, ing the emission intensity of the coexisting element together when the multi-component standard solution is prepared, it with measurement of the target element. Separately, the should be confirmed beforehand that any trouble such as matrix matching method or the mathematical spectral sepa- coprecipitation does not occur. ration technique, by which the spectra of coexisting element The confirmation test of the target element should be done superimposed to the analytical line are separated, can also be according to the section 6.1.1, and the impurity content of used for the correction. each element in the sample specimen is roughly estimated by 5.3.2. Background interference the ratio of emission intensity of the sample solution and the The background interference indicates the following effect standard solution at the analytical line (1 point calibration that analytical results are affected by the increase of back- method). ground due to the emission lines caused by the element high- 6.2. Quantitative analysis ly contained in the sample. In this case, this effect will be re- The quantitative evaluation of inorganic impurities in the moved by applying the following correction method. Based sample specimen is usually done by measuring the emission on the background behavior before and after the analytical intensity during a certain time of integration. line, estimate the background intensity at the position of the 6.2.1. Calibration curve method analytical line, and obtain an intrinsic emission intensity of Regarding to the target element, not less than 4 kinds of the target element by subtraction of this background inten- the calibration standard solutions with different concentra- sity from the apparent emission intensity. tion are prepared. By using these calibration standards, plot Furthermore where the pretreatment of organic sample is the emission intensity at the analytical line versus the concen- not complete, the molecular band spectra (NO, OH, NH, tration to obtain the calibration curve. After measuring the CH, etc.) generated by elements of N, O, H, C in the sample emission intensity of the sample solution, the concentration solution may happen to affect the intensity of the analytical of the target element is determined by the calibration curve. line. This kind of interference is moderately reduced by the 6.2.2. Internal standard method selection of the observation way and the adjustments of the Regarding to the target element, not less than 4 kinds of observation height, the radio frequency power output, and calibration standard solutions with different concentration, the flow rate of carrier gas, etc. in which a definite concentration of internal standard is contained, are prepared. Usually, yttrium (Y) is used as an inter- 6. Qualitative and quantitative analysis nal standard. By using these calibration standards, plot the 6.1. Qualitative analysis ratio of emission intensity of the target element and the in- 6.1.1. Identification of inorganic impurities such as metal ternal standard versus the concentration to obtain the elements calibration curve. In the preparation of the sample solution, In JP, the confirmation test for drug substances is usually the internal standard element is added to give the same con- specified by applying the method of spectral analysis, by centration as the calibration standards. After measuring the which the structural features of the target molecule can be ratio of the emission intensity of the target element and the totally found. Drug substances frequently contain specific internal standard, the concentration of the target element is elements such as N, S, P, halogens, and metals etc. in their determined by the calibration curve. molecule, which are required for their existence in the in- Applying this method to inorganic impurity analysis, it dividual specification. Where the existence is not confirmed should be confirmed that the internal standard element is not by such as spectral analyses, it is required for their confirma- contained in the sample. As for the internal standard ele- tion by using chemical reactions. Thus the present method ment, it is expected that changes of the emission intensity can be applied to their confirmation test, except for nitrogen due to the operational conditions and the solution character- (N). istics, are similar to those of the target element, and the When a few of wavelengths and their relative intensity of selection of emission line is necessary so as not to give the the target element in the sample solution coincide with those spectral interference to the analytical line. of the standard solution, the existence of the target element 6.2.3. Standard addition method is confirmed. In place of the standard solution, the spectra Take not less than 4 sample solutions of equivalent library attached to the individual instrument or the wave- volume, add the target element to these solutions to prepare length table provided by the academic society, can also be the calibration standard solutions with different concentra- used for the confirmation. tions containing zero addition. After measuring the emission 6.1.2. Profile analysis intensity of these calibration standards, plot the emission in- If the target elements such as metal catalysts, inorganic tensity at the analytical line versus the concentration. The elements, and other high toxicity elements such as Pb and concentration of the target element is determined to be the As, which should be ordinarily controlled in view of the drug concentration, at which the regression line and the abscissa safety, are decided to be controlled, the profile analysis of cross each other. those inorganic impurities can be performed by the present This method can be applied only to the case of analysis method as a part of the good manufacturing practice of drug with no spectral interference, or where the background and substances. the spectral interference are exactly corrected, and the rela- Although the analytical line of the individual element can tion between the emission intensity and the concentration be selected referring to Table 1, an appropriate another keeps good linearity. emission line can also be available, when the spectral interferences are supposed to affect the analytical results. The References standard solution of the individual element is adjusted to a 1) Japanese Industrial Standard: General Rules for Atomic JP XVI General Information / Physics and Chemistry 2141

Emission Spectrometry (JIS K 0116), Japanese Standard maceutical field include qualitative or quantitative evalua- Association (2003). tion of ingredients, additives or water contents of active 2) US Pharmacopeia 31 (2008), <730> PLASMA SPEC- substances or preparations. Furthermore, near-infrared TROCHEMISTRY. spectrometry can also be used for evaluation of physical 3) European Pharmacopoeia 6.0 (2008), 2.2.57 INDUC- conditions of substances, such as crystal forms, crystallinity, TIVELY COUPLED PLASMA-ATOMIC EMISSION particle diameters. It is also possible to perform spectrome- SPECTROMETRY. try on samples that are located in a remote location away 4) Data Book on Analytical Chemistry, Ed. by The Japan from equipment main units, without sampling, by using Society for Analytical Chemistry, pp. 88 – 90 (2004), optical fibers. It can therefore be used as an effective means Maruzen Press. to perform pharmaceutical manufacturing process control 5) European Medicines Agency: Guideline on the specifica- online. tion limits for residues of metal catalysts or metal rea- 1. Equipment gents (2008). Near-infrared spectrophotometers can either be a distributed near-infrared spectrophotometer or a Fourier transform near-infrared spectrophotometer1). Interference filter- Near Infrared Spectrometry type near-infrared spectrophotometers that use interference filter in the spectrometry section are also available, however, Near infrared spectrometry (NIR) is one of spectroscopic this type of equipment is hardly used in the field of phar- methods used to qualitatively and quantitatively evaluate maceutical quality control. substances from analysis of data obtained by determining 1.1. Distributed near-infrared spectrophotometer their absorption spectrum of light in the near-infrared range. This equipment is comprised of light source section, sam- The near-infrared range lies between the visible light and ple section, spectrometry section, photometry section, signal infrared light, typically of wavelengths (wave numbers) be- processing section, data processing section, display-record- tween 750 and 2500 nm (13333 – 4000 cm－1). The absorption output section. Halogen lamps, tungsten lamps, light emit- of near-infrared light occurs due to harmonic overtones ting diodes and other such devices that can emit high inten- from normal vibration or combination tones in the infrared sity near-infrared light in a stable manner are used in the range (4000 to 400 cm－1), primarily absorption of O–H, light source section. The sample section is comprised of a N–H, C–H and S–H that involve hydrogen atoms, in par- sample cell and a sample holder. Equipment that have an ticular. For instance the asymmetrical stretching vibration of optical fiber section that is comprised of optical fibers and a N–H occurs in the vicinity of 3400 cm－1, but the absorption collimator are equipped with a function for transmitting due to the first harmonic overtone occurs in the vicinity of light to sample section, which is remotely located away from 6600 cm－1 (wavelength 1515 nm), which is near double 3400 the spectrophotometer main unit. Quartz is ordinarily used cm－1. as material for optical fibers. Absorption in the near-infrared range is far weaker than The spectrometry section is intended to extract light of re- absorption due to normal vibration that occurs in the in- quired wavelength, using dispersive devices and is comprised frared range. Furthermore, in comparison with visible light, of slits, mirrors and dispersive devices. Potential dispersive near-infrared light has longer wavelength, which makes it devices include prisms, diffraction grating, acousto-optical possible for the light to penetrate to a depth of several mm tunable filters (AOTF), or liquid crystal tunable filters into solid specimens including fine particles. This method is (LCTF). often utilized as a nondestructive analysis, as changes occur- The photometry section is comprised of detectors and am- ring with absorbed light spectrum (transmitted light or plifiers. Sensors include semiconductor detectors (silicon, reflected light) in this process provide physical and chemical lead sulfide, indium-gallium-arsenic, indium-antimony), as information pertaining to specimens. well as photomultiplier tubes. Detecting methods that use Conventional spectrometry, such as calibration curve semiconductor detectors generally perform detections with method, is used as a method for analyzing near-infrared single elements, but there are also occasions where array- absorption spectrum whenever applicable. Ordinarily, type detectors that use multiple elements are used. Such de- however, chemometrics methods are used for analysis. tectors are capable of simultaneously detecting multiple Chemometrics ordinarily involve quantification of chemical wavelengths (wave numbers). The signal processing section data, as well as numerical and statistical procedures for separates signals required for measurements from output sig- computerization of information. Chemometrics for near- nals fed by amplifiers and then outputs such isolated signals. infrared spectrometry includes various types of multivariate The data processing section performs data conversions and analysis such as multiple regression analysis, to perform spectral analysis, etc. The display-record-output section out- qualitative or quantitative evaluation of active substances. puts data, analysis results and data processing results to a Near-infrared spectrometry is used as a rapid and nondes- printer. tructive method of analysis that replaces conventional and 1.2. Fourier transform near-infrared spectrophotometer established analysis methods for water determinations or The configuration of the equipment is fundamentally substance verifications. It is necessary to perform a compari- same as that of the distributed-type equipment described in son test to evaluate this method against an existing analysis Section 1.1, except for the spectrometry section and the sig- method, to verify that this method is equivalent to such nal processing section. existing analysis method, before using this analysis method The spectrometry section is comprised of interferometers, as a quality evaluation test method in routine tests. sampling signal generators, detectors, amplifiers, A/D con- Applications of near-infrared spectrometry in the phar- version devices, etc. Interferometers include Michelson inter- 2142 Physics and Chemistry / General Information JP XVI ferometers, transept interferometers and polarization inter- Ar ＝ log (1/r) ＝ log (Ir/I) ferometers. The signal processing section is equipped with The intensity of diffuse reflectance spectrum can also be functions that are required for spectrometer, as well as a expressed with the Kubelka-Munk (K-M) function. The K-M function for translating acquired interference waveform function is derived, based on the existence of a sample with (interferogram) into absorption spectrum by Fourier trans- sufficient thickness, and expressed in terms of light scatter- formation. ing coefficient, which is determined by absorptivity, grain 2. Determination size, shape and fill condition (compression). There are three types of measurement methods that are This method is applied to solid samples, including fine used with near-infrared spectrometry: transmittance particles, and requires a diffuse reflector. method, diffuse reflectance method and transmittance 2.3. Transmittance reflectance method reflectance method. The selection of measurement methods The transmittance reflectance method is a combination of relies on the shape of samples and applications. The trans- the transmittance method and reflectance method. A mirror mittance method or diffuse reflectance method is used for is used to re-reflect a light that has passed through a sample solid samples, including fine particles. The transmittance in order to take a measurement of transmittance reflectance method or transmittance reflectance method is used for liq- rate, T * (z). Light path must be twice the thickness of the uid samples. sample. On the other hand, the light reflected off a mirror 2.1. Transmittance method and enters into a detector is used as the control light. When The degree of decay for incident light intensity as the light this method is applied to suspended samples, however, a from a light source passes through a sample, is represented metal plate or a ceramic reflector with rough surface that as transmittance rate T (z) or absorbance A with the trans- causes diffuse reflectance is used instead of a mirror. mittance method. A sample is placed in the light path be- Transmittance reflectance absorbance (A*) is obtained by tween a light source and a detector, the arrangement of the following formula with this method: which is ordinarily same as that of the spectroscopic method. T * ＝ 100t*

T ＝ 100t t* ＝ I/IT －acl T ＝ I/I0 ＝ 10 I: Intensity of transmitted and reflected light, in cases I0: Incident light intensity where a sample is placed I: Transmitted light intensity lT: Intensity of reflected light, in cases where is no sample a: Absorptivity A* ＝ log (1/t*) c: Solution concentration l: Layer length (sample thickness) This is a method that is applied to solid samples, including fine particles, as well as liquids and suspended samples. The A ＝－log t ＝ log (1/t) ＝ log (I /I) ＝ acl 0 thickness of a sample must be adjusted when applying this This method is applied for taking measurements of sam- method to a solid sample. Ordinarily adjustment is made by ples that are liquids and solutions. Quartz glass cells and setting absorbance to 0.1 – 2 (transmittance of 79 – 1z), flowcellsareused,withthelayerlengthof1–5mmalong. which provides the best linearity and S/N ratio of detector. Furthermore, this method can also be applied for taking A cell with appropriate layer length, according to the grain measurements of samples that are solids, including fine par- size of the fine particle, must be selected when applying the ticles. It is also known as diffuse transmittance method. method to a fine particle sample. Selecting appropriate layer length is critical for this method, 3. Factors that affect spectrum since the transmitted light intensity varies depending on Following items must be considered as factors that can af- grain sizes and surface conditions of samples. fect spectrum when applying near-infrared spectrometry, 2.2. Diffuse reflectance method particularly when conducting quantitative analysis. The ratio of the reflection light intensity I, emitted from (i) Sample temperature: A significant change (wave- the sample in a wide reflectance range and a control reflec- length shift, for example) can occur when the temperature tion light intensity I emitted from surface of a substance, is r varies by a several degree (9C).Caremustbetaken,particu- expressed as reflectance R (z) with the diffuse reflectance larly when the sample is a solution or contains water. method. The near-infrared light penetrates to a depth of (ii) Water or residual solvent: Water or residual solvent several mm into solid samples, including fine particles. In contents of a sample, as well as water (humidity) in the en- that process, transmission, refraction, reflection and disper- vironment wherein measurements are taken, can potentially sion are repeated, and diffusion takes place, but a portion of significantly affect absorption band of the near-infrared the diffused light is emitted again from the surface of the range. sample and captured by a detector. The spectrum for the (iii) Sample thickness: The thickness of a sample is a diffuse reflectance absorbance (A ) can ordinarily be ob- r factor for spectral changes and therefore needs to be contained by plotting logarithm of inverse numbers for reflec- trolled at a certain thickness. A sample may be considered to tance (1/r) against wavelengths (wave numbers). be of adequate thickness for the diffuse reflectance method, R ＝ 100r however, if the thickness is less than a certain amount, for

r ＝ I/Ir example, the sample may have to be placed on a support I: Reflection light intensity of light, diffuse reflected off plate with high reflectance to take measurements by the the sample transmittance reflectance method.

Ir: Control reflection light intensity of light emitted from (iv) Fill condition of sample: The condition of sample surface of reference substance fill can potentially affect spectrum, when taking measure- JP XVI General Information / Physics and Chemistry 2143 ments of samples that are solids or fine particles. Care must standard plates with no less than 4 levels of concentration be taken when filling samples in a cell, to ensure that a cer- within the reflectance of 10 – 90z must be used. When tain amount is filled through a specific procedure. measurements are expected to be taken with absorbance of (v) Optical characteristics of samples: When a sample no less than 1.0, it is necessary to add standard plates with is physically, chemically or optically uneven, relatively large reflectance of either 2z or 5z or both. beam size must be used, multiple samples must be used, In order to plot absorbance (AOBS) of such standard plates measurements must be taken at multiple points on the same at locations in the vicinity of wavelengths 1200 nm, 1600 nm sample, or a sample must be pulverized to ensure averaging and 2000 nm against absorbance (AREF) assigned to each of the sample. Grain size, fill condition, as well as roughness standard plate, verifications must be made to ensure that the of surface can also affect fine particle samples. gradient of linearity obtained are ordinarily within the range (vi) Crystal forms: Variations in crystal structures 1.0 ± 0.05 for each of these wavelengths and 0 ± 0.05 for (crystal forms) can also affect spectrum. In cases where mul- ordinate intercept. tiple crystal forms exist, it is necessary to have consideration 4.3. Spectrophotometric noise for characteristics of samples to be considered and care must The spectrophotometric noise of the equipment can be be taken to ensure that even standard samples for calibration checked using appropriate reflectance standard plates, such curve method have diversified distributions similar to that of as white-colored reflecting ceramic tiles or reflective ther- samples that are subject to analysis. moplastic resin (such as polytetrafluoroethylene). (vii) Temporal changes in characteristics of samples: 4.3.1. High flux noise Samples can potentially undergo chemical, physical or Spectrophotometric noise is evaluated by using standard optical property changes, due to passing of time or storage plates with high reflectance, such as reflectance of 99z. after sampling, and such changes affect spectrum in a subtle Standard plates are used to take measurements for both sam- manner. For instance even with identical samples, if elapsed ples and control samples. Generally, the average value ob- times differ, then their characteristics of near-infrared spec- tained from calculation of mean square root (RMS) of noise trum can vary significantly. In creating calibration curves, for each 100 nm segments in the wavelength range of 1200 – therefore, measurements must be taken offline in a laborato- 2200 nm ordinarily must not be more than 0.3 × 10－3 and ry or online in manufacturing process (or inline) and samples individual values must not exceed 0.8 × 10－3. for calibration curves must be prepared with adequate con- RMS ＝ s1/N・S(A － A )2t1/2 siderations for the passing of time before measurements are i m taken. N: Number of measurement points per segment A : Absorbance at each measurement point of segment 4. Control of equipment performance2,3) i A : Average absorbance for segment 4.1. Accuracy of wavelengths (wave numbers) m The accuracy of wavelengths (wave numbers) of an equip- 4.3.2. Low flux noise ment is derived from the deviation of substances for which Spectrophotometric noise is evaluated by using standard peak absorption wavelengths (wave numbers) have been plates with low reflectance, such as reflectance of 10z, defined, such as polystyrene, mixture of rare earth oxides when the amount of light is low. In such cases, light source, (dysprosium, holmium and erbium; 1:1:1) or steam, from optical system, detector and electronic circuit systems all the figures indicated on the equipment. Tolerance figures in have some impact on noise. Similar to the cases of high flux the vicinity of 3 peaks are ordinarily set in the following noise, generally, the average value obtained from calculation manner: of RMS for each 100 nm segments in the wavelength range 1200 ± 1 nm (8300 ± 8cm－1) of 1200 – 2200 nm ordinarily must not be more than 1.0 × 1600 ± 1 nm (6250 ± 4cm－1) 10－3 and individual values must not exceed 2.0 × 10－3. 2000 ± 1.5 nm (5000 ± 4cm－1) 5. Application to qualitative or quantitative analysis Since the location of absorption peaks vary, depending on Unlike in the infrared range, mainly harmonic overtones the substance used as reference, absorption peaks of wave- and combinations manifest as spectrum in the near-infrared lengths (wave numbers) that are closest to the above 3 peaks range. Such absorbance spectrums are often observed as are selected for suitability evaluations. A mixture of rare overlay of absorption bands of functional groups and atomic earth oxides, for instance, would indicate characteristic groups. The near-infrared spectrometry, therefore, differs absorption peaks at 1261 nm, 1681 nm and 1971 nm. from conventional analysis methods and it is usually neces- Absorption peaks at 1155 nm, 1417 nm, 1649 nm, 2352 sary to establish analysis methods that correspond to each nm (layer length: 1.0 mm) can be used, when taking meas- application, by preparing model analysis methods using urements with transmittance method that involve the use of methodologies of chemometrics, such as multivariate dichloromethane as reference. The absorption peak of steam analysis. at 7306.7 cm－1 can be used with a Fourier transformation- Characteristics of near-infrared absorption spectrum must type spectrophotometer, as its wave number resolution be emphasized and effects of complexities of spectrums, as ability is high. well as overlay of absorption bands must be reduced by Other substances can also be used as reference, so long as performing mathematical preprocesses, such as primary or their adequacy for the purpose can be verified. secondary spectral differentiation processes or normaliza- 4.2. Spectroscopic linearity tions, which becomes one of vital procedures in establishing Appropriate standard plates, such as plate-shaped poly- analysis methods that use methodologies of chemometrics. mer impregnated with varying concentrations of carbon While there are many chemometrics methodologies and (carbon-doped polymer standards), can be used to evaluate mathematical preprocessing methods for data, appropriate spectroscopic linearity. In order to verify linearity, however, combinations must be selected that suit the purposes of 2144 Physics and Chemistry / General Information JP XVI intended analysis. Chemometrics methodologies for obtaining quantitative Evaluation of validity based on analysis parameters is models include multiple regression analysis method, main ordinarily required for the analysis validation when estab- ingredient regression analysis method and PLS (partial least lishing a near-infrared analysis method. Selection of squares) regression analysis method. parameters that are appropriate for applications must be In cases where the composition of a sample is simple, con- made for its intended use. Furthermore, following issues centrations of ingredients in the sample that are subject to must be considered, in conformity with attributes of the analysis can be calculated, by plotting a calibration curve near-infrared spectrometry. using the absorbance of a specific wavelength (wave number) (i) Whether or not wavelengths (wave numbers) intended or the correlating relationship between the parameters and for the particular analysis method, are suitable for evalua- concentration, using samples for preparation of calibration tion of characteristics of a sample in performing analysis curves with known concentrations (calibration curve under given conditions. method). (ii) Whether or not the method is adequately robust to 6. Reference deal with variables such as handling of samples (for instance 1) General Rules for Near-infrared Spectrophotometric fill condition for fine particle samples, etc.) and configura- Analysis, JIS K 0134 (2002), Japanese Industrial Stand- tion matrix. ards (iii) Whether or not about the same level of accuracy or 2) Near-infrared Spectrophotometry, 2.2.40, European precision can be obtained, in comparison with the existing Pharmacopoeia 5.0 (2005) and established analysis methods, which are available as 3) Near-infrared Spectrophotometry, <1119>,USPhar- standards. macopoeia 30 (2007) (iv) Sustaining and managing performance of an analysis method, once established, are critical. Continuous and systematic maintenance and inspection work must therefore be implemented. Furthermore, it must be determined whether pH Test for Gastrointestinal or not appropriate evaluation procedures are available to deal with change controls or implementation of re-validation Medicine on changes made in manufacturing processes or raw mate- In this test, medicine for the stomach and bowels, which is rials, as well as changes arising from replacement of major said to control stomach acid, is stirred in a fixed amount of components in equipment. the 0.1 mol/L hydrochloric acid for a fixed duration, and (v) Whether or not there are appropriate evaluation the pH value of this solution is obtained. The pH value of a procedures in place to verify validity of transferring im- stomach medicine will be based on the dose and the dosage plementation of an analysis, which presupposes the use of a of the medicine (when the dosage varies, a minimum dosage specific equipment, from such originally intended equipment is used) and expressed in the pH value obtained from the test to another equipment (model transfer) for the purpose of performed by the following procedure. sharing the analysis method. 5.1. Qualitative analysis 1. Preparation of Sample Qualitative analysis, such as verification of substances, is Solid medicine which conforms to the general regulations performed after preparing a reference library that includes for medicine (the powdered medicine section) can be used as inter-lot variations within tolerance range and chemometrics a sample. When the medicine is in separate packages, the methodologies, such as multivariate analysis, have been contentof20ormorepackagesisaccuratelyweighedtocal- established. Minute quality characteristic variations between culate the average mass for one dose and mixed evenly to lots can also be estimated by using this method. make a sample. For granules and similar types in separate Furthermore, multivariate analysis includes direct analysis packages, among the solid medicine which does not conform methods that consider wavelengths (wave numbers) and to the general regulations for medicine (the powdered medi- absorptions as variables, such as wavelength correlation cine section), the content of 20 or more packages is accu- method, residual sum of squares, range sum of squares, rately weighed to calculate the average mass for one dose along with factor analysis method, cluster analysis method, and is then powdered to make sample. For granules and discriminant analysis method, as well as SIMCA (soft in- similar types not in separate packages, among solid medicine dependent modeling of class analogy). which does not conform to the general regulations for medi- It is also possible to consider the overall near-infrared cine (the powdered medicine section), 20 doses or more are absorption spectrum as a single pattern and to identify powdered to make a sample. For capsules and tablets, 20 parameters obtained by applying multivariate analysis doses or more are weighed accurately to calculate the methods or characteristic wavelength (wave number) peaks average mass for one dose or average mass and then pow- of the sample substance as indices for monitoring, for the deredtomakeasample. purpose of manufacturing process control for active sub- Liquid medicine is generously mixed to make a sample. stances or preparations. 2. Procedure 5.2. Quantitative analysis Put 50 mL of the 0.1 mol/L hydrochloric acid with the Quantitative analysis uses spectrums of sample groups and molarity coefficient adjusted to 1.000, or equivalent 0.1 analysis values obtained through the existing and established mol/L hydrochloric acid with its volume accurately meas- analysis methods, to obtain quantitative models with ured in a 100-mL beaker. Stir this solution with a magnetic methodologies of chemometrics. These are used to calculate stirrer and a magnetic stirrer rotator (35 mm length, 8 mm concentrations of individual ingredients and material values diameter) at the speed of about 300 revolutions per minute. of samples being measured, using conversion formulas. JP XVI General Information / Physics and Chemistry 2145

While stirring, add the accurately weighed one-dose sample. level based on the data when suitability of the method for After 10 minutes, measure the pH value of the solution using the evaluation of quality of the drug was verified, and the the pH Determination. The solution temperature should be precision necessary for the quality test''. maintained at 37 ± 29C throughout this operation. Based on the above description, an allowable limit of system repeatability for 6 replicate injections should be set in consideration with the following descriptions. However, in the case that the test method prescribed in the JP monograph System Suitability is used for the test, the allowable limit of system repeatability prescribed in the monograph should be applied. In order to ensure the reliability on the results of drug ana- (i) Assay for drug substance (for drug substance with the lyses, it is essential to verify that the test method to be ap- content nearby 100z): An adequate allowable limit should plied to the test, including the method prescribed in the be set at the level that the chromatographic system is able to Japanese Pharmacopoeia (JP), can give the results adequate give the precision suitable for the evaluation of variation in for its intended use using the analytical system in the labora- the content of active ingredient within and among the batch- tory in which the test is to be performed, then to carry out es of drug substance. For example, the allowable limit of system suitability testing for confirming that the analytical ``not more than 1.0z'' is usually recommended for the drug system maintains the state suitable for the quality test. substances whose width of content specification are not 1. Definition and role of system suitability more than 5z, as is in the case of content specification of ``System Suitability'' is the concept for ensuring that the 98.0 – 102.0z which is often observed in the assay using liq- performance of the analytical system is as suitable for the uid chromatography. analysis of the drug as was at the time when the verification (ii) Assay for drug products: An adequate allowable of the test method was performed using the system. limit should be set considering the width of content specifi- Usually, system suitability testing should be carried out at cation of the drug product and the allowable limit prescribed every series of drug analysis. The test procedures and accep- in the assay of drug substance (when the drug product is ana- tance criteria of system suitability testing must be prescribed lyzed by a method with the same chromatographic condi- in the test methods of drugs. The results of drug analyses are tions as those used for the analysis of drug substance). not acceptable unless the requirements of system suitability (iii) Purity test for related substances: An adequate have been met. allowable limit should be set considering the concentration System suitability testing is an integral part of test of active ingredients in the solution used for the system suita- methods using analytical instruments, and based on the con- bility testing. In the case that a solution with active ingredi- cept that the equipments, electronic data processing systems, ent concentration of 0.5 – 1.0z is used for the test of system analytical operations, samples to be analyzed and operators repeatability, an allowable limit of ``not more than 2.0z'' is constitute an integral system that can be evaluated, when the usually recommended. test procedures and acceptance criteria of system suitability Recommendations for allowable limits described above testing are prescribed in the test methods. should not be applicable to gas chromatography. 2.1.2. Method for decreasing the number of replicate injec- 2. Points to consider in setting system suitability tions without losing the quality of system repeatability test- Parameters of system suitability testing to be prescribed in ing the test method depend on the intended use and type of ana- It is described in the section of system suitability in ``Liq- lytical method. Since system suitability testing is to be car- uid Chromatography'' that ``In principle, total number of ried out in a routine manner, it is preferable to select the replicate injections should be 6. However, in the case that a parameters necessary for ensuring that the analytical system long time is necessary for one analysis, such as the analysis maintains the state suitable for the analysis of the drug and using the gradient method, or the analysis of samples con- to prescribe its test procedure able to carry out easily and taining late eluting components, it may be acceptable to rapidly. decrease the number of replicate injections by adopting new For example, in the case of quantitative purity tests using allowable limit of ``System repeatability'' which can guaran- liquid chromatography or gas chromatography, the evalua- tee a level of ``System repeatability'' equivalent to that at 6 tion of parameters such as ``System performance'' (to con- replicate injections.'' firm the ability to analyze target substance specifically), In consideration of the above description, a method for ``System repeatability'' (to confirm that the degree of varia- decreasing the number of replicate injections without losing tion in the analytical results of target substance in replicate the quality of system repeatability testing is adopted. One injections is within the allowable limit) and ``Test for re- can set the test for system repeatability with reduced number quired detectability'' (to confirm the linearity of chromato- of replicate injections by utilizing this method, if necessary, graphic response around the specification limit) are usually and can also apply it as an alternative for the method required. prescribed in a monograph. The followings are supplements to the section of system The following table shows the allowable limits to be at- suitability prescribed in ``Liquid Chromatography''. tained in the test at 3 – 5 replicate injections (n ＝ 3–5)to 2.1. System repeatability of HPLC and GC keep the quality test equivalent to that of test at n ＝ 6. 2.1.1. Allowable limit of system repeatability However, it should be kept in mind that since decrease in It is described in the section of system suitability in ``Liq- the number of replicate injections results in increase in the uid Chromatography'' that ``In principle, total number of weight of each injection, it becomes more important to per- replicate injections should be 6'', and ``The allowable limit form the test by the experienced operator, and to maintain of ``System repeatability'' should be set at an appropriate the equipment in a suitable state. 2146 Physics and Chemistry / General Information JP XVI

Table Allowablelimitstobeattainedinthe test at 3 – 5 replicate injections (n ＝ 3 – 5) to keep the quality of test equivalent to that of test at n ＝ 6*

Allowable limit (RSD)

Allowable limit prescribed in the test 1z 2z 3z 4z 5z 10z of n ＝ 6

n ＝ 50.88z 1.76z 2.64z 3.52z 4.40z 8.81z Allowable limit to be n ＝ 40.72z 1.43z 2.15z 2.86z 3.58z 7.16z attained n ＝ 30.47z 0.95z 1.42z 1.89z 2.37z 4.73z

* The probability for inadequate analytical systems to meet the requirements of system suitability testing, is supposed to be 5z.

3. Points to consider at the change of analytical system of aluminum have recently been reported in several coun- (Change control of analytical system) tries, testing methods for trace amounts of aluminum con- When the test method and analytical system verified is taminating TPNs are required for the official standard. The continuously used for the quality test without any change, it following three analytical methods are available: High- is sufficient to confirm the compliance to the requirements Performance Liquid Chromatography using a fluorescence of system suitability at every series of drug analysis. photometric detector (HPLC with fluorescence detection), However, when the test is performed for a long period, a Inductivity Coupled Plasma-Atomic Emission Spectrometry situation in which some changes in the analytical system are (ICP-AES method), Inductivity Coupled Plasma-Mass inevitable, may occur. These changes don't affect the quality Spectrometry (ICP-MS method). Detection sensitivity by of the product itself, but they affect the scale in the evalua- HPLC with fluorescence detection is about 1 mg/L (ppb), tion of product quality. If the change in the analytical system while ICP-AES fitted with special apparatus and ICP-MS may induce a significant deviation of the scale, it may lead to have higher sensitivity. the acceptance of products with inadequate quality and/or Since TPNs are nutrient preparations, they contain many the rejection of products with adequate quality. Thus, at the nutrients such as sugars, amino acids, electrolytes, etc., in time of change in the analytical system, it is necessary to various compositions. Thus, care is needed in the selection check whether the change is appropriate or not, to avoid the of a suitable analytical method, because these coexisting deviation of the scale in the evaluation of product quality. components may affect the measurement of trace amounts In the case of the change of test method, it is required to of aluminum. perform an adequate validation depending on the extent of In view of the general availability of HPLC apparatus, the the change. present general information describes procedures for the de- On the other hand, in the case of the change of analytical termination of trace levels of aluminum in TPNs by means system in a laboratory, such as renewal of apparatus or of HPLC with a fluorescence photometric detector, using column of liquid chromatography, and the change of opera- two kinds of fluorescent chelating agents, i.e., Quinolinol tor, it is necessary to perform at least system suitability test- complexing method, Lumogallion complexing method. ing using the system after change, and to confirm that the 1. Quinolinol complexing method equivalency of the results before and after change. After forming a complex of aluminum ion in the sample In the case that equivalent results would not be obtained solution with quinolinol, the assay for aluminum by HPLC after change, for example, when a renewal of column of liq- fitted with a fluorescence photometer is performed. uid chromatograph may induce a significant change of 1.1. Preparation of sample solution elution pattern, such as the reversal of elution order between Pipet 1 mL of the sample (TPNs) exactly, and after adding target ingredient of the test and substance for checking reso- 10 mL of water for aluminum test, make up the sample solu- lution, it is required to perform a revalidation of the analyti- tion to 10 mL exactly by adding the mobile phase. cal system for the test using new column, since it is uncertain 1.2. Preparation of a series of standard solutions for whether the specificity and/or other validation characteris- calibration curve tics necessary for estimating target ingredient is kept or not. Pipet 1 mL of water for aluminum test exactly, and after adding 10 mL each of standard solutions of aluminum (1)– (5), make up the standard solutions for calibration curve to Test for Trace Amounts of 10 mL (Aluminum concentration: 0, 1.25, 2.5, 5.0, and 10.0 ppb). Aluminum in Trans Parenteral 1.3. Standard testing method Nutrition (TPN) Solutions Pipet 0.1 mL each of the sample solution and standard solutions, and perform the test by HPLC under the following Trans parenteral nutrition solutions (TPNs) are nutrient conditions. Calculate the aluminum cotent in the sample so- preparations for intravenous injection. Since toxic effects to lution using a calibration curve method. the central nervous system, bone, etc. due to trace amounts Operating conditions— JP XVI General Information / Physics and Chemistry 2147

Detector: Fluorescence photometer(excitation wavelength: tion curve method. 380 nm, emission wavelength: 520 nm) Operating conditions— Column: A stainless steel column 4.6 mm in inside diame- Detector: Fluorescence photometer(excitation wavelength ter and 15 cm in length, packed with phenylsilanized silica 505 nm, emission wavelength 574 nm) gel for liquid chromatography (5 mm in particle diameter). Column: A stainless steel column 6.0 mm in inside diame- Column temperature: A constant temperature of about ter and 10 cm in length, packed with octylsilanized silica gel 409C. for liquid chromatography (5 mm in particle diameter). Mobile phase: A mixture of 8-quinolinol in acetonitrile (3 Column temperature: A constant temperature of about in 100) and diluted 0.5 mol/L ammonium acetate TS (2 in 5) 409C. (1:1). Mobile phase: Take 100 mL of 2-propanol, and add a Flow rate: Adjust the flow rate so that the retention time diluted 1 mol/L acetic acid-sodium acetate buffer solution of of aluminum/8-quinolinol complex is about 9 minutes. pH 5.0 (1 in 10) to make 1000 mL. System suitability— Flow rate: Adjust the flow rate so that the retention time The correlation coefficient of the calibration curve, which of aluminum/lumogallion complex is about 5 minutes. is prepared using a series of standard solutions, is not less System suitability— than 0.99. The correlation coefficient of the calibration curve, which Furthermore there is an alternative method, in which the is prepared using a series of standard solutions, is not less chelating agent 8-quinolinol is not included in the mobile than 0.99. phase. In this method also, aluminum is detected as a com- 3. Notes plex with 8-quinolinol in the sample solution by using HPLC (i) Regarding water, solvents, reagents, vessels and other fitted with fluorescence photometer. But it is necessary to tools used for the examination, select those not contaminat- form a more stable aluminum/8-quinolinol complex in the ed with aluminum. Further, keep the testing environment sample solution, because the chelating agent is not included clean and free from dust in the testing room. in the mobile phase. Further, since the analytical wavelength (ii) Before the measurement, it is necessary to confirm for the fluorescence detection is different from that in the that the characteristic properties of the sample do not affect standard method, excitation WL: 370 nm, emission WL: 504 the formation of the complex. nm, the detection sensitivity is different. Thus, it is appropri- (iii) Reference substances of river water for analysis of ate to obtain the calibration curve between 0 – 25 ppb of alu- trace elements, distributed by the Japan Society for Analyti- minum. Other than the above- mentioned differences, the cal Chemistry, contain certified amounts of aluminum: size of column, column temperature, and the mobile phase JSAC 0301-1 and JSAC 0302 (a known amount of alumi- are also different from those used in the standard method, so num is artificially added to JSAC 0301-1). suitable analytical conditions should be established for performing precise and reproducible examinations of trace 4. Standard Solutions, Reagents and Test Solutions amounts of aluminum in the sample specimen. Other than the standard solutions, reagents and test solutions specified in the Japanese Pharmacopoeia, those 2. Lumogallion complexing method described below can be used in this test. After forming a complex of aluminum ion in the sample (i) N,N-Bis(2-hydroxyethyl)-2-aminoethane sulfonic acid specimen with the fluorescent reagent of lumogallion, the so- C H NO S White crystals or powder. lution is examined by HPLC fitted with a fluorescence pho- 6 15 5 (ii) Hydrochloric acid for aluminum test Same as the tometer. reagent Hydrochloric acid. Further, it contains not more 2.1. Preparation of sample solution than 1 ppb of aluminum. Pipet 70 mL of the sample specimen (TPN) exactly, add (iii) Lumogallion [5-Chloro-2-hydroxy-3(2,4-dihydrox- 0.15 mL of lumogallion hydrochloric acid TS and 0.6 mL of yphenylazo)benzenesulfonic acid] C H ClN O SRed-brown buffer solution for aluminum test, pH 7.2 exactly, then mix 12 9 2 6 to dark brown powder. Further, it contains not more than 1 the solution. After this solution has been allowed to stand ppm of aluminum. for 4 hours at 409C, it can be used for the measurement as a (iv) Lumogallion hydrochloric acid TS Dissolve 0.86 g sample solution. of lumogallion in 300 mL of 2-propanol, and add 350 mL of 2.2. Preparation of a series of standard solutions for diluted Hydrochloric acid for aluminum test (9 in 50) and calibration curve Water for aluminum test to make 1000 mL exactly. Pipet 1 mL each of standard aluminum solutions (1) – (5) (v) Nitric acid for aluminum test Same as the reagent exactly, and add diluted nitric acid for aluminum test (1 in Nitric acid. Further, it contains not more than 1 ppb of alu- 100) to make exactly 100 mL. Pipet 70 mL each of these solu- minum. tions exactly, and add exactly 0.15 mL of lumogallion hy- (vi) pH buffer solution for aluminum test, pH 7.2 Dis- drochloric acid TS and exactly 0.6 mL of buffer solution for solve 106.6 g of N,N-bis(2-hydroxyethyl)-2-aminoethane sul- aluminum test, pH 7.2 then allow to stand for 4 hours at fonic acid in 800 mL of Water for aluminum test, adjust the 409C to make a series of standard solutions for obtaining the pH7.2byusingTetramethylammonium hydroxide aqueous calibration curve (Aluminum: 0, 1.07, 2.13, 4.27, and 8.54 solution,andaddWater for aluminum test to make 1000 ppb). mL. 2.3. Standard examination method (vii) Standard aluminum solution Pipet a constant Take 0.1 mL each of the sample solution and standard volume each of Water for aluminum test or the Standard aluminum solutions for the calibration curve, and perform aluminum stock solution, dilute and adjust the aluminum HPLC analysis under the following conditions. Calculate the concentration to 0, 1.25, 2.5, 5.0, and 10 ppm by using aluminum content in the sample solution by using a calibra- diluted Nitric acid for aluminum test (1 in 100), to make 2148 Physics and Chemistry / General Information JP XVI

Standard aluminum solutions (1) – (5). 2. Validation characteristics

(viii) Tetramethylammonium hydroxide TS (CH3)4NOH The definition of typical validation characteristics to be It is a 25z aqueous solution, prepared for aluminum test. assessed in validation of analytical procedures and examples Further, it contains not more than 1 ppb of aluminum. of assessing procedures are given below. (ix) Water for aluminum test It contains not more than The terminology and definitions of the validation charac- 1 ppb of aluminum. teristics may possibly vary depending upon the fields to which analytical procedures are applied. The terminology and definitions shown in this document are established for the purpose of the Japanese Pharmacopoeia. Typical Validation of Analytical Procedures methods for assessing the validation characteristics are shown in the item of assessment. Various kinds of methods The validation of an analytical procedure is the process of to determine the validation characteristics have been confirming that the analytical procedure employed for a test proposed and any methods that are widely accepted will be of pharmaceutics is suitable for its intended use. In other accepted for the present purpose. However, since values of word, the validation of an analytical procedure requires us the validation characteristics may possibly depend upon to demonstrate scientifically that risks in decision by testing methods of determination, it is required to present the caused by errors from analytical steps are acceptably small. methods of determining the validation characteristics, the The performance of an analytical procedure is established by data and calculation methods in sufficient detail. various kinds of validation characteristics. The validity of a Although robustness is not listed as a validation character- proposed analytical procedure can be shown by demonstrat- istic, it should be considered during the development of ana- ing experimentally that the validation characteristics of the lytical procedures. Studying the robustness may help to im- analytical procedure satisfy the standards set up according to prove analytical procedures and to establish appropriate the acceptable limits of testing. analytical conditions including precautions. When an analytical procedure is to be newly carried in the 2.1. Accuracy/Trueness Japanese Pharmacopoeia, when a test carried in the 2.1.1. Definition Japanese Pharmacopoeia is to be revised, and when the test The accuracy is a measure of the bias of observed values carried in the Japanese Pharmacopoeia is to be replaced with obtained by an analytical procedure. The accuracy is ex- a new test according to regulations in general notices, ana- pressed as the difference between the average value obtained lytical procedures employed for these tests should be validat- from a large series of observed values and the true value. ed according to this document. 2.1.2. Assessment 1. Required data for analytical procedures to be carried in The estimate of accuracy of an analytical method is ex- the Japanese Pharmacopoeia pressed as the difference between the total mean of observed 1.1. Outline values obtained during investigation of the reproducibility This section should provide a brief explanation of the and the true value. The theoretical value is used as the true principle of a proposed analytical procedure, identify the value (e.g., in the case of titration methods, etc.). When necessity of the analytical procedure and its advantage com- there is no theoretical value or it is difficult to obtain a theo- pared with other procedures, and summarize the validation. retical value even though it exists, a certified value or a con- When an analytical procedure is revised, the limitation of the sensus value may be used as the true value. When an analyti- current analytical procedure and the advantage offered by cal procedure for a drug product is considered, the observed the new analytical procedure should be described. value of the standard solution of the drug substance may be 1.2. Analytical procedure used as the consensus value. This section should contain a complete description of the It may be inferred from specificity data that an analytical analytical procedure to enable skilled persons to evaluate procedure is unbiased. correctly the analytical procedure and replicate it if neces- Theestimateofaccuracyanda95z confidence interval of sary. Analytical procedures include all important operating the accuracy should be calculated using the standard error procedures for performing analyses, the preparation of based on the reproducibility (intermediate precision). It standard samples, reagents and test solutions, precautions, should be confirmed that the confidence interval includes procedures to verify system suitability (e.g. the verification zero or that the upper or lower confidence limits are within of the separating performance of a chromatographic sys- the range of the accuracy required of the analytical proce- tem), formulas to obtain results, the number of replications dure. and so forth. Any instruments and apparatus that are not 2.2. Precision stated in the Japanese Pharmacopoeia should be described in 2.2.1. Definition detail. The physical, chemical or biological characteristics of The precision is a measure of the closeness of agreement any new reference standards should be clarified and their tes- between observed values obtained independently from multi- ting methods should be established. ple samplings of a homogenous sample and is expressed as 1.3. Data showing the validity of analytical procedures the variance, standard deviation or relative standard devia- This section should provide complete data showing the tion (coefficient of variation) of observed values. validity of the analytical procedures. This includes the ex- The precision should be considered at three levels with perimental designs to determine the validation characteris- different repetition conditions; repeatability, intermediate tics, experimental data, calculation results and results of precision and reproducibility. hypothesis tests. (i) Repeatability/Intra-assay precision The repeatability expresses the precision of observed values obtained from multiple samplings of a homogenous JP XVI General Information / Physics and Chemistry 2149 sample over a short time interval within a laboratory, by the 2.4. Detection limit same analyst, using the same apparatus and instruments, lots 2.4.1. Definition of reagents and so forth (repeatability conditions). The detection limit is the lowest amount or concentration (ii) Intermediate precision of the analyte in a sample that is detectable, but not neces- The intermediate precision expresses the precision of ob- sarily quantifiable. served values obtained from multiple samplings of a 2.4.2. Assessment homogenous sample by changing a part of or all of the oper- The detection limit should be normally determined so that ating conditions including analysts, experimental dates, ap- producer's and consumer's risks are less than 5z. The detec- paratus and instruments and lots of reagents within a labora- tion limit may be calculated using the standard deviation of tory (intermediate precision condition). responses of blank samples or samples containing an analyte (iii) Reproducibility close to the detection limit and the slope of the calibration The reproducibility expresses the precision of observed curve close to the detection limit. The following equation is values obtained from multiple samplings of a homogenous an example to determine the detection limit using the stand- sample in different laboratories (reproducibility condition). ard deviation of responses of blank samples and the slope of 2.2.2. Assessment the calibration curve. A sufficient volume of a homogenous sample should be DL ＝ 3.3s/slope prepared before studying the precision. The solution is assumed to be homogenous. When it is difficult to obtain a DL: detection limit homogenous sample, the following samples may be used as s: the standard deviation of responses of blank samples homogenous samples; e.g., a large amount of drug products slope: slope of the calibration curve or mixture of drug substance and vehicles that are crushed The noise level may be used as the standard deviation of and mixed well until they can be assumed to be homogenous. responses of blank samples in chromatographic methods. It Suitable experimental designs such as one-way layout may should be ensured that the detection limit of the analytical be employed when more than one level of precision is to be procedure is lower than the specified limit for testing. investigated simultaneously. A sufficient number of repeti- 2.5. Quantitation limit tions, levels of operating conditions and laboratories should 2.5.1. Definition be employed. Sources of variations affecting analytical The quantitation limit is the lowest amount or concentra- results should be evaluated as thoroughly as possible through tion of the analyte in a sample that can be determined. The the validation. precision expressed as the relative standard deviation of sam- It is required to show the variance, standard deviation and ples containing an analyte at the quantitation limit is usually relative standard deviation (coefficient of variation) of each 10z. level of precision. The 90z confidence interval of the vari- 2.5.2. Assessment ance and corresponding intervals of the standard deviation The quantitation limit may be calculated using the stand- and relative standard deviation should also be established. ard deviation of responses of blank samples or samples con- The validity of the proposed analytical procedure for its in- taining an analyte close to the quantitation limit and the tended use may be confirmed by comparing obtained values slope of the calibration curve close to the quantitation limit. with the required values of the analytical procedure. The following equation is an example to determine the quan- Whether the proposed analytical procedure is acceptable titation limit using the standard deviation of responses of may normally be decided based on the reproducibility. blank samples and the slope of the calibration curve. 2.3. Specificity 2.3.1. Definition QL ＝ 10s/slope The specificity is the ability of an analytical procedure to QL: quantitation limit measure accurately an analyte in the presence of components s: the standard deviation of responses of blank samples thatmaybeexpectedtobepresentinthesamplematrix.The slope: slope of the calibration curve specificity is a measure of discriminating ability. Lack of specificity of an analytical procedure may be compensated The noise level may be used as the standard deviation of by other supporting analytical procedures. responses of blank samples in chromatographic methods. It 2.3.2. Assessment should be ensured that the quantitation limit of the analyti- It should be confirmed that the proposed analytical proce- cal procedure is lower than the specified limit for testing. dure can identify an analyte or that it can accurately measure 2.6. Linearity the amount or concentration of an analyte in a sample. The 2.6.1. Definition method to confirm the specificity depends very much upon The linearity is the ability of an analytical procedure to the purpose of the analytical procedure. For example, the elicit responses linearly related to the amount or concentra- specificity may be assessed by comparing analytical results tion of an analyte in samples. A well-defined mathematical obtained from a sample containing the analyte only with transformation may sometimes be necessary to obtain a results obtained from samples containing excipients, related linear relationship. substances or degradation products, and including or exclud- 2.6.2. Assessment ing the analyte. If reference standards of impurities are un- Responses are obtained after analyzing samples with vari- available, samples that are expected to contain impurities or ous amounts or concentrations of an analyte according to degradation products may be used (e.g. samples after ac- described operating procedures. The linearity may be eval- celerated or stress tests). uated in terms of the correlation coefficient, and the slope and y-intercept of the regression line. It may be also helpful for evaluating the linearity to plot residual errors from the 2150 Physics and Chemistry / General Information JP XVI regression line against the amount or concentration and to ingredients, and major components in pharmaceuticals. confirm that there is no particular tendency in the graph. (Additives such as stabilizing agents and preservatives are Samples with five different amounts or concentrations of an included in major components.) Tests for determining per- analyte should be usually investigated. formance of pharmaceuticals, such as dissolution testing. 2.7. Range 4. Terminology used in the validation of analytical proce- 2.7.1. Definition dures The range for the validation of analytical procedures is the (i) Analytical procedure: This document covers analyti- interval between the lower and upper limits of the amount or cal procedures applied to identification, and ones that pro- concentration of an analyte providing sufficient accuracy vides responses depending upon the amount or concentra- and precision. The range for the validation of analytical tion of analytes in samples. procedures for an analytical procedure with linearity is the (ii) Laboratory: The laboratory means an experimental interval between the lower and upper limits providing room or facility where tests are performed. In this document sufficient accuracy, precision and linearity. different laboratories are expected to perform an analytical 2.7.2. Assessment procedure using different analysts, different experimental When the range for the validation of analytical procedures apparatus and instruments, different lots of reagents and so is investigated, 80 to 120z of specified limits of testing forth. should be usually considered. The accuracy, precision and (iii) Number of replications: The number of replications linearity should be evaluated using samples containing the is one that is described in analytical procedures. An observed lower and upper limits and in the middle of the range. value is often obtained by more than one measurement in 3. Categories of tests employing analytical procedures order to achieve good precision of analytical procedures. Tests covered with this document are roughly classified Analytical procedures including the number of replications into three categories shown below according to their pur- should be validated. This is different from repetition in the poses. The table lists the normally required validation char- validation of analytical procedures to obtain accuracy or acteristics to be evaluated in the validation of analytical precision. procedures used in these tests. This list should be considered (iv) Observed value: The value of a characteristic ob- to represent typical validation characteristics. A different ap- tained as the result of performing an analytical procedure. proach to validating analytical procedures should be consi- (v) Consumer's risk: This is the probability that products dered depending upon the characteristics of analytical proce- out of the specification of tests are decided to be accepted dures and their intended use. after testing. It is usually expressed as b, and is called the (i) Type I Identification. Tests for identifying major probability of type II error or the probability of false nega- components in pharmaceuticals according to their character- tive in impurity tests. istics. (vi) Producer's risk: This is the probability that products (ii) Type II Impurity tests. Tests for determination of satisfying the specification of tests are decided to be rejected impurities in pharmaceuticals. after testing. It is usually expressed as a, and is called the (iii) Type III Tests for assaying drug substances, active probability of type I error or the probability of false positive in impurity tests. (vii) Robustness: The robustness is a measure of the capacity to remain unaffected by small but deliberate varia- Table Lists of validation characteristics required to be tions in analytical conditions. The stability of observed evaluated in tests of each type values may be studied by changing various analytical condi- Type of test Type I Type II Type III tions within suitable ranges including pH values of solutions, reaction temperature, reaction time or amount of reagents Validation Quantitation Limit added. When observed values are unstable, the analytical characteristics test test procedure should be improved. Results of studying robust- Accuracy/Trueness －＋－＋ ness may be reflected in the developed analytical procedure Precision as precautions or significant digits describing analytical con- Repeatability －＋－＋ ditions. Intermediate precision －－* －－* (viii) Test: Tests mean various tests described in general Reproducibility －＋* －＋* tests and official monographs in the Japanese Phar- Specificity** ＋＋＋＋ macopoeia such as impurity tests and assay. They includes Detection limit －－＋－ sampling methods, specification limits and analytical proce- Quantitation limit －＋－－ dures. Linearity －＋－＋ Range －＋－＋

－ Usually need not to be evaluated. ＋ Usually need to be evaluated. * Either intermediate precision or reproducibility should be evaluated depending upon circumstances in which analytical procedures or tests are performed. The latter should be normally evaluated in the validation of analytical procedures proposed to be included in the Japanese Pharmacopoeia. ** The lack of the specificity of an analytical procedure may be compensated by other relevant analytical procedures JP XVI General Information / Solid-state Properties 2151

distribution and associated data analysis and reporting. The particles can enter the laser beam in 2 positions. In the G2 Solid-state Properties conventional case the particles enter the parallel beam before the collecting lens and within its working distance. In so- called reversed Fourier optics the particles enter behind the Laser Diffraction Measurement of collecting lens and thus, in a converging beam. The advantage of the conventional set-up is that a reasonable path Particle Size length for the sample is allowed within the working distance of the lens. The second set-up allows only small path lengths This test is harmonized with the European Pharmacopoeia but enables measurement of scattered light at lager angles, and the U.S. Pharmacopoeia. which is useful when submicron particles are present. The laser light diffraction technique used for the determi- The interaction of the incident light beam and the ensem- nation of particle-size distribution is based on the analysis of ble of dispersed particles results in a scattering pattern with the diffraction pattern produced when particles are exposed different light intensities at various angles. The total angular to a beam of monochromatic light. Historically, the early intensity distribution, consisting of both direct and scattered laser diffraction instruments only used scattering at small light, is then focused onto a multi-element detector by a lens angles. However, the technique has since been broadened to or a series of lenses. These lenses create a scattering pattern include laser light scattering in a wider range and application that, within limits, does not depend on the location of the of the Mie theory, in addition to the Fraunhofer approxima- particles in the light beam. Hence, the continuous angular tion and anomalous diffraction. intensity distribution is converted into a discrete spatial in- The technique cannot distinguish between scattering by tensity distribution on a set of detector elements. single particles and scattering by clusters of primary parti- It is assumed that the measured scattering pattern of the cles, i.e. by agglomerates or aggregates. As most particulate particle ensemble is identical to the sum of the patterns from samples contain agglomerates or aggregates and as the focus all individual single scattering particles presented in random of interest is generally on the size distribution of primary relative positions. Note that only a limited angular range of particles, the clusters are usually dispersed into primary par- scattered light is collected by the lens(es) and, therefore, by ticles before measurement. the detector. For non-spherical particles, an equivalent sphere-size dis- 3. Development of the method tribution is obtained because the technique assumes spherical The measurement of particle size by laser diffraction can particles in its optical model. The resulting particle-size dis- give reproducible data, even in the sub-micron region, tribution may differ from those obtained by methods based provided the instrument used and the sample tested are care- on other physical principles (e.g. sedimentation, sieving). fully controlled to limit variability of the test conditions (e.g. This chapter provides guidance for the measurement of dispersion medium, method of preparation of the sample size distributions of particles in different dispersed systems, dispersion). for example, powders, sprays, aerosols, suspensions, emul- Traditionally, the measurement of particle size using laser sions, and gas bubbles in liquids, through analysis of their diffraction has been limited to particles in the range of ap- angular light-scattering patterns. It does not address specific proximately 0.1 mm to 3 mm. Because of recent advances in requirements of particle-size measurement of specific lens and equipment design, newer instruments are capable of products. exceeding this range routinely. With the validation report the 1. Principle user demonstrates the applicability of the method for its in- A representative sample, dispersed at an adequate concen- tended use. tration in a suitable liquid or gas, is passed through a beam 3.1. Sampling of monochromatic light, usually a laser. The light scattered The sampling technique must be adequate to obtain a by the particles at various angles is measured by a multi- representative sample of a suitable volume for the particle- element detector. Numerical values representing the scatter- size measurement. Sample splitting techniques such as rotating pattern are then recorded for subsequent analysis. These ing riffler or the cone and quartering method may be ap- scattering pattern values are then transformed, using an plied. appropriate optical model and mathematical procedure, to 3.2. Evaluation of the dispersion procedure yield the proportion of total volume to a discrete number of Inspect the sample to be analyzed, visually or with the aid size classes, forming a volumetric particle-size distribution. of a microscope, to estimate its size range and particle shape. The dispersion procedure must be adjusted to the purpose of 2. Instrument the measurement. The purpose may be such that it is prefera- The instrument is located in an environment where it is not ble to deagglomerate clusters into primary particles as far as affected by electrical noise, mechanical vibrations, tempera- possible, or it may be desirable to retain clusters as intact as ture fluctuations, humidity ordirectbrightlight.Anexam- possible. In this sense, the particles of interest may be either ple of a set-up of a laser light diffraction instrument is given primary particles or clusters. in Fig. 1. Other equipment may be used. For the development of a method it is highly advisable to The instrument comprises a laser light source, beam check that comminution of the particles does not occur, and processing optics, a sample measurement region (or cell), a conversely, that dispersion of particles or clusters is satisfac- Fourier lens, and a multi-element detector for measuring the tory. This can usually be done by changing the dispersing scattered light pattern. A data system is also required for energy and monitoring the change of the particle-size distri- deconvolution of the scattering data into a volumetric size bution. The measured size distribution must not change sig- 2152 Solid-state Properties / General Information JP XVI

Fig. 1 Example of a set-up of a laser light diffraction instrument

nificantly when the sample is well dispersed and the particles test material; are neither fragile nor soluble. Moreover, if the manufactur- (iii) be non-solvent of the test material (pure liquid or ing process (e.g. crystallization, milling) of the material has pre-filtered, saturated solution); changed, the applicability of the method must by verified (iv) not alter the size of the test materials (e.g. by solubil- (e.g. by microscopic comparison). ity, solubility enhancement, or recrystallization effects); Sprays, aerosols and gas bubbles in a liquid should be (v) favor easily formation and stability of the dispersion; measured directly, provided that their concentration is ade- (vi) be compatible with the materials used in the instru- quate, because sampling or dilution generally alters the par- ment (such as O-rings, gaskets, tubing, etc.); ticle-size distribution. (vii) possess a suitable viscosity to facilitate recircula- In other cases (such as emulsions, pastes and powders), tion, stirring and filtration. representative samples may be dispersed in suitable liquids. Surfactants and/or dispersing aids are often used to wet Dispersing aids (wetting agents, stabilizers) and/or mechani- the particles and to stabilize the dispersion. For weak acids cal forces (e.g. agitation, sonication) are often applied for and weak bases, buffering of the dispersing medium at low deagglomeration or deaggregation of clusters and stabiliza- or high pH respectively can assist in identifying a suitable tion of the dispersion. For these liquid dispersions, a recir- dispersant. culating system is most commonly used, consisting of an op- A preliminary check of the dispersion quality can be tical measuring cell, a dispersion bath usually equipped with performed by visual or microscopic inspection. It is also stirrer and ultrasonic elements, a pump, and tubing. Non- possible to take fractional samples out of a well-mixed stock recirculating, stirred cells are useful when only small dispersion. Such stock dispersions are formed by adding a amounts of a sample are available or when special dispersion liquid to the sample while mixing it with, for example, a liquids are used. glass rod, a spatula or a vortex mixer. Care must be taken to Dry powders can also be converted into aerosols through ensure the transfer of a representative sample and that the use of suitable dry powder dispersers, which apply settling of larger particles does not occur. Therefore a sam- mechanical force for deagglomeration or deaggregation. ple paste is prepared or sampling is carried out quickly from Generally, the dispersers use the energy of compressed gas or a suspension maintained under agitation. the differential pressure of a vacuum to disperse the particles 3.4. Optimization of the gas dispersion to an aerosol, which is blown through the measuring zone, For sprays and dry powder dispersions, a compressed gas usually into the inlet of a vacuum unit that collects the parti- free from oil, water and particles may be used. To remove cles. However, for free flowing, coarser particles or granules such materials from the compressed gas, a dryer with a filter the effect of gravity may be sufficient to disperse the parti- can be used. Any vacuum unit should be located away from cles adequately. the measurement zone, so that its output does not disturb the If the maximum particle size of the sample exceeds the measurement. measuring range of the instrument, the material that is too 3.5. Determination of the concentration range coarse can be removed by sieving and the mass and percen- In order to produce an acceptable signal-to-noise ratio in tage of removed material are reported. However, after pre- the detector, the particle concentration in the dispersion sieving, note that the sample is no longer representative, un- must exceed a minimum level. Likewise, it must be below a less otherwise proven. maximum level in order to avoid multiple scattering. The 3.3. Optimization of the liquid dispersion concentration range is influenced by the width of the laser Liquids, surfactants, and dispersing aids used to disperse beam, the path length of the measurement zone, the optical powders must: properties of the particles, and the sensitivity of the detector (i) be transparent at the laser wavelength and practically elements. free from air bubbles or particles; In view of the above, measurements must be performed at (ii) have a refractive index that differs from that of the different particle concentrations to determine the appropri- JP XVI General Information / Solid-state Properties 2153 ate concentration range for any typical sample of material. robust/fragile, width of its size distribution, etc.), whereas (Note: in different instruments, particle concentrations are the required repeatability depends on the purpose of the usually represented by differently scaled and differently measurement. Mandatory limits cannot be specified in this named numbers, e.g. obscuration, optical concentration, chapter, as repeatabilities (different sample preparations) proportional number of total mass). may vary appreciably from one substance to another. 3.6. Determination of the measuring time However, it is good practice to aim at acceptance criteria for The time of measurement, the reading time of the detector repeatability such as RSD (z) ≦ 10z [n ＝ 6] for any cen- and the acquisition frequency is determined experimentally tral value of the distribution (e.g. for x50). Values at the sides in accordance with the required precision. Generally, the of the distribution (e.g. x10 and x90) are oriented towards less time for measurement permits a large number of detector stringent acceptance criteria such as RSD ≦ 15z [n ＝ 6]. scans or sweeps at short time intervals. Below 10 mm, these values must be doubled. Robustness may 3.7. Selection of an appropriate optical model be tested during the selection and optimization of the disper- Most instruments use either the Fraunhofer or the Mie sion media and forces. The change of the dispersing energy theory, though other approximation theories are sometimes may be monitored by the change in the particle-size distribu- applied for calculation of the scattering matrix. The choice tion. of the theoretical model depends on the intended application 4. Measurement and the different assumptions (size, absorbance, refractive 4.1. Precautions index, roughness, crystal orientation, mixture, etc.) made (i) never look into the direct path of the laser beam or its for the test material. If the refractive index values (real and reflections; imaginary parts for the used wavelength) are not exactly (ii) earth all instrument components to prevent ignition known, then the Fraunhofer approximation or the Mie of solvents or dust explosions; theory with a realistic estimate of the refractive index can be (iii) check the instrument set-up (e.g. warm-up, required used. The former has the advantages that it is simple and it measuring range and lens, appropriate working distance, does not need refractive index values; the latter usually pro- position of the detector, no direct bright daylight); vides less-biased particle-size distributions for small parti- (iv) in the case of wet dispersions, avoid air bubbles, cles. For instance, if the Fraunhofer model is used for sam- evaporation of liquid, schlieren or other inhomogeneities in ples containing an appreciable amount of small, transparent the dispersion; similarly, avoid improper mass-flow from the particles, a significantly larges amount of small particles may disperser or turbulent air-flow in the case of dry dispersions; be calculated. In order to obtain traceable results, it is essen- such effects can cause erroneous particle-size distributions. tial to document the refractive index values used, since small 4.2. Measurement of the light scattering of dispersed sam- differences in the values assumed for the real and imaginary ple(s) part of the complex refractive index may cause significant After proper alignment of the optical part of the instru- differences in the resulting particle-size distributions. Small ment, a blank measurement of the particle-free dispersion values of the imaginary part of the refractive index (about medium must be performed using the same method as that 0.01 – 0.1i) are often applied to allow the correction of the used for the measurement of the sample. The background absorbance for the surface roughness of the particles. It signal must be below an appropriate threshold. The detector should be noted, in general, that the optical properties of the data are saved in order to substract them later from the data substance to be tested, as well as the structure (e.g. shape, obtained with the sample. The sample dispersion is measured surface roughness and porosity) bear upon the final result. according to the developed method. 3.8. Validation For each detector element, an average signal is calculated, Typically, the validity of a procedure may be assessed by sometimes together with its standard deviation. The magni- the evaluation of its specificity, linearity, range, accuracy, tude of the signal from each detector element depends upon precision and robustness. In particle-size analysis by laser the detection area, the light intensity and the quantum ef- light diffraction, specificity as defined by ICH is not applica- ficiency. The co-ordinates (size and position) of the detector ble as it is not possible to discriminate different components elements together with the focal distance of the lens deter- into a sample, as is neither possible to discriminate between mine the range of scattering angles for each element. Most agglomerates from dispersed particles unless properly instruments also measure the intensity of the central (unscat- complemented by microscopic techniques. Exploring a liner tered) laser beam. The ratio of the intensity of a dispersed relationship between concentration and response, or a sample to that in its absence (the blank measurement) indi- mathematical model for interpolation, is not applicable to cates the proportion of scattered light and hence the particle this procedure. Rather than evaluating linearity, this method concentration. requires the definition of a concentration range within which 4.3. Conversion of scattering pattern into particle-size dis- the result of the measurements does not vary significantly. tribution Concentrations below that range produce an error due to a This deconvolution step is the inverse of the calculation of poor signal to noise ratio, while concentrations above that a scattering pattern for a given particle-size distribution. The range produce an error due to multiple scattering. The range assumption of spherical particle shape is particularly im- depends mostly in the instrument hardware. Accuracy portant as most algorithms use the mathematical solution for should be confirmed through an appropriate instrument scattering from spherical particles. Furthermore, the meas- qualification and comparison with microscopy, while preci- ured data always contain some random and systematic sion may be assessed by means of a repeatability determina- errors, which may vitiate the size distributions. Several tion. mathematical procedures have been developed for use in the The attainable repeatability of the method mainly depends available instruments. They contain some weighting of devi- on the characteristics of the material (milled/not milled, 2154 Solid-state Properties / General Information JP XVI ations between measured and calculated scattering patterns dered to meet the requirements if the mean value of x50 from (e.g. least squares), some constraints (e.g. non-negativity for at least 3 independent measurements does not deviate by amounts of particles), and/or some smoothing of the size more than 3z from the certified range of values of the certi- distribution curve. fied reference material. The mean values for x10 and x90 must The algorithms used are specific to each make and model not deviate by more than 5z from the certified range of of equipment, and are proprietary. The differences in the values. Below 10 mm, these values must be doubled. algorithms between different instruments may give rise to Although the use of materials consisting of spherical differences in the calculated particle-size distributions. particles is preferable, non-spherical particles may also be 4.4. Replicates employed. Preferably, these particles have certified or typi- The number of replicate measurements (with individual cal values from laser diffraction analysis performed accord- sample preparations) to be performed, depends on the ing to an agreed, detailed operating procedure. The use of required measurement precision. It is recommended to set reference values from methods other than laser diffraction this number in a substance-specific method. may cause a significant bias. Thereasonforthisbiasisthat the different principles inherent in the various methods may 5. Reporting of results lead to different sphere-equivalent diameters for the same The particle-size distribution data are usually reported as non-spherical particle. cumulative undersize distribution and/or as density distribu- Although the use of certified reference materials is tion by volume. The symbol x is used to denote the particle preferred, other well-defined reference materials may also be size, which in turn is defined as the diameter of a volume- employed. They consist of substances of typical composition equivalent sphere. Q3(x) denotes the volume fraction under- and particle-size distribution for a specified class of sub- size at the particle size x. In a graphical representation, x is stances. Their particle-size distribution has proven to be plotted on the abscissa and the dependent variable Q3 on the stable over time. The results must comply with previously ordinate. Most common characteristic values are calculated determined data, with the same precision and bias as for the from the particle-size distribution by interpolation. The certified reference material. particle sizes at the undersize values of 10z,50z,and90z 6.2. Qualification of the system (denoted as x , x ,andx respectively) are frequently used. 10 50 90 In addition to the calibration, the performance of the x is also known as the median particle size. It is recognized 50 instrument must be qualified at regular time intervals or as that the symbol d is also widely used to designate the particle frequently as appropriate. This can be undertaken using any size, thus the symbol x may be replaced by d. suitable reference material as mentioned in the previous Moreover, sufficient information must be documented paragraph. about the sample, the sample preparation, the dispersion The qualification of the system is based on the concept conditions, and the cell type. As the results depend on the that the equipment, electronics, software and analytical particular instrument, data analysis program, and optical operations constitute an integral system, which can be eval- model used, these details must also be documented. uated as an entity. Thus the entire measurement procedure is 6. Control of the instrument performance examined, including sample collection, sample dispersion, Use the instrument according to the manufacturer's in- sample transport through the measuring zone, and the meas- structions and carry out the prescribed qualifications at an urement and deconvolution procedure. It is essential that the appropriate frequency, according to the use of the instru- total operational procedure is fully described. ment and substances to be tested. In general, unless otherwise specified in the individual 6.1. Calibration monograph, the response of a laser diffraction instrument is

Laser diffraction systems, although assuming idealized considered to meet the requirements if the x50 value does not properties of the particles, are based on first principles of deviate by more than 10z from the range of values of the laser light scattering. Thus, calibration in the strict sense is reference material. If optionally the values at the sides of the not required. However, it is still necessary to confirm that distribution are evaluated (e.g. x10 and x90), then these values the instrument is operating correctly. This can be undertaken must not deviate by more than 15z from the certified range using any certified reference material that is acceptable in of values. Below 10 mm, these values must be doubled. industrial practice. The entire measurement procedure is Note 1: For calibration of the instrument stricter requirements are examined, including sample collection, sample dispersion, laid down in 6.1. Calibration. sample transport through the measuring zone, measurement, Note 2: Laser Diffraction Measurement of Particle Size complies and the deconvolution procedure. It is essential that the total with ISO13320-1 (1999) and 9276-1 (1998). operational procedure is fully described. The preferred certified reference materials consist of spherical particles of a known distribution. They must be certified as to the mass-percentage size distribution by an Powder Fineness absolute technique, if available, and used in conjunction with an agreed, detailed operation procedure. It is essential This classification is harmonized with the European Phar- that the real and imaginary parts of the complex refractive macopoeia and the U.S. Pharmacopoeia. index of the material are indicated if the Mie theory is A simple descriptive classification of powder fineness is applied in data analysis. The representation of the particle- provided in this chapter. Sieving is most suitable where a size distribution by volume will equal that of the distribution majority of the particles are larger than about 75 mm, by mass, provided that the density of the particles is the although it can be used for some powders having smaller same for all size fractions. particle sizes where the method can be validated. Light The response of a laser diffraction instrument is consi- JP XVI General Information / Solid-state Properties 2155 diffraction is also a widely used technique for measuring the the pharmaceutical literature, attempting to correlate the size of a wide range of particles. various measures of powder flow to manufacturing proper- Where the cumulative distribution has been determined by ties. The development of such a variety of test methods was analytical sieving or by application of other methods, parti- inevitable; powder behavior is multifaceted and thus compli- clesizemaybecharacterized in the following manner: cates the effort to characterize powder flow. The purpose of this chapter is to review the methods for characterizing pow- x : particle size corresponding to 90z of the cumulative 90 der flow that have appeared most frequently in the phar- undersize distribution maceutical literature. In addition, while it is clear that no x : median particle size (ie: 50z of the particles are 50 single and simple test method can adequately characterize smaller and 50z of the particles are larger) the flow properties of pharmaceutical powders, this chapter x : particle size corresponding to 10z of the cumulative 10 proposes the standardization of test methods that may be undersize distribution valuable during pharmaceutical development. It is recognized that the symbol d is also widely used to Four commonly reported methods for testing powder flow designate these values. Therefore, the symbols d90, d50, d10 are (1) angle of repose, (2) compressibility index or Hausner may be used. ratio, (3) flow rate through an orifice, and (4) shear cell. In The following parameters may be defined based on the addition, numerous variations of each of these basic cumulative distribution. methods are available. Given the number of test methods

Qr(x): cumulative distribution of particles with a dimen- and variations, standardizing the test methodology, where sion less than or equal to x where the subscript r reflects the possible, would be advantageous. distribution type With this goal in mind, the most frequently used methods are discussed below. Important experimental considerations are identified and recommendations are made regarding r Distribution type standardization of the methods. In general, any method of measuring powder flow should be practical, useful, 0 Number reproducible, sensitive, and yield meaningful results. It bears 1Length repeating that no one simple powder flow method will adequately or completely characterize the wide range of flow 2Area properties experienced in the pharmaceutical industry. An 3 Volume appropriate strategy may well be the use of multiple standardized test methods to characterize the various aspects of powder flow as needed by the pharmaceutical scientist. Therefore, by definition: 1. Angle of repose The angle of repose has been used in several branches of Qr(x) ＝ 0.90 when x ＝ x90 Qr(x) ＝ 0.50 when x ＝ x50 science to characterize the flow properties of solids. Angle of repose is a characteristic related to interparticulate friction, Qr(x) ＝ 0.10 when x ＝ x10 or resistane to movement between particles. Angle of repose An alternative but less informative method of classifying test results are reported to be very dependent upon the powder fineness is by use of the descriptive terms in the fol- method used. Experimental difficulties arise due to segrega- lowing table. tion of material and consolidation or aeration of the powder as the cone is formed. Despite its difficulties, the method continues to be used in the pharmaceutical industry, and a Classification of powders by fineness number of examples demonstrating its value in predicting Descriptive Cumulative distribution by volume manufacturing problems appear in the literature. x50 (mm) term basis, Q3 (x) The angle of repose is the constant, three-dimensional angle (relative to the horizontal base) assumed by a cone-like Coarse ＞355 Q3 (355)＜0.50 pile of material formed by any of several different methods (described briefly below). Moderately 180–355 Q3 (180)＜0.50 and Q3 (355)≧0.50 1.1. Basic methods for angle of repose fine A variety of angle of repose test methods are reported in

Fine 125–180 Q3 (125)＜0.50 and Q3 (180)≧0.50 the literature. The most common methods for determining the static angle of repose can be classified based on two im- Very fine ≦125 Q3(125)≧0.50 portant experimental variables: (i) The height of the ``funnel'' through which the powder passes may be fixed relative to the base, or the height may be varied as the pile forms. Powder Flow (ii) The base upon which the pile forms may be of fixed diameter or the diameter of the powder cone may be allowed This test is harmonized with the European Pharmacopoeia to vary as the pile forms. and the U.S. Pharmacopeia. 1.2. Variations in angle of repose methods In addition to the above methods, variations of them have The widespread use of powders in the pharmaceutical in- been used to some extent. dustry has generated a variety of methods for characterizing (i) Drained angle of repose: This is determined by powder flow. Not surprisingly, scores of references appear in 2156 Solid-state Properties / General Information JP XVI allowing an excess quantity of material positioned above a ly or reproducibly prepared, this method is not appropriate. fixed diameter base to ``drain'' from the container. Forma- Determine the angle of repose by measuring the height of the tion of a cone of powder on the fixed diameter base allows cone of powder and calculating the angle of repose, a, from determination of the drained angle of repose. the following equation: (ii) Dynamic angle of repose: This is determined by fill- tan a ＝ height/(base × 0.5) ing a cylinder (with a clear, flat cover on one end) and rotat- 2. Compressibility index and Hausner ratio ing it at a specified speed. The dynamic angle of repose is the In recent years the compressibility index and the closely angle (relative to the horizontal) formed by the flowing related Hausner ratio have become the simple, fast and powder. The internal angle of kinetic friction is defined by popular methods of predicting powder flow characteristics. the plane separating those particles sliding down the top The compressibility index bas been proposed as an indirect layer of the powder and those particles that are rotating with measure of bulk density, size and shape, surface area, mois- the drum (with roughened surface). ture content, and cohesiveness of materials because all of 1.3. Angle of repose general scale of flowability these can influence the observed compressibility index. The While there is some variation in the qualitative description compressibility index and the Hausner ratio are determined of powder flow using the angle of repose, much of the phar- by measuring both the bulk volume and tapped volume of a maceutical literature appears to be consistent with the classi- powder. fication by Carr1), which is shown in Table 1. There are ex- 2.1. Basic methods for compressibility index and Hausner amples of formulations with an angle of repose in the range ratio of 40 to 50 degrees that manufactured satisfactorily. When While there are some variations in the method of deter- the angle of repose exceeds 50 degrees, the flow is rarely ac- mining the compressibility index and Hausner ratio, the bas- ceptable for manufacturing purposes. ic procedure is to measure (1) the unsettled apparent volume,

Vo, and (2) the final tapped volume, Vf, of the powder after Table 1 Flow properties and corresponding tapping the material until no further volume changes occur. angles of repose1) The compressibility index and the Hausner ratio are calculated as follows: Flow property Angle of repose (degrees) Compressibility Index ＝ (Vo － Vf)/Vo × 100 Excellent 25 – 30 Hausner Ratio ＝ Vo/Vf Good 31 – 35 Fair aid not needed 36 – 40 Alternatively, the compressibility index and Hausner ratio Passable may hang up 41 – 45 may be calculated using measured values for bulk density Poor most agitate, 46 – 55 (rbulk) and tapped density (rtapped)asfollows: vibrate Compressibility Index ＝ (rtapped － rbulk)/rtapped × 100 Very poor 56 – 65 Hausner Ratio ＝ rtapped/rbulk Very, very poor ＞66 In a variation of these methods, the rate of consolidation is sometimes measured rather than, or in addition to, the 1.4. Experimental considerations for angle of repose change in volume that occurs on tapping. For the compres- Angle of repose is not an intrinsic property of the powder, sibility index and the Hausner ratio, the generally accepted that is to say, it is very much dependent upon the method scale of flowability is given in Table 2. used to form the cone of powder. On this subject, the existing literature raises these important considerations: Table 2 Scale of flowability1) (i) The peak of the cone of powder can be distorted by the impact of powder from above. By carefully building the Compressibility Flow character Hausner ratio powder cone, the distortion caused by impact can be index (z) minimized. ≦10 Excellent 1.00 – 1.11 (ii) The nature of the base upon which the powder cone 11–15 Good 1.12–1.18 is formed influences the angle of repose. It is recommended 16–20 Fair 1.19–1.25 that the powder cone be formed on a ``common base'', 21–25 Passable 1.26–1.34 which can be achieved by forming the cone of powder on a 26–31 Poor 1.35–1.45 layer of powder. This can be done by using a base of fixed 32 – 37 Very poor 1.46 – 1.59 diameter with a protruding outer edge to retain a layer of ＞38 Very, very poor ＞1.60 powder upon which the cone is formed. 1.5. Recommended procedure for angle of repose Form the angle of repose on a fixed base with a retaining 2.2. Experimental considerations for the compressibility lip to retain a layer of powder on the base. The base should index and Hausner ratio be free of vibration. Vary the height of the funnel to care- Compressibility index and Hausner ratio are not intrinsic fully build up a symmetrical cone of powder. Care should be properties of the powder, that is to say, they are dependent taken to prevent vibration as the funnel is moved. The upon the methodology used. The existing literature points funnel height should be maintained approximately 2–4cm out several important considerations affecting the determi- from the top of the powder pile as it is being formed in order nation of the (1) unsettled apparent volume, Vo, (2) the final to minimize the impact of falling powder on the tip of the tapped volume, Vf, (3) the bulk density, rbulk,and(4)the cone. If a symmetrical cone of powder cannot be successful- tapped density, rtapped: JP XVI General Information / Solid-state Properties 2157

(i) The diameter of the cylinder used between published results is difficult. (ii) The number of times the powder is tapped to achieve 3.4. Experimental considerations for flow through an the tapped density orifice (iii) The mass of material used in the test Flow rate through an orifice is not an intrinsic property of (iv) Rotation of the sample during tapping the powder. It is very much dependent upon the methodolo- 2.3. Recommended procedure for compressibility index gy used. The existing literature points out several important and Hausner ratio considerations affecting these methods: Use a 250-mL volumetric cylinder with a test sample (i) The diameter and shape of the orifice weight of 100 g. Smaller weights and volumes may be used, (ii) The type of container material (metal, glass, plastic) but variations in the method should be described with the (iii) The diameter and height of the powder bed. results. An average of three determinations is recommended. 3.5. Recommended procedure for flow through an orifice Flow rate through an orifice can be used only for materials 3. Flow through an orifice that have some capacity to flow. It is not useful for cohesive The flow rate of a material depends upon many factors, materials. Provided that the height of the powder bed (the some of which are particle-related and some related to the `head' of powder) is much greater than the diameter of the process. Monitoring the rate of flow of material through an orifice, the flow rate is virtually independent of the powder orifice has been proposed as a better measure of powder head. Use a cylinder as the container because the cylinder flowability. Of particular significance is the utility of material should have little effect on flow. This configuration monitoring flow continuously since pulsating flow patterns results in flow rate being determined by the movement of have been observed even for free flowing materials. Changes powder over powder rather than powder along the wall of in flow rate as the container empties can also be observed. the container. Powder flow rate often increases when the Empirical equations relating flow rate to the diameter of the height of the powder column is less than two times the diam- opening, particle size, and particle density have been deter- eter of the column. The orifice should be circular and the mined. However, determining the flow rate through an cylinder should be free of vibration. General guidelines for orifice is useful only with free-flowing materials. dimensions of the cylinder are as follows: The flow rate through an orifice is generally measured as (i) Diameter of opening ＞6 times the diameter of the the mass per time flowing from any of a number of types of particles containers (cylinders, funnels, hoppers). Measurement of the (ii) Diameter of the cylinder ＞2 times the diameter of flow rate can be in discrete increments or continuous. the opening 3.1. Basic methods for flow through an orifice Use of a hopper as the container may be appropriate and There are a variety of methods described in the literature. representative of flow in a production situation. It is not ad- The most common for determining the flow rate through an visable to use a funnel, particularly one with a stem, because orifice can be classified based on three important experimen- flow rate will be determined by the size and length of the tal variables: stem as well as the friction between the stem and the powder. (1) The type of container used to contain the powder. A truncated cone may be appropriate, but flow will be in- Common containers are cylinders, funnels and hoppers from fluenced by the powder—wall friction coefficient, thus, production equipment. selection of an appropriate construction material is im- (2) The size and shape of the orifice used. The orifice di- portant. ameter and shape are critical factors in determining powder For the opening in the cylinder, use a flat-faced bottom flow rate. plate with the option to vary orifice diameter to provide (3) The method of measuring powder flow rate. Flow maximum flexibility and better ensure a powder-over-pow- rate can be measured continuously using an electronic der flow pattern. Rate measurement can be either discrete or balance and with some sort of recording device (strip chart continuous. Continuous measurement using an electronic recorder, computer). It can also be measured in discrete sam- balance can more effectively detect momentary flow rate ples (for example, the time it takes for 100 g of powder to variations. pass through the orifice to the nearest tenth of a second or the amount of powder passing through the orifice in 10 4. Shear cell methods seconds to the nearest tenth of a gram). In an effort to put powder flow studies and hopper design 3.2. Variations in methods for flow through an orifice on a more fundamental basis, a variety of powder shear Either mass flow rate or volume flow rate can be deter- testers and methods that permit more thorough and precisely mined. Mass flow rate is the easier of the methods, but it defined assessment of powder flow properties have been de- biases the results in favor of high-density materials. Since die veloped. Shear cell methodology has been used extensively in fill is volumetric, determining volume flow rate may be the study of pharmaceutical materials. From these methods, preferable. A vibrator is occasionally attached to facilitate a wide variety of parameters can be obtained, including the flow from the container, however, this appears to complicate yield loci representing the shear stress-shear strain relation- interpretation of results. A moving orifice device has been ship, the angle of internal friction, the unconfined yield proposed to more closely simulate rotary press conditions. strength, the tensile strength, and a variety of derived The minimum diameter orifice through which powder flows parameters such as the flow factor and other flowability in- can also be identified. dices. Because of the ability to more precisely control experi- 3.3. General scale of flowability for flow through an mental parameters, flow properties can also be determined orifice as a function of consolidation load, time, and other environ- No general scale is available because flow rate is critically mental conditions. The methods have been successfully used dependent on the method used to measure it. Comparison to determine critical hopper and bin parameters. 2158 Solid-state Properties / General Information JP XVI

4.1. Basic methods for shear cell Generally, the densities of liquid or gas are affected only One type of shear cell is the cylindrical shear cell which is by temperature and pressure, but the solid or powder density split horizontally, forming a shear plane between the lower is affected by the state of aggregation of the molecules or the stationary base and the upper movable portion of the shear particles. Therefore, the solid or powder densities naturally cell ring. After powder bed consolidation in the shear cell vary depending on crystal structure or crystallinity of the (using a well-defined procedure), the force necessary to shear substance concerned, and also varies depending on the the powder bed by moving the upper ring is determined. method of preparation or handling if the sample is amor- Annular shear cell designs offer some advantages over the phous form or partially amorphous. Consequently, even in a cylindrical shear cell design, including the need for less mate- case that two solids or powders are chemically identical, it rial. A disadvantage, however, is that because of its design, may be possible that the different figures of density are ob- the powder bed is not sheared as uniformly because material tained if their crystal structures are different. As the solid or on the outside of the annulus is sheared more than material powder particle densities are important physical properties in the inner region. A third type of shear cell (plate-type) for the powdered pharmaceutical drugs or the powdered raw consists of a thin sandwich of powder between a lower materials of drugs, the Japanese Pharmacopoeia specifies stationary rough surface and an upper rough surface that is each density determination as ``Powder Particle Density De- moveable. termination'' for the particle density and as ``Determination All of the shear cell methods have their advantages and of Bulk and Tapped Densities'' for the bulk density. disadvantages, but a detailed review is beyond the scope of The solid or powder densities are expressed in mass per this chapter. As with the other methods for characterizing unit volume (kg/m3), and generally expressed in g/cm3 (1 g/ powder flow, many variations are described in the literature. cm3 ＝ 1000 kg/m3). A significant advantage of shear cell methodology in general Crystal Density is a greater degree of experimental control. The methodology The crystal density of a substance is the average mass per generally is rather time-consuming and requires significant unit volume, exclusive of all voids that are not a fundamen- amounts of material and a well-trained operator. tal part of the molecular packing arrangement. It is an in- 4.2. Recommendations for shear cell trinsic property concerning the specific crystal structure of The many existing shear cell configurations and test the substance, and is not affected by the method of determi- methods provide a wealth of data and can be used very effec- nation. The crystal density can be determined either by cal- tively to characterize powder flow. They are also helpful in culation or by simple measurement. the design of equipment such as hoppers and bins. A. The calculated crystal density is obtained using: Because of the diversity of available equipment and ex- 1) For example, the crystallographic data (volume and perimental procedures, no specific recommendations regard- composition of the unit cell) obtained by indexing ing methodology are presented in this chapter. It is recom- the perfect crystal X-ray diffraction data from mended that the results of powder flow characterization single crystal or the powder X-ray diffraction data. using shear cell methodology include a complete description 2) Molecular mass of the substance. of equipment and methodology used. B. The measured crystal density is obtained as the mass to volume ratio after measuring the single crystal mass and References volume. 1) Carr R.L.: Evaluating flow properties of solids. Chem. Eng. 1965; 72: 163–168. Particle Density The particle density takes account both the crystal density and the intraparticulate porosity (sealed and/or experimentally non-accessible open pores) as a part of the particle Solid and Particle Densities volume. The particle density depends on the value of the volume determined, and the volume in turn depends on the Densityofasolidorapowderasastateofaggregation method of measurement. Particle density can be determined has different definitions depending on the way of including either by gas displacement pycnometry or mercury porosi- of the interparticulate and intraparticulate voids that exist metry, but the Japanese Pharmacopoeia specifies the pycno- between the particles or inside the powder. Different figures metry as the ``Powder Particle Density Determination''. are obtained in each case, and there are different practical A. The pycnometric density is obtained by assuming that meanings. Generally, there are three levels of definitions of the volume of the gas displaced, which is measured with the solid or powder density. the gas displacement pycnometer, is equivalent to that (1) Crystal density: It is assumed that the system is ho- of a known mass of the powder. In pycnometric density mogeneous with no intraparticulate void. Crystal density is measurements, any volume with the open pores accessi- also called true density. ble to the gas is not included as a part of volume of the (2) Particle density: The sealed pores or the experimen- powder, but the sealed pores or pores inaccessible to the tally non-accessible open pores is also included as a part of gas is included as a part of the volume of the powder. the volumes of the solid or the powder. Due to the high diffusivity of helium which can pene- (3) Bulk density: The interparticulate void formed in the trate to most open pores, it is recommendable as the powder bed is also included as a part of the volumes of the measurement gas of particle density. Therefore, the py- solid or the powder. Bulk density is also called apparent den- cnometric particle density of a finely milled powder is sity. Generally, the powder densities at loose packing and at generally not very different from the crystal density. tapping are defined as the bulk density and the tapped den- Hence, the particle density by this method is the best es- sity, respectively. timate of the true density of an amorphous or partially JP XVI General Information / Biotechnological/Biological Products 2159

crystalline sample, and can be widely used for manufac- bonded amino acid residues organized as a linear polymer. turing control of the processed pharmaceutical powder The sequence of the amino acids in a protein or peptide samples. determines the properties of the molecule. Proteins are B. The mercury porosimetric density is also called granular considered large molecules that commonly exist as folded density. This method also includes the sealed pores as a structures with a specific conformation, while peptides are part of the volumes of the solid or the powder, but ex- smaller and may consist of only a few amino acids. Amino cludes the volume only from the open pores larger than acid analysis can be used to quantify protein and peptides, to some size limit. This pore size limit or minimal access determine the identity of proteins or peptides based on their diameter depends on the maximal mercury intrusion amino acid composition, to support protein and peptide pressure applied during the measurement and under structure analysis, to evaluate fragmentation strategies for normal operating pressure, the mercury does not pene- peptide mapping, and to detect atypical amino acids that trate the finenest pores accessible to helium. Since this might be present in a protein or peptide. It is necessary to method is capable of measuring the density which cor- hydrolyze a protein/peptide to its individual amino acid responds to the pore size limit at each mercury intrusion constituents before amino acid analysis. Following protein/ pressure, the various granular densities can be obtained peptide hydrolysis, the amino acid analysis procedure can be from one sample. the same as that practiced for free amino acids in other pharmaceutical preparations. The amino acid constituents of the Bulk Density and Tapped Density test sample are typically derivatized for analysis. The bulk density of a powder includes the contribution of interparticulate void volume as a part of the volume of the Apparatus powder. Therefore, the bulk density depends on both the Methods used for amino acid analysis are usually based on powder particle density and the space arrangement of parti- a chromatographic separation of the amino acids present in cles in the power bed. Further, since the slightest disturbance the test sample. Current techniques take advantage of the of the bed may result in variation of the space arrangement, automated chromatographic instrumentation designed for it is often very difficult to determine the bulk density with analytical methodologies. An amino acid analysis instrument good reproducibility. Therefore, it is essential to specify how will typically be a low-pressure or high-pressure liquid chro- the determination was made upon reporting the bulk density. matograph capable of generating mobile phase gradients The Japanese Pharmacopoeia specifies ``Determination of that separate the amino acid analytes on a chromatographic BulkandTappedDensities''. column. The instrument must have postcolumn derivatiza- A. The bulk density is determined by measuring the appar- tion capability, unless the sample is analyzed using precol- ent volume of a known mass of powder sample that has umn derivatization. The detector is usually an ultraviolet- been passed through a screen in a graduated cylinder visible or fluorescence detector depending on the derivatiza- (constant mass method). Separately, the Phar- tion method used. A recording device (e.g., integrator) is macopoeia specifies the method of determining bulk used for transforming the analog signal from the detector density by measuring the mass of powder in a vessel and for quantitation. It is preferred that instrumentation be having a known volume (constant volume method). dedicated particularly foraminoacidanalysis. B. The tapped density is obtained by mechanically tapping General Precautions a measuring cylinder containing a powder sample. After Background contamination is always a concern for the determining the initial bulk volume, carry out tapping analyst in performing amino acid analysis. High purity rea- under a fixed measurement condition (tapping rate and gents are necessary (e.g., low purity hydrochloric acid can drop height), and the measurement is carried out contribute to glycine contamination). Analytical reagents are repeatedly until the bulk volume variation obtained at changed routinely every few weeks using only high-pressure consecutive two measurements is within an acceptable liquid chromatography (HPLC) grade solvents. Potential range (constant mass method). Separately, the Phar- microbial contamination and foreign material that might be macopoeia specifies the method of determining the present in the solvents are reduced by filtering solvents be- tapped density by measuring the mass of a fixed volume fore use, keeping solvent reservoirs covered, and not placing of the tapped powder (constant volume method). amino acid analysis instrumentation in direct sunlight. Laboratory practices can determine the quality of the amino acid analysis. Place the instrumentation in a low G3 Biotechnological/Biological traffic area of the laboratory. Keep the laboratory clean. Clean and calibrate pipets according to a maintenance sched- Products ule. Keep pipet tips in a covered box; the analysts may not handle pipet tips with their hands. The analysts may wear powder-free latex or equivalent gloves. Limit the number of Amino Acid Analysis times a test sample vial is opened and closed because dust can contribute to elevated levels of glycine, serine, and This test is harmonized with the European Pharmacopoeia alanine. and the U.S. Pharmacopeia. A well-maintained instrument is necessary for acceptable amino acid analysis results. If the instrument is used on a Amino acid analysis refers to the methodology used to de- routine basis, it is to be checked daily for leaks, detector and termine the amino acid composition or content of proteins, lamp stability, and the ability of the column to maintain peptides, and other pharmaceutical preparations. Proteins resolution of the individual amino acids. Clean or replace all and peptides are macromolecules consisting of covalently instrument filters and other maintenance items on a routine 2160 Biotechnological/Biological Products / General Information JP XVI schedule. to determine the variation due to sample handling. Often the amino acid composition of a standard protein (e.g., bovine Reference Standard Material serum albumin) is analyzed as part of the repeatability evalu- Acceptable amino acid standards are commercially availa- ation. By evaluating the replicate variation (i.e., RSD), the ble for amino acid analysis and typically consist of an aque- laboratory can establish analytical limits to ensure that the ous mixture of amino acids. When determining amino acid analyses from the laboratory are under control. It is desira- composition, protein or peptide standards are analyzed with ble to establish the lowest practical variation limits to ensure the test material as a control to demonstrate the integrity of the best results. Areas to focus on to lower the variability of the entire procedure. Highly purified bovine serum albumin the amino acid analysis include sample preparation, high has been used as a protein standard for this purpose. background spectral interference due to quality of reagents Calibration of Instrumentation and/or laboratory practices, instrument performance and Calibration of amino acid analysis instrumentation typi- maintenance, data analysis and interpretation, and analyst cally involves analyzing the amino acid standard, which con- performance and habits. All parameters involved are fully sists of a mixture of amino acids at a number of concentra- investigated in the scope of the validation work. tions, to determine the response factor and range of analysis Sample Preparation for each amino acid. The concentration of each amino acid Accurate results from amino acid analysis require purified in the standard is known. In the calibration procedure, the protein and peptide samples. Buffer components (e.g., salts, analyst dilutes the amino acid standard to several different urea, detergents) can interfere with the amino acid analysis analyte levels within the expected linear range of the amino and are removed from the sample before analysis. Methods acid analysis technique. Then, replicates at each of the that utilize postcolumn derivatization of the amino acids are different analyte levels can be analyzed. Peak areas obtained generally not affected by buffer components to the extent for each amino acid are plotted versus the known concentra- seen with precolumn derivatization methods. It is desirable tion for each of the amino acids in the standard dilution. to limit the number of sample manipulations to reduce These results will allow the analyst to determine the range of potential background contamination, to improve analyte amino acid concentrations where the peak area of a given recovery, and to reduce labor. Common techniques used to aminoacidisanapproximately linear function of the amino remove buffer components from protein samples include the acid concentration. It is important that the analyst prepare following methods: (1) injecting the protein sample onto a the samples for amino acid analysis so that they are within reversed-phase HPLC system, eluting the protein with a the analytical limits (e.g., linear working range) of the tech- volatile solvent containing a sufficient organic component, nique employed in order to obtain accurate and repeatable and drying the sample in a vacuum centrifuge; (2) dialysis results. against a volatile buffer or water; (3) centrifugal ultrafiltra- Four to six amino acid standard levels are analyzed to de- tion for buffer replacement with a volatile buffer or water; termine a response factor for each amino acid. The response (4) precipitating the protein from the buffer using an organic factor is calculated as the average peak area or peak height solvent (e.g., acetone); and (5) gel filtration. per nmol of amino acid present in the standard. A calibration file consisting of the response factor for each amino Internal Standards acid is prepared and used to calculate the concentration of It is recommended that an internal standard be used to each amino acid present in the test sample. This calculation monitor physical and chemical losses and variations during involves dividing the peak area corresponding to a given amino acid analysis. An accurately known amount of inter- amino acid by the response factor for that amino acid to give nal standard can be added to a protein solution prior to hy- the nmol of the amino acid. For routine analysis, a single- drolysis. The recovery of the internal standard gives the gen- point calibration may be sufficient; however, the calibration eral recovery of the amino acids of the protein solution. Free file is updated frequently and tested by the analysis of ana- amino acids, however, do not behave in the same way as lytical controls to ensure its integrity. protein-bound amino acids during hydrolysis because their rates of release or destruction are variable. Therefore, the Repeatability use of an internal standard to correct for losses during hy- Consistent high quality amino acid analysis results from drolysis may give unreliable results. It will be necessary to an analytical laboratory require attention to the repeatability take this point under consideration when interpreting the of the assay. During analysis of the chromatographic separa- results. Internal standards can also be added to the mixture tion of the amino acids or their derivatives, numerous peaks of amino acids after hydrolysis to correct for differences in can be observed on the chromatogram that correspond to the sample application and changes in reagent stability and flow amino acids. The large numberofpeaksmakesitnecessary rates. Ideally, an internal standard is an unnaturally occur- to have an amino acid analysis system that can repeatedly ring primary amino acid that is commercially available and identify the peaks based on retention time and integrate the inexpensive. It should also be stable during hydrolysis, its peak areas for quantitation. A typical repeatability evalua- response factor should be linear with concentration, and it tion involves preparing a standard amino acid solution and needs to elute with a unique retention time without overlap- analyzing many replicates (i.e., six analyses or more) of the ping other amino acids. Commonly used amino acid stand- same standard solution. The relative standard deviation ards include norleucine, nitrotyrosine, and a-aminobutyric (RSD) is determined for the retention time and integrated acid. peak area of each amino acid. An evaluation of the repeatability is expanded to include multiple assays conducted over Protein Hydrolysis several days by different analysts. Multiple assays include Hydrolysis of protein and peptide samples is necessary for the preparation of standard dilutions from starting materials amino acid analysis of these molecules. The glassware used JP XVI General Information / Biotechnological/Biological Products 2161 for hydrolysis must be very clean to avoid erroneous results. investigated for each individual protein/peptide sample. The Glove powders and fingerprints on hydrolysis tubes may microwave hydrolysis technique typically requires only a few cause contamination. To clean glass hydrolysis tubes, boil minutes, but even a deviation of one minute may give inade- tubes for 1 hour in 1 mol/L hydrochloric acid or soak tubes quate results (e.g., incomplete hydrolysis or destruction of in concentrated nitric acid or in a mixture of concentrated labile amino acids). Complete proteolysis, using a mixture of hydrochloric acid and concentrated nitric acid (1:1). Clean proteases, has been used but can be complicated, requires hydrolysis tubes are rinsed with high-purity water followed the proper controls, and is typically more applicable to pep- by a rinse with HPLC grade methanol, dried overnight in an tides than proteins. oven, and stored covered until use. Alternatively, pyrolysis Note: During initial analyses of an unknown protein, ex- of clean glassware at 5009C for 4 hours may also be used to periments with various hydrolysis time and temperature con- eliminate contamination from hydrolysis tubes. Adequate ditions are conducted to determine the optimal conditions. disposable laboratory material can also be used. Method 1 Acid hydrolysis is the most common method for hydrolyz- Acid hydrolysis using hydrochloric acid containing phenol ing a protein sample before amino acid analysis. The acid is the most common procedure used for protein/peptide hy- hydrolysis technique can contribute to the variation of the drolysis preceding amino acid analysis. The addition of analysis due to complete or partial destruction of several phenol to the reaction prevents the halogenation of tyrosine. amino acids. Tryptophan is destroyed; serine and threonine Hydrolysis Solution 6 mol/L hydrochloric acid contain- are partially destroyed; methionine might undergo oxida- ing 0.1z to 1.0z of phenol. tion; and cysteine is typically recovered as cystine (but cys- Procedure— tine recovery is usually poor because of partial destruction or Liquid Phase Hydrolysis Place the protein or peptide reduction to cysteine). Application of adequate vacuum sample in a hydrolysis tube, and dry. [Note: The sample is (≦less than 200 mm of mercury or 26.7 Pa) or introduction dried so that water in the sample will not dilute the acid used of an inert gas (argon) in the headspace of the reaction vessel for the hydrolysis.] Add 200 mLofHydrolysis Solution per can reduce the level of oxidative destruction. In peptide 500 mg of lyophilized protein. Freeze the sample tube in a dry bonds involving isoleucine and valine the amido bonds of ice-acetone bath, and flame seal in vacuum. Samples are Ile-Ile, Val-Val, Ile-Val, and Val-Ile are partially cleaved; typically hydrolyzed at 1109C for 24 hours in vacuum or and asparagine and glutamine are deamidated, resulting in inert atmosphere to prevent oxidation. Longer hydrolysis aspartic acid and glutamic acid, respectively. The loss of times (e.g., 48 and 72 hours) are investigated if there is a tryptophan, asparagine, and glutamine during an acid hy- concern that the protein is not completely hydrolyzed. drolysis limits quantitation to 17 amino acids. Some of the Vapor Phase Hydrolysis This is one of the most common hydrolysis techniques described are used to address these acid hydrolysis procedures, and it is preferred for microanal- concerns. Some of the hydrolysis techniques described (i.e., ysis when only small amounts of the sample are available. Methods 4-11) may cause modifications to other amino Contamination of the sample from the acid reagent is also acids. Therefore, the benefits of using a given hydrolysis minimized by using vapor phase hydrolysis. Place vials con- technique are weighed against the concerns with the tech- taining the dried samples in a vessel that contains an ap- nique and are tested adequately before employing a method propriate amount of Hydrolysis Solution.TheHydrolysis other than acid hydrolysis. Solution does not come in contact with the test sample. A time-course study (i.e., aminoacidanalysisatacidhy- Apply an inert atmosphere or vacuum (≦less than 200 mmof drolysis times of 24, 48, and 72 hours) is often employed to mercury or 26.7 Pa) to the headspace of the vessel, and heat analyze the starting concentration of amino acids that are to about 1109C for a 24-hour hydrolysis time. Acid vapor partially destroyed or slow to cleave. By plotting the ob- hydrolyzes the dried sample. Any condensation of the acid in served concentration of labile amino acids (i.e., serine and the sample vials is minimized. After hydrolysis, dry the test threonine) versus hydrolysis time, the line can be extrapo- sample in vacuum to remove any residual acid. lated to the origin to determine the starting concentration of these amino acids. Time-course hydrolysis studies are also Method 2 used with amino acids that are slow to cleave (e.g., isoleucine Tryptophan oxidation during hydrolysis is decreased by and valine). During the hydrolysis time course, the analyst using mercaptoethanesulfonic acid (MESA) as the reducing will observe a plateau in these residues. The level of this acid. plateau is taken as the residue concentration. If the hydroly- Hydrolysis Solution 2.5 mol/L MESA solution. sis time is too long, the residue concentration of the sample Vapor Phase Hydrolysis About 1 to 100 mg of the pro- will begin to decrease, indicating destruction by the hydroly- tein/peptide under test is dried in a hydrolysis tube. The hy- sis conditions. drolysis tube is placed in a larger tube with about 200 mLof An acceptable alternative to the time-course study is to the Hydrolysis Solution. The larger tube is sealed in vacuum subject an amino acid calibration standard to the same hy- (about 50 mm of mercury or 6.7 Pa) to vaporize the Hydroly- drolysis conditions as the test sample. The amino acid in free sis Solution. The hydrolysis tube is heated to 1709Cto1859C form may not completely represent the rate of destruction of for about 12.5 minutes. After hydrolysis, the hydrolysis tube labile amino acids within a peptide or protein during the hy- is dried in vacuum for 15 minutes to remove the residual drolysis. This is especially true for peptide bonds that are acid. slow to cleave (e.g., Ile-Val bonds). However, this technique Method 3 will allow the analyst to account for some residue destruc- Tryptophan oxidation during hydrolysis is prevented by tion. Microwave acid hydrolysis has been used and is rapid using thioglycolic acid (TGA) as the reducing acid. but requires special equipment as well as special precautions. Hydrolysis Solution A solution containing 7 mol/L hy- The optimal conditions for microwave hydrolysis must be 2162 Biotechnological/Biological Products / General Information JP XVI drochloric acid, 10z of trifluoroacetic acid, 20z of thiogly- phan are also modified, a complete compositional analysis is colic acid, and 1z of phenol. not obtained with this technique. Vapor Phase Hydrolysis About 10 to 50 mg of the pro- Method 7 tein/peptide under test is dried in a sample tube. The sample Cysteine-cystine reduction and alkylation is accomplished tube is placed in a larger tube with about 200 mLofthe by a vapor phase pyridylethylation reaction. Hydrolysis Solution. The larger tube is sealed in vacuum Reducing Solution Transfer 83.3 mL of pyridine, 16.7 mL (about 50 mm of mercury or 6.7 Pa) to vaporize the TGA. of 4-vinylpyridine, 16.7 mL of tributylphosphine, and 83.3 The sample tube is heated to 1669C for about 15 to 30 mL of water to a suitable container, and mix. minutes. After hydrolysis, the sample tube is dried in vacu- Procedure Add the protein/peptide (between 1 and 100 um for 5 minutes to remove the residual acid. Recovery of mg) to a hydrolysis tube, and place in a larger tube. Transfer tryptophan by this method may be dependent on the amount the Reducing Solution to the large tube, seal in vacuum of sample present. (about 50 mm of mercury or 6.7 Pa), and incubate at about Method 4 1009C for 5 minutes. Then remove the inner hydrolysis tube, Cysteine-cystine and methionine oxidation is performed and dry it in a vacuum desiccator for 15 minutes to remove with performic acid before the protein hydrolysis. residual reagents. The pyridylethylated protein/peptide can Oxidation Solution The performic acid is prepared fresh then be acid hydrolyzed using previously described proce- by mixing formic acid and 30 percent hydrogen peroxide dures. The pyridylethylation reaction is performed simul- (9:1), and incubated at room temperature for 1 hour. taneously with a protein standard sample containing 1 to 8 Procedure The protein/peptide sample is dissolved in 20 mol of cysteine to improve accuracy in the pyridylethyl- mL of formic acid, and heated at 509C for 5 minutes; then cysteine recovery. Longer incubation times for the pyri- 100 mLoftheOxidation Solution is added. The oxidation is dylethylation reaction can cause modifications to the a- allowed to proceed for 10 to 30 minutes. In this reaction, amino terminal group and the e-amino group of lysine in the cysteine is converted to cysteic acid and methionine is con- protein. verted to methionine sulfone. The excess reagent is removed Method 8 from the sample in a vacuum centrifuge. This technique may Cysteine-cystine reduction and alkylation is accomplished cause modifications to tyrosine residues in the presence of by a liquid phase pyridylethylation reaction. halides. The oxidized protein can then be acid hydrolyzed Stock Solutions Prepare and filter three solutions: 1 using Method 1 or Method 2. mol/L Tris hydrochloride (pH 8.5) containing 4 mmol/L Method 5 disodium dihydrogen ethylendiamine tetraacetate (Stock So- Cysteine-cystine oxidation is accomplished during the liq- lution A), 8 mol/L guanidine hydrochloride (Stock Solution uid phase hydrolysis with sodium azide. B), and 10z of 2-mercaptoethanol in water (Stock Solution Hydrolysis Solution 6 mol/L hydrochloric acid contain- C). ing 0.2z of phenol, to which is added sodium azide to ob- Reducing Solution Prepare a mixture of Stock Solution tain a final concentration of 0.2z (w/v). The added phenol B and Stock Solution A (3:1) to obtain a buffered solution prevents halogenation of tyrosine. of 6 mol/L guanidine hydrochloride in 0.25 mol/L Tris hy- Liquid Phase Hydrolysis The protein/peptide hydrolysis drochloride. is conducted at about 1109C for 24 hours. During the hy- Procedure Dissolve about 10 mg of the test sample in 50 drolysis, the cysteine-cystine present in the sample is con- mLoftheReducing Solution, and add about 2.5 mLofStock verted to cysteic acid by the sodium azide present in the Solution C. Store under nitrogen or argon for 2 hours at Hydrolysis Solution. This technique allows better tyrosine room temperature in the dark. To achieve the pyridylethyla- recovery than Method 4, but it is not quantitative for tion reaction, add about 2 mL of 4-vinylpyridine to the pro- methionine. Methionine is converted to a mixture of the par- tein solution, and incubate for an additional 2 hours at room ent methionine and its two oxidative products, methionine temperature in the dark. The protein/peptide is desalted by sulfoxide and methionine sulfone. collecting the protein/peptide fraction from a reversed-phase HPLC separation. The collected sample can be dried in a Method 6 vacuum centrifuge before acid hydrolysis. Cysteine-cystine oxidation is accomplished with dimethyl sulfoxide (DMSO). Method 9 Hydrolysis Solution 6 mol/L hydrochloric acid contain- Cysteine-cystine reduction and alkylation is accomplished ing 0.1z to 1.0z of phenol, to which DMSO is added to by a liquid phase carboxymethylation reaction. obtain a final concentration of 2z (v/v). Stock Solutions Prepare as directed for Method 8. Vapor Phase Hydrolysis The protein/peptide hydrolysis Carboxymethylation Solution Prepare a solution con- is conducted at about 1109C for 24 hours. During the hy- taining 100 mg of iodoacetamide per mL of ethanol (95). drolysis, the cysteine-cystine present in the sample is con- Buffer Solution Use the Reducing Solution, prepared as verted to cysteic acid by the DMSO present in the Hydrolysis directed for Method 8. Solution. As an approach to limit variability and compensate Procedure Dissolve the test sample in 50 mLofthe for partial destruction, it is recommended to evaluate the Buffer Solution, and add about 2.5 mLofStock Solution C. cysteic acid recovery from oxidative hydrolyses of standard Store under nitrogen or argon for 2 hours at room tempera- proteins containing 1 to 8 mol of cysteine. The response fac- ture in the dark. Add the Carboxymethylation Solution in a tors from protein/peptide hydrolysates are typically about ratio 1.5 fold per total theoretical content of thiols, and in- 30z lower than those for nonhydrolyzed cysteic acid stand- cubate for an additional 30 minutes at room temperature in ards. Because histidine, methionine, tyrosine, and trypto- the dark. [Note: If the thiol content of the protein is JP XVI General Information / Biotechnological/Biological Products 2163 unknown, then add 5 mL of 100 mmol/L iodoacetamide for drolysis without BTI will have to be performed if the analyst every 20 nmol of protein present.] The reaction is stopped by is interested in the composition of these other amino acid adding excess of 2-mercaptoethanol. The protein/peptide is residues of the protein/peptide.] desalted by collecting the protein/peptide fraction from a Methodologies of Amino Acid Analysis General Principles reversed-phase HPLC separation. The collected sample can Many amino acid analysis techniques exist, and the choice be dried in a vacuum centrifuge before acid hydrolysis. The of any one technique often depends on the sensitivity re- S-carboxyamidomethyl-cysteine formed will be converted to quired from the assay. In general, about one-half of the S-carboxymethylcysteine during acid hydrolysis. amino acid analysis techniques employedrelyonthesepara- Method 10 tion of the free amino acids by ion-exchange chromatogra- Cysteine-cystine is reacted with dithiodiglycolic acid or phy followed by postcolumn derivatization (e.g., with nin- dithiodipropionic acid to produce a mixed disulfide. [Note: hydrin or o-phthalaldehyde). Postcolumn detection tech- The choice of dithiodiglycolic acid or dithiodipropionic acid niques can be used with samples that contain small amounts depends on the required resolutionoftheaminoacidanaly- of buffer components, such as salts and urea, and generally sis method.] require between 5 and 10 mg of protein sample per analysis. Reducing Solution A solution containing 10 mg of The remaining amino acid techniques typically involve pre- dithiodiglycolic acid (or dithiodipropionic acid) per mL of column derivatization of the free amino acids (e.g., phenyl 0.2 mol/L sodium hydroxide. isothiocyanate; 6-aminoquinolyl-N-hydroxysuccinimidyl Procedure Transfer about 20 mg of the test sample to a carbamate or o-phthalaldehyde; (dimethylamino)azoben- hydrolysis tube, and add 5 mLoftheReducing Solution. zenesulfonyl chloride; 9-fluorenylmethylchloroformate; and, Add 10 mL of isopropyl alcohol, and then remove all of the 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole) followed by rever- sample liquid by vacuum centrifugation. The sample is then sed-phase HPLC. Precolumn derivatization techniques are hydrolyzed using Method 1. This method has the advantage very sensitive and usually require between 0.5 and 1.0 mgof that other amino acid residues are not derivatized by side protein sample per analysis but may be influenced by buffer reactions, and the sample does not need to be desalted prior salts in the samples. Precolumn derivatization techniques to hydrolysis. may also result in multiple derivatives of a given amino acid, which complicates the result interpretation. Postcolumn Method 11 derivatization techniques are generally influenced less by Asparagine and glutamine are converted to aspartic acid performance variation of the assay than precolumn derivati- and glutamic acid, respectively, during acid hydrolysis. zation techniques. Asparagine and aspartic acid residues are added and repre- The following Methods may be used for quantitative sented by Asx, while glutamine and glutamic acid residues amino acid analysis. Instruments and reagents for these are added and represented by Glx. Proteins/peptides can be procedures are available commercially. Furthermore, many reacted with bis(1,1-trifluoroacetoxy)iodobenzene (BTI) to modifications of these methodologies exist with different convert the asparagine and glutamine residues to diamino- reagent preparations, reaction procedures, chromatographic propionic acid and diaminobutyric acid residues, respec- systems, etc. Specific parameters may vary according to the tively, upon acid hydrolysis. These conversions allow the exact equipment and procedure used. Many laboratories will analyst to determine the asparagine and glutamine content of utilize more than one amino acid analysis technique to a protein/peptide in the presence of aspartic acid and glu- exploit the advantages offered by each. In each of these tamic acid residues. Methods, the analog signal is visualized by means of a data Reducing Solutions Prepare and filter three solutions: a acquisition system, and the peak areas are integrated for solution of 10 mmol/L trifluoroacetic acid (Solution A), a quantification purposes. solution of 5 mol/L guanidine hydrochloride and 10 mmol/ L trifluoroacetic acid (Solution B), and a freshly prepared Method 1—Postcolumn Ninhydrin Detection General solution of N,N-dimethylformamide containing 36 mg of Principle BTI per mL (Solution C). Ion-exchange chromatography with postcolumn ninhydrin Procedure In a clean hydrolysis tube, transfer about 200 detection is one of the most common methods employed for mg of the test sample, and add 2 mL of Solution A or Solu- quantitative amino acid analysis. As a rule, a Li-based tion B and 2 mL of Solution C. Seal the hydrolysis tube in cation-exchange system is employed for the analysis of the vacuum. Heat the sample at 609C for 4 hours in the dark. more complex physiological samples, and the faster Na- The sample is then dialyzed with water to remove the excess based cation-exchange system is used for the more simplistic reagents. Extract the dialyzed sample three times with equal amino acid mixtures obtained with protein hydrolysates volumes of n-butyl acetate, and then lyophilize. The protein (typically containing 17 amino acid components). Separation can then be acid hydrolyzed using previously described of the amino acids on an ion-exchange column is accom- procedures. The a,b-diaminopropionic and a,g-diaminobu- plished through a combination of changes in pH and cation tyric acid residues do not typically resolve from the lysine strength. A temperature gradient is often employed to residues upon ion-exchange chromatography based on enhance separation. amino acid analysis. Therefore, when using ion-exchange as When the amino acid reacts with ninhydrin, the reactant the mode of amino acid separation, the asparagine and has characteristic purple or yellow color. Amino acids, ex- glutamine contents are the quantitative difference in the cept imino acid, give a purple color, and show the maximum aspartic acid and glutamic acid content assayed with absorption at 570 nm. The imino acids such as proline give a underivatized and BTI-derivatized acid hydrolysis. [Note: yellow color, and show the maximum absorption at 440 nm. The threonine, methionine, cysteine, tyrosine, and histidine The postcolumn reaction between ninhydrin and amino acid assayed content can be altered by BTI derivatization; a hy- eluted from column is monitored at 440 and 570 nm, and the 2164 Biotechnological/Biological Products / General Information JP XVI chromatogram obtained is used for the determination of Detection limit is considered to be 1 pmol for most of the amino acid composition. amino acid derivatives. Response linearity is obtained in the Detection limit is considered to be 10 pmol for most of the range of 20 to 500 pmol with correlation coefficients amino acid derivatives, but 50 pmol for proline. Response exceeding 0.999. To obtain good compositional data, sam- linearity is obtained in the range of 20 to 500 pmol with cor- ples larger than 500 ng of protein/peptide before hydrolysis relation coefficients exceeding 0.999. To obtain good com- is best suited for this amino acid analysis of proteins/ position data, samples larger than 1 mg before hydrolysis are peptides. best suited for this amino acid analysis of protein/peptide. Method 4—Precolumn AQC Derivatization General Method 2—Postcolumn OPA Fluorometric Detection Principle General Principle Precolumn derivatization of amino acids with 6-amino- o-Phthalaldehyde (OPA) reacts with primary amines in quinolyl-N-hydroxysuccinimidyl carbamate (AQC) followed the presence of thiol compound, to form highly fluorescent by reversed-phase HPLC separation with fluorometric detec- isoindole products. This reaction is utilized for the postcol- tion is used. umn derivatization in analysis of amino acids by ion- AQC reacts with amino acids to form stable, fluorescent exchange chromatography. The rule of the separation is the unsymmetric urea derivatives (AQC-amino acids) which are same as Method 1. Instruments and reagents for this form of readily amenable to analysis by reversed-phase HPLC. amino acid analysis are available commercially. Many Therefore, precolumn derivatization of amino acids with modifications of this methodology exist. AQC followed by reversed-phase HPLC separation is used Although OPA does not react with secondary amines to analyze the amino acid composition. (imino acids such as proline) to form fluorescent substances, Separation of the AQC-amino acids on an ODS column is the oxidation with sodium hypochlorite allows secondary accomplished through a combination of changes in concen- amines to react with OPA. The procedure employs a trations of acetonitrile and salt. Selective fluorescence detec- strongly acidic cation-exchange column for separation of tion of the derivatives with excitation wavelength at 250 nm free amino acids followed by postcolumn oxidation with and emission wavelength at 395 nm allows for the direct sodium hypochlorite and postcolumn derivatization using injection of the reaction mixture with no significant interfer- OPA and thiol compound such as N-acetyl-L-cysteine and 2- ence from the only major fluorescent reagent by-product, mercaptoethanol. The derivatization of primary amino acids 6-aminoquinoline. Excess reagent is rapidly hydrolyzed are not noticeably affected by the continuous supply of (t1/2 ＜ 15 seconds) to yield 6-aminoquinoline, N-hydroxy- sodium hypochlorite. succinimide and carbon dioxide, and after 1 minute no fur- Separation of the amino acids on an ion-exchange column ther derivatization can take place. is accomplished through a combination of changes in pH Peak areas for AQC-amino acids are essentially un- and cation strength. After postcolumn derivatization of changed for at least 1 week at room temperature, and the eluted amino acids with OPA, the reactant passes through derivatives have more than sufficient stability to allow for the fluorometric detector. Fluorescence intensity of OPA- overnight automated chromatographic analysis. derivatized amino acids are monitored with an excitation Detection limit is considered to be ranging from ca. 40 to wavelength of 348 nm and an emission wavelength of 450 320 fmol for each amino acid, except for Cys. Detection nm. limit for Cys is approximately 800 fmol. Response linearity Detection limit is considered to be a few tens of picomole is obtained in the range of 2.5 to 200 mmol/L with correla- level for most of the amino acid derivatives. Response tion coefficients exceeding 0.999. Good compositional data linearity is obtained in the range of a few picomole level to a could be obtained from the analysis of derivatized protein few tens of nanomole level. To obtain good compositional hydrolysates containing as little as 30 ng of protein/peptide. data, the starting with greater than 500 ng of sample before Method 5—Precolumn OPA Derivatization General hydrolysis is best suited for the amino acid analysis of pro- Principle tein/peptide. Precolumn derivatization of amino acids with o-phthalal- Method 3—Precolumn PITC Derivatization General dehyde (OPA) followed by reversed-phase HPLC separation Principle with fluorometric detection is used. This technique does not Phenylisothiocyanate (PITC) reacts with amino acids to detect amino acids that exist as secondary amines (e.g., form phenylthiocarbamyl (PTC) derivatives which can be proline). detected with high sensitivity at 245 nm. Therefore, precol- OPA in conjunction with a thiol reagent reacts with umn derivatization of amino acids with PITC followed by a primary amine groups to form highly fluorescent isoindole reversed-phase HPLC separation with UV detection is used products. 2-Mercaptoethanol or 3-mercaptopropionic acid to analyze the amino acid composition. can be used as thiol. OPA itself does not fluoresce and con- After the reagent is removed under vacuum, the deriva- sequently produces no interfering peaks. In addition, its tized amino acids can be stored dry and frozen for several solubility and stability in aqueous solution, along with the weeks with no significant degradation. If the solution for in- rapid kinetics for the reaction, make it amenable to automat- jection is kept cold, no noticeable loss in chromatographic ed derivatization and analysis using an autosampler to mix response occurs after three days. the sample with the reagent. However, lack of reactivity with Separation of the PTC-amino acids on a reversed-phase secondary amino acids has been a predominant drawback. HPLC with ODS column is accomplished through a combi- This method does not detect amino acids that exist as nation of changes in concentrations of acetonitrile and secondary amines (e.g., proline). To compensate for this buffer ionic strength. PTC-amino acids eluted from the drawback, this technique may be combined with another column are monitored at 254 nm. technique described in Method 7 or Method 8. JP XVI General Information / Biotechnological/Biological Products 2165

Precolumn derivatization of amino acids with OPA is fol- the reagent excess and fluorescent side-products can be lowed by a reversed-phase HPLC separation. Because of the eliminated without loss of FMOC-amino acids. instability of the OPA-amino acid derivative, HPLC separa- FMOC-amino acids are separated by a reversed-phase tion and analysis are performed immediately following HPLC using an ODS column. The separation is carried out derivatization. The liquid chromatograph is equipped with a by gradient elution varied linearly from a mixture of aceto- fluorometric detector for the detection of derivatized amino nitrile methanol and acetic acid buffer (10:40:50) to a mix- acids. Fluorescence intensity of OPA-derivatized amino ture of acetonitrile and acetic acid buffer (50:50), and 20 acids is monitored with an excitation wavelength of 348 nm amino acid derivatives are separated in 20 minutes. Each and an emission wavelength of 450 nm. derivative eluted from the column is monitored by a fluoro- Detection limits as low as 50 fmol via fluorescence have metric detector set at an excitation wavelength of 260 nm been reported, although the practical limit of analysis andanemissionwavelengthof313nm. remains at 1 pmol. The detection limit is in the low fmol range. A linearity range of 0.1 to 50 mmol/L is obtained for most of the amino Method 6—Precolumn DABS-Cl Derivatization General acids. Principle Precolumn derivatization of amino acids with Method 8—Precolumn NBD-F Derivatization General (dimethylamino)azobenzenesulfonyl chloride (DABS-Cl) Principle followed by reversed-phase HPLC separation with visible Precolumn derivatization of amino acids with 7-fluoro-4- light detection is used. nitrobenzo-2-oxa-1.3-diazole (NBD-F) followed by reversed- DABS-Cl is a chromophoric reagent employed for the phase HPLC separation with fluorometric detection is used. labeling of amino acids. Amino acids labeled with DABS-Cl NBD-F reacts with both primary and secondary amino (DABS-amino acids) are highly stable and show the maxi- acids to form highly fluorescent products. Amino acids are mum absorption at 436 nm. derivatized with NBD-F by heating to 609C for 5 minutes. DABS-amino acids, all 19 naturally occurring amino acids NBD-amino acid derivatives are separated on an ODS derivatives, can be separated on an ODS column of a rever- column of a reversed-phase HPLC by employing gradient sed-phase HPLC by employing gradient systems consisting elution system consisting of acetonitrile and aqueous buffer of acetonitrile and aqueous buffer mixture. Separated mixture, and 17 amino acid derivatives are separated in 35 DABS-amino acids eluted from the column are detected at minutes. e-Aminocaproic acid can be used as an internal 436 nm in the visible region. standard, because it is eluted in a clean chromatographic This Method can analyze the imino acids such as proline region. Each derivative eluted from the column is monitored together with the amino acids at the same degree of sensi- by a fluorometric detector set at an excitation wavelength of tivity, DABS-Cl derivatization method permits the simul- 480 nm and an emission wavelength of 530 nm. taneous quantification of tryptophan residues by previous The sensitivity of this method is almost the same as for hydrolysis of the protein/peptide with sulfonic acids such as precolumn OPA derivatization method (Method 5), exclud- mercaptoethanesulfonic acid, p-toluenesulfonic acid or ing proline to which OPA is not reactive, and might be ad- methanesulfonic acid described under Method 2 in ``Protein vantageous for NBD-F against OPA. The detection limit for Hydrolysis''. The other acid-labile residues, asparagine and each amino acid is about 10 fmol. Profile analysis was glutamine, can also be analysed by previous conversion into achieved for about 1.5 mg of protein hydrolysates in the final diaminopropionic acid and diaminobutyric acid, respec- precolumn labeling reaction mixture for HPLC. tively, by treatment of protein/peptide with BTI described Data Calculation and Analysis under Method 11 in ``Protein Hydrolysis''. When determining the amino acid content of a protein/ The non-proteinogenic amino acid, norleucine cannot be peptide hydrolysate, it should be noted that the acid hydroly- used as internal standard in this method, as this compound is sis step destroys tryptophan and cysteine. Serine and threo- eluted in a chromatographic region crowded with peaks of nine are partially destroyed by acid hydrolysis, while isoleu- primary amino acids. Nitrotyrosine can be used as an inter- cine and valine residues may be only partially cleaved. nal standard, because it is eluted in a clean region. Methionine can undergo oxidation during acid hydrolysis, Detection limit of DABS-amino acid is about 1 pmol. As and some amino acids (e.g., glycine and serine) are common little as 2 to 5 pmol of an individual DABS-amino acid can contaminants. Application of adequate vacuum (≦0.0267 be quantitatively analysed with reliability, and only 10 to 30 kPa) or introduction of inert gas (argon) in the headspace of ng of the dabsylated protein hydrolysate is required for each the reaction vessel during vapor phase hydrolysis can reduce analysis. the level of oxidative destruction. Therefore, the quantitative Method 7—Precolumn FMOC-Cl Derivatization General results obtained for cysteine, tryptophan, threonine, isoleu- Principle cine, valine, methionine, glycine, and serine from a protein/ Precolumn derivatization of amino acids with 9- peptide hydrolysate may be variable and may warrant fur- fluorenylmethyl chloroformate (FMOC-Cl) followed by ther investigation and consideration. reversed-phase HPLC separation with fluorometric detec- Calculations tion is used. Amino Acid Mole Percent This is the number of specific FMOC-Cl reacts with both primary and secondary amino amino acid residues per 100 residues in a protein. This result acids to form highly fluorescent products. The reaction of may be useful for evaluating aminoacidanalysisdatawhen FMOC-Cl with amino acid proceeds under mild conditions the molecular weight of the protein under investigation is in aqueous solution and is completed in 30 seconds. The unknown. This information can be used to corroborate the derivatives are stable, only the histidine derivative showing identity of a protein/peptide and has other applications. any breakdown. Although FMOC-Cl is fluorescent itself, 2166 Biotechnological/Biological Products / General Information JP XVI

Carefully identify and integrate the peaks obtained as di- determine the amino acid composition of the sample by rected for each Procedure. Calculate the mole percent for analysis. each amino acid present in the test sample by the formula: Calculate the relative compositional error, in percentage,

100rU/r, by the formula: in which rU is the peak response, in nmol, of the amino acid 100m/mS, under test; and r is the sum of peak responses, in nmol, for in which m is the experimentally determined quantity, in all amino acids present in the test sample. Comparison of the nmol per amino acid residue, of the amino acid under test; mole percent of the amino acids under test to data from and mS is the known residue value for that amino acid. The known proteins can help establish or corroborate the identity average relative compositional error is the average of the of the sample protein. absolute values of the relative compositional errors of the Unknown Protein Samples This data analysis technique individual amino acids, typically excluding tryptophan and can be used to estimate the protein concentration of an cysteine from this calculation. The average relative composi- unknown protein sample using the amino acid analysis data. tional error can provide important information on the stabil- Calculate the mass, in mg, of each recovered amino acid by ity of analysis run over time. The agreement in the amino the formula: acid composition between the protein sample and the known

mMW/1000, composition can be used to corroborate the identity and in which m is the recovered quantity, in nmol, of the amino purity of the protein in the sample. acid under test; and MW is the average molecular weight for that amino acid, corrected for the weight of the water molecule that was eliminated during peptide bond formation. The sum of the masses of the recovered amino acids will give an Basic Requirements for Viral Safety estimate of the total mass of the protein analyzed after ap- of Biotechnological/Biological propriate correction for partially and completely destroyed amino acids. If the molecular weight of the unknown protein Products listed in Japanese is available (i.e., by SDS-PAGE analysis or mass spectros- Pharmacopoeia copy), the amino acid composition of the unknown protein can be predicted. Calculate the number of residues of each Introduction aminoacidbytheformula: The primary role of specification of biotechnological/ m/(1000M/MWT), biological products listed in Japanese Pharmacopoeia (JP) is in which m is the recovered quantity, in nmol, of the amino not only for securing quality control or consistency of the acid under test; M is the total mass, in mg, of the protein; quality but also for assuring their efficacy and safety. In the and MWT is the molecular weight of the unknown protein. meantime, the requirements to assure quality and safety of Known Protein Samples This data analysis technique can drugs have come to be quite strict recently, and a rigid atti- be used to investigate the amino acid composition and pro- tude addressing safety assurance is expected for biotechno- tein concentration of a protein sample of known molecular logical/biological products. The key points for quality and weight and amino acid composition using the amino acid safety assurance of biotechnological/biological products are analysis data. When the composition of the protein being selection and appropriate evaluation of source material, ap- analyzed is known, one can exploit the fact that some amino propriate evaluation of manufacturing process and main- acids are recovered well, while other amino acid recoveries tenance of manufacturing consistency, and control of spe- may be compromised because of complete or partial destruc- cific physical properties of the products. Now, how to assure tion (e.g., tryptophan, cysteine, threonine, serine, methio- quality and safety of such drugs within a scope of JP has nine), incomplete bond cleavage (i.e., for isoleucine and come to be questioned. This General Information describes valine) and free amino acid contamination (i.e., by glycine what sorts of approaches are available to overcome these and serine). issues. Because those amino acids that are recovered best It is desired that quality and safety assurance of JP listed represent the protein, these amino acids are chosen to quan- products are achieved by state-of-the-art methods and con- tify the amount of protein. Well-recovered amino acids cepts which reflect progress of science and accumulation of are, typically, aspartate-asparagine, glutamate-glutamine, experiences. This General Information challenges to show alanine, leucine, phenylalanine, lysine, and arginine. This the highest level of current scientific speculation. It is ex- list can be modified based on experience with one's own pected that this information will contribute to promotion of analysis system. Divide the quantity, in nmol, of each of the scientific understanding of quality and safety assurance of well-recovered amino acids by the expected number of not only JP listed products but also the other biotechnologi- residues for that amino acid to obtain the protein content cal/biological products and to promotion of active discus- based on each well-recovered amino acid. Average the pro- sion of each Official Monograph in JP. tein content results calculated. The protein content determined for each of the well-recovered amino acids should be 1. Fundamental measures to ensure viral safety of JP listed evenly distributed about the mean. Discard protein content biotechnological/biological products values for those amino acids that have an unacceptable de- The biotechnological/biological product JP includes the viation from the mean. Typically ≧greater than 5z varia- products derived from living tissue and body fluid (urine, tion from the mean is considered unacceptable. Recalculate blood, etc.) of mammals, etc. Protein drugs derived from the mean protein content from the remaining values to ob- cell lines of human or animal origin (e.g., recombinant DNA tain the protein content of the sample. Divide the content of drug, cell culture drug) are also included. The fundamental each amino acid by the calculated mean protein content to measures required for comprehensive viral safety of JP listed JP XVI General Information / Biotechnological/Biological Products 2167 biotechnological/biological products are as follows: 1) ac- urine, and protein drugs derived from cell line of human or quaintance of possible virus contamination (source of con- animal origin (Table 1). tamination); 2) careful examination of eligibility of raw ma- 3.1. Purpose terials and their sources, e.g. human/animal, and thorough The purpose of this document is to propose the compre- analysis and screening of the sample chosen as a substrate hensive concepts of the measures to be taken for ensuring for drug production (e.g., pooled body fluid, cell bank, etc.) viral safety of biotechnological/biological products derived to determine any virus contamination and determination of from living tissue or body fluid of mammals, etc. and of type and nature of the virus, if contaminated; 3) evaluation protein drugs derived from cell lines of human or animal to determine virus titer and virus-like particles hazardous to origin. That is to say, this document describes the measures human, if exists; 4) selection of production related material and the points of concern on the items, such as  considera- (e.g., reagent, immune antibody column) free from infecti- tion of the source of virus contamination;  appropriate ous or pathogenic virus; 5) performance of virus free test at evaluation on eligibility at selecting the raw material and on an appropriate stage of manufacturing including the final qualification of its source, e.g. human or animal;  virus product, if necessary; 6) adoption of effective viral clearance test, and its analysis and evaluation at a stage of cell sub- method in the manufacturing process to remove/inactivate strate for drug production;  appropriate evaluation to virus. Combined method sometimes achieves higher level of choose product related materials derived from living organ- clearance; 7) development of a deliberate viral clearance isms (e.g. reagent, immune antibody column, etc.);  con- scheme; 8) performance of the test to evaluate viral removal duct of necessary virus test on the product at an appropriate and inactivation. It is considered that the stepwise and sup- stage of manufacturing;  development of viral clearance plemental adoption of the said measures will contribute to test scheme;  performance and evaluation of viral clear- ensure viral safety and its improvement. ance test. This document is also purposed to comprehensively describe in details that supplemental and combining 2. Safety assurance measures described in the Official adoption of the said measures will contribute to secure viral Monograph and this General Information safety and its improvement. As mentioned in above 1, this General Information 3.2. Background describes, in package, points to be concerned with and con- One of the most important issues to be cautioned for crete information on the measures taken for viral safety of safety of a biological product, which is directly derived from JP listed products. Except where any specific caution is human or animal, or of a protein drug, which is derived provided in Official Monograph of a product in question, from cell line of human or animal origin (recombinant DNA Official Monograph provides in general that ``Any raw ma- derived product, cell culture derived product, etc.), is risk of terial, substrate for drug production and production related virus contamination. Virus contamination may cause serious material used for production of drug should be derived from situation at clinical use once it occurs. Virus contamination healthy animals and should be shown to be free of latent may be from a raw material or from a cell substrate for drug virus which is infectious or pathogenic to human'', ``Cell production, or may be from an adventitious factor intro- line and culture method well evaluatedinaspectsofap- duced to the manufacturing process. propriateness and rationality on viral safety are used for JP listed biological drugs or protein drugs derived from production, and the presence of infectious or pathogenic la- cell line have achieved drastic contribution to the medical tent virus to human in process related materials derived from society, and to date, there has not been any evidence of any living organisms should be denied''. and ``biotechnological/ safety problem on them caused by virus. But, social require- biological drug should be produced through a manufactur- ment of health hazard prevention is strong, and it is now ing process which is capable of removing infectious or very important to prevent accidental incidence, taking pathogenic virus'', etc., to raise awareness on viral safety security measures carefully supported by scientific ration- and on necessity to conduct test and process evaluation for ality. It is always great concern among the persons involved viral safety. that under what sort of viewpoint and to what extent we 3. Items and contents described in this General Information have to pursue for ensuring viral safety of a biotechnolo- As for viral safety of protein drug derived from cell line of gical/biological product. Before discussing these issues, two human or animal origin, there is a Notice in Japan entitled fundamental points have to be reconfirmed. One is that; we ``Viral safety evaluation of biotechnology products derived have to consider scientific, medical, and social profiles a from cell lines of human or animal origin'' (Iyakushin No. drug has. In other words, ``Medicine is a social asset which is 329 issued on February 22, 2000 by Director, Evaluation and utilized in medical practice paying attention to the risk and Licensing Division, Pharmaceutical and Medical Safety benefit from the standpoints of science and society''. It is the Bureau, Ministry of Health and Welfare) to reflect the inter- destine and the mission of the medical/pharmaceutical nationally harmonized ICH Guideline, and as for blood society to realize prompt and stable supply of such a social plasma protein fraction preparations, there is a document asset, drug, among the medical work front to bring gospel to entitled ``Guideline for ensuring viral safety of blood plasma the patients. protein fraction preparations''. This General Information The other is that; issue of viral safety is independent from for ensuring viral safety of JP listed biotechnological/biosafety of the components of a drug per se (narrow sense of logical products has been written, referencing the contents of safety). It is important to consider that this is the matter of those guidelines, to cover general points and their details to general safety of drug (broad sense of safety). In case of a be concerned for ensuring viral safety of not only JP listed drug which has been used for a long time in the medical biotechnological/biological products but also all products front, such as a JP listed product, its broad sense of safety is whichwouldbelistedinJPinfuture,i.e.,biological considered to have been established epidemiologically, and products derived from living tissue and body fluid, such as its usage past records have a great meaning. However, 2168 Biotechnological/Biological Products / General Information JP XVI

Table 1 Items described in General Information tion to the measures to take for prevention, while evaluating for Viral Safety Assurance of JP listed the accumulated results. Biotechnological/Biological Product Adopting strict regulations and conducting tests at maximum level to the extent theoretically considered may be the I. Introduction ways off assuring safety, but applying such way generally, 1. Fundamental measures to ensure viral safety of JP list- without sufficient scientific review of the ways and evalua- ed biotechnological/biological products tion of usage results, causes excessive requirement of regula- 2. Safety assurance measures described in the Official tion and test not having scientific rationality. As the results, Monograph and this General Information effective and prompt supply of an important drug, already 3. Items and contents described in this General Informa- having enough accumulation of experiences, to the medical tion work front will be hampered, and the drug, a social asset, II. General Matters may not to be utilized effectively. Medicine is a sword used 1. Purpose in medical field having double-edge named effectiveness and 2. Background safety. Effectiveness and safety factors have to be derived as 3. Unknown risk on the measures taken for ensuring viral the fruits of leading edge of science, and relatively evaluated safety on a balance sheet of usefulness. Usefulness evaluation 4. Applicable range should not be unbalanced in a way that too much emphasis 5. Possible viral contamination to a JP listed biotechno- is placed on safety concern without back-up of appropriate logical/biological product (source of virus contamina- scientific rationality. A drug can play an important role as a tion) social asset only when well balanced appropriate scientific 6. Basis for ensuring viral safety usefulness evaluation in addition to social concern of the age 7. Limit of virus test are given. In other words, drug is a common asset utilized by 8. Roles of viral clearance studies society for medication as a fruit of science of the age, and III. Raw material/substrate for drug production the key point of its utilization lies on a balance of risk and 1. Issues relating to animal species and its region as a benefit produced from scientific and social evaluation. So, source of raw materialsubstrate for drug production those factors have to be taken into account when target and and countermeasures to be taken thereto pursuance levels for ensuring viral safety of a JP listed 2. Qualification evaluation test on human or animal as a biotechnological/biological product are reviewed. source of raw material/substrate for drug production And, in general, the risk and benefit of drugs should be IV. Points of concern with respect to manufacturing and considered with the relative comparison to alternative drugs virus testing or medical treatment. The usefulness of certain drug should 1. Virus test conducted in advance of purification process be reviewed finally after the competitive assessment on the 2. Virus test as an acceptance test of an intermediate risk and benefit on the alternative drugs, relevant drugs and/ material, etc. or alternative medical treatment. 3. Virus test on a final product Under such background, the purpose of this article is to V. Process evaluation on viral clearance describe the scientific and rational measures to be taken for 1. Rationale, objective and general items to be concerned ensuring viral safety of JP listed biotechnological/biological with respect to viral clearance process evaluation products. Giving scientific and rational measures mean that; 2. Selection of virus appropriate and effective measures, elaborated from the cur- 3. Design of viral clearance studies rent scientific level, are given to the issues assumable under 4. Interpretation of viral clearance studies the current scientific knowledge. In other words, possible 1) Evaluation of viral clearance factor contaminant virus is assumed to have the natures of genus, 2) Calculation of viral clearance index morph, particle size, physical/chemical properties, etc. 3) Interpretation of results and items to be concerned at which are within the range of knowledge of existing evaluation virology, and is those assumed to exist in human and animal, VI. Statistics tissue and body fluid, which are the source of biotechnolo- 1. Statistical considerations for assessing virus assays gical/biological product, reagent, material, additives, etc. 2. Reproducibility and confidence limit of viral clearance Accordingly, viral clearance studies using a detection studies method which target those viruses have to be designed. VII. Re-evaluation of viral clearance 3.3. Unknown risk on the measures taken for ensuring VIII. Measurement for viral clearance studies viral safety 1. Measurement of virus infective titer There are known and unknown risks. 2. Testing by nucleic-acid amplification test (NAT) It is easy to determine a test method and an evaluation IX. Reporting and preservation standard on the known risk, which exists in the drug per se X. Others (pharmaceutical component) or inevitably exists due to a quality threshold, and quantification of such risk is possible. In other words, it is easy to evaluate the known risk on a different from safety of drug per se (its components), taking balance sheet in relation to the benefit, and we can say that into account any possibility of virus contamination, we have valuation even in this respect has been established to some to say that only the results accumulated can not always extent. assure viral safety of a drug used in future. Accordingly, the On the other hand, as for the unknown risk which is in- basis for securing broad sense of viral safety of JP listed evitable for ensuring viral safety, the subject of the risk can biotechnological/biological products is to pay every atten- not be defined and quantitative concept is hard to introduce, JP XVI General Information / Biotechnological/Biological Products 2169 and, therefore, taking a counter measure and evaluating its tures, such as type of nucleic acid, with/without envelope, effect are not so easy. Therefore, this is the subject to be particle size, physical/chemical properties, etc., would be challenged calling upon wisdom of the related parties among enough to simulate every sort of the virus already known, the society of drug. and will be a ``good measure for ensuring safety''. Talking about the unknown risk, there are view points The issue of ``the sort of viruses that exist in the world is that say `Ìt is risky because it is unknown.'' and ``What are unknown'' may be a future study item, but it is not an ap- the unknowns, and how do we cope with them in ensuring propriate subject for the viral clearance test. Further, even if safety?''. the subject of `ùnknown viruses, which have a particle size The view of `Ìt is risky because it is unknown.'' is already smaller than that of currently known viruses, may exists'' or nothing but a sort of evaluation result, and directly connects `ùnknown viruses, which have special physical/chemical to a final decision if it can be used as a drug. Such evalu- properties that can not be matched to any of the currently ation/decision has to be made based upon a rational, scien- known viruses, may exists'' is set up as an armchair theory, tific or social judgment. any experimental work can not be pursued under the current For example, in the case that `Ìn a manufacturing process scientific level, since such virus model is not available. Fur- of drug, virus, virus-like particle or retrovirus was detected, ther, any viral clearance test performed by using the current- but its identification could not be confirmed, and, therefore, ly available methods and technologies will be meaningless its risk can not be denied.'', the evaluation of `Ìt is risky ``for ensuring safety'', since particle size or natures of such because it is unknown.'' is scientifically rational and reason- speculated virus are unknown. Likewise, any counter meas- able. On the other hand, however, if we reach a decision of ures can not be taken on the subject of `ùnknown virus, `Ìt is risky because it is unknown.'' due to the reason that which can not be detected by currently available screening `Ìn a manufacturing process of drug, virus, virus-like parti- method, may exist'', and conducting any virus detection test cle or retrovirus was not detected, but there is a `concern' at any stage will be useless ``for ensuring safety''. that something unknown may exist.'', it can not be said that The requirement of regulations or tests excessively over such evaluation is based upon a rational, scientific or social scientific rationality will raise human, economical and tem- judgment. It goes without saying that the utmost care has to poral burden to the pharmaceutical companies, and will ad- be taken for viral safety, but the substance of `concern' has versely affect prompt, effective and economical supply of a to be at least clearly explainable. Otherwise, the `concern' drug to the medical front. As drug is a sort of social asset, may result in causing contradiction in the meaningful mis- which has to be scientifically evaluated, how to assure max- sion to utilize a social asset, drug, in medical practice. imization of its safety by means of scientifically rational ap- From scientific view point, we should not be narrow mind- proach at minimum human, economical and time resources ed by saying `ìt is risky'' because ``there is a `concern' that is important. something unknown may exists'', but challenge to clarify the It is also important to reconfirm that achievement of those subject of ``What is unknown, and how to cope with it for issues is on the premise that appropriate measures are taken ensuring safety'' using wisdom. What is important at the on the supply source of drugs. For example, in a case of `Ìn time is to define ``what is unknown'' based upon current a manufacturing process of drug, virus, virus-like particle or scientific knowledge. Only through this way, is it possible retrovirus was not detected, but there is a `concern' that for us to elaborate the measures for ensuring safety. something unknown may exist.'', appropriateness of the Once we chase up the substance of unknown risk for viral test, which resulted in the judgment that ``virus, virus-like safety without premises of ``what is unknown'', `ùnknown'' particle or retrovirus was not detected in a process of drug will be an endless question because it theoretically remains production'', should be a prerequisite premise when judged unresolved forever. If this kind of approach is taken, the by science standard ate the time. If there is any question on issue and the measure can not be scientifically connected to the premise, it is quite natural that the question of ``there is a each other, which will result in the excessive requirement of `concern' that unknown something may exist.'' will be effec- regulation and of test to be conducted. Yet, it is unlikely that tive. the measure which has no relation with science will be effec- 3.4. Applicable range tive to the subject of ``What is unknown is unknown.'' This General Information is on JP listed biological For example, ``what is unknown'' at the `èvaluation of a products, derived from living tissue or body fluid, and pro- purification process which can completely clear up every tein drugs, derived from human or animal cell line, that in virus that contaminated in a manufacturing process'' should Japan. In the case of protein drugs derived from human or be the subject of ``what sort of existing virus that contami- animal cell line, the products developed and approved before nated is unknown'', not on the subject of ``what sort of enforcement of the Notice Iyakushin No. 329 entitled ``Viral virus that exist in the world is unknown. In the former sub- safety evaluation of biotechnology products derived from ject, the premise of the study is based on all the knowledge cell lines of human or animal origin'' should have been treat- on viruses including DNA/RNA-virus, virus with/without ed under the Notice had there been one, and it is inevitable envelope, particle size, physical/chemical properties, etc. that some products approved after the Notice might not have The premise is that the virus contaminated should be within been sufficiently treated. It is expected that such biodrug will range of existing wisdom and knowledge of virus such as be sufficiently examined to meet such General Information species, type, nature, etc., even though the virus that con- before being listed in JP. On the other hand, blood prepara- taminated is unknown. Under such premise, when evaluations listed in the biological products standard and covered tion is made on a purification process to decide its capability by ``Guideline for securing safety of blood plasma protein of clearing a derived virus, which is within the range of exist- fraction preparations against virus'', are out of the scope of ing wisdom and learning, specific viral clearance studies this General Information. Further, in case of a relatively designed to combine a few model viruses with different na- lower molecular biogenous substance, such as amino acid, 2170 Biotechnological/Biological Products / General Information JP XVI saccharide and glycerin, and of gelatin, which is even classi- a raw material/substrate for drug production in old times fied as infectious or pathogenic polymer, there are cases that are Hepatitis A Virus (HAV) or Hepatitis C Virus (HCV) viral contamination can not be considered due to its contamination in blood protein fraction preparations. It is manufacturing or purification process, and that potent viral also well known that Human Immunodeficiency Virus (HIV) inactivation/removal procedure that can not be applied to infection caused by blood plasma protein fraction prepara- protein, can be used, and, therefore, it is considered reason- tions occurred in 1980s. The aim of this General Information able to omit such substances from the subject for applica- is to show concrete guidelines for comprehensive viral safety tion. However, some part of this General Information may assurance of the JP listed biotechnological/biological prod- be used as reference. Further, a comprehensive assurance ucts. The pathogenic infectious viruses, currently known to measure for viral safety is recommendable on a biotechnolo- contaminate to raw materials, etc. of drug and have to be gical/biological product not listed in JP using this document cautioned, are HIV, HAV, Hepatitis B Virus (HBV), HCV, as a reference so long as it is similar to the biotechnological/ Human T-Lymphotropic Virus (HTLV-I/II), Human Par- biological product JP. vovirus B19, Cytomegalovirus (CMV), etc. Biotechnolo- 3.5. Possible viral contamination to a JP listed biotech- gical/biological products produced from raw material/cell nological/biological product (source of virus contamination) substrate derived from tissue or body fluid of human or Promoting awareness of virus contamination to a JP listed animal origin always have a risk of contamination of patho- biotechnological/biological product (source of virus con- genic or other latent virus. Therefore, safety measures tamination) and citing countermeasure are important for should be thoroughly taken. There is also the case that a eradicating any possible virus contamination and raising material, other than the biological component such as raw probability of safety assurance. Many biotechnological/ material/substrate, causes virus contamination. Using enzy- biological products are produced from a ``substrate'' which matic or monoclonal antibody column or using albumin etc. is derived from human or animal tissue, body fluid, etc. as as a stabilizer, is the example of the case, in which caution an origin/raw material, and in purification or pharmaceuti- has to be taken on risk of virus contamination from the cal processing of such products column materials or addi- source animal or cell. Further, there is a possibility of con- tives, which are living organism origin, are occasionally tamination from environment or personnel in charge of used. Accordingly, enough safety measures should be taken production or at handling of the product. So, caution has to against diffusion of the contaminant virus. Further, as men- be taken on these respects as well. tioned in Notice Iyakushin No. 329, any protein drug de- In case of protein drugs derived from cell line of human or rived from cell lines of human or animal origin should be animal origin, there may be cases where latent or persistent carefully examined with respect to the risk of virus contami- infectious viruses (e.g., herpesvirus) or endogenous retro- nation through the cell line, the cell substrate for drug viruses exist in the cell. Further, adventitious viruses may be production, and through the manufacturing process applied introduced through the routes such as: 1) derivation of a cell thereafter. line from an infected animal; 2) use of a virus to drive a cell ``Substrate for drug production'' is defined as a starting line; 3) use of a contaminated biological reagent (e.g., material which is at a stage where it is deemed to be in a animal serum components); 4) contamination during cell position to ensure quality/safety of an active substance. The handling. In the manufacturing process of drug, an adven- ``substrate for drug production'' is sometimes tissue, body titious virus may contaminate to the final product through fluid, etc. of human or animal per se and pooled material the routes, such as 1) contamination through a reagent of liv- such as urine, and sometimes a material after some treat- ing being origin, such as serum component, which is used for ment. In many cases, it is considered rational that starting culturing, etc.; 2) use of a virus for introduction of a specific point of full-scale test, evaluation and control should be at gene expression to code an objective protein; 3) contamina- the stage of ``substrate for drug production''. The more tion through a reagent used for purification such as strict levels of test, evaluation and control achieved at the monoclonal antibody affinity column; 4) contamination stage of ``substrate for drug production'' can more ration- through an additive used for formulation production; 5) alize evaluation and control of the raw material or individual contamination at handling of cell and culture media, etc. It level of upper stream. On the contrary, strict evaluation and is reported that monitoring of cell culture parameter may be control of the raw material or individual level at an upper helpful for early detection of an adventitious viral contami- stream stage can rationalize tests, evaluation or quality con- nation. trol at the stage of ``substrate for drug production''. 3.6. Basis for ensuring viral safety The measures taken for ensuring viral safety on a Viral safety of a biotechnological/biological product pro- biotechnological/biological product currently listed in JP duced from a raw material/substrate, which derived from can be assumed from the provisions of manufacturing tissue, body fluid, cell line, etc. of human or animal origin, method, specification and test methods of each preparation. can be achieved by supplemental and appropriate adoption However, unitary principles or information with respect to of the following plural methods. the measures to be taken for ensuring viral safety, totally (1) Acquaintance of possible virus contamination (source reviewing the entire process up to the final product rationally of contamination). and comprehensively, including source/raw material/sub- (2) Careful examination of eligibility of the raw material strate, purification process, etc. have not been clarified. The and its source, i.e., human or animal, thorough analysis most important thing for ensuring viral safety is to take and screening of the sample chosen as the substrate for thorough measures to eliminate the risk of virus contamina- drug production to determine virus contamination and tion at any stage of source animal, raw material and sub- through examination of the type of virus and its nature, strate. Although not the cases of a biotechnological/biologi- if contaminated. cal product, known examples of a virus contamination from (3) Evaluation to determine hazardous properties of the vi- JP XVI General Information / Biotechnological/Biological Products 2171

rus or virus-like particle to human, if exists. ance, etc., with an aim to determine removal/inactivation (4) Choosing a product related material of living organism capability of the virus that can not be detected in a raw mate- origin (e.g., reagent, immune anti-body column, etc.) rial or contingently contaminated. which is free from infectious or pathogenic virus. As mentioned above, the role of viral clearance study is to (5) Conduct virus free test at an appropriate stage of speculate removal/inactivation capability of a process manufacturing including the final product, if necessary. through a model test, and it contributes to give scientific (6) Adoption of an effective method to remove/inactivate basis to assure that a biotechnological/biological product of the virus in the manufacturing process for viral clear- human or animal origin has reached an acceptable level in ance. Combined processes sometimes achieve higher aspect of viral safety. level of viral clearance. At a viral clearance study, it is necessary to adopt an ap- (7) Develop a deliberate viral clearance scheme. propriate approach method which is definitive and rational (8) Conduct test and evaluation to confirm removal/inacti- and can assure viral safety of a final product, taking into vation of the virus. consideration the source and the properties of the raw mate- Manufacturer is responsible for explaining rationality of rial/substrate as well as the manufacturing process. the way of approach adopted among the comprehensive 4. Raw material/substrate for drug production strategy for viral safety on each product and its manufactur- 4.1. Issues relating to animal species and its region as a ing process. At the time, the approach described in this source of raw material/substrate for drug production and General Information shall be applicable as far as possible. countermeasures to be taken thereto 3.7. Limit of virus test For manufacturing JP listed biotechnological/biological Virus test has to be conducted to define existence of virus, products, which require measures for viral safety, a raw ma- but it should be noted that virus test alone can not reach a terial/substrate derived mainly from human, bovine, swine conclusion of inexistence of virus nor sufficient to secure or equine is used, and it is obvious that such human and safety of the product. Examples of a virus not being detected animal has to be healthy nature. A wild animal should be are as follows: 1) Due to statistical reason, there is an inher- avoided, and it is recommendedtouseanimalsderivedfrom ent quantitative limit, such as detection sensitivity at lower a colony controlled by an appropriate SPF (Specific concentration depends upon the sample size. 2) Generally, Pathogen-Free) condition and bred under a well deigned every virus test has a detection limit, and any negative result hygienic control, including appropriate control for preven- of a virus test can not completely deny existence of a virus. tion of microbial contamination and contamination 3) A virus test applied is not always appropriate in terms of monitoring system. If a meat standard for food is available, specificity or sensitivity for detection of a virus which exists an animal meeting this standard has to be used. The type of in the tissue or body fluid of human or animal origin. virus to be concerned about depend on animal species, but it Virus testing method is improved as science and tech- may be possible to narrow down the virus for investigation nology progress, and it is important to apply scientifically by means of examining the hygiene control, applicability of the most advanced technology at the time of testing so that it a meat standard for food, etc. On the other hand, even with can be possible to raise the assurance level of virus detection. the animals of the same species, a different approach may be It should be noted, however, that the limit as mentioned necessary depending upon the region where the specimen for above can not always be completely overcome. Further, risk a raw material/substrate is taken. For example, in case of of virus contamination in a manufacturing process can not obtaining raw material/substrate from blood or other spe- be completely denied, and, therefore, it is necessary to cific region, it is necessary to be aware of the risk level, virus elaborate the countermeasure taken these effects into ac- multiplication risk, etc. which may specifically exists de- count. pending upon its region. Such approach may be different Reliable assurance of viral free final product can not be from those applied to body waste such as urine, milk, etc. as obtained only by negative test results on the raw material/ a source of raw material/substrate. Further, caution has to substrate for drug production or on the product in general, be taken on transmissible spongiform encephalopathy (TSE) it is also necessary to demonstrate inactivation/removal when pituitary gland, etc. is used as a raw material. This capability of the purification process. report does not include detailed explanation on TSE, but 3.8. Roles of viral clearance studies recommendations are to use raw material derived from 1) Under the premises as mentioned in the preceding clause animals originated in the countries (area) where incidence of that there is a limit of a virus test, that there is a possibility TSE has not been reported; 2) animals not infected by TSE; of existence of latent virus in a raw material/substrate for or 3) species of animal which has not been reported on TSE. drug production and that there is a risk of entry of a non- It is recommended to discuss the matters concerned with endogenous virus in a manufacturing process, one of the TSE with the regulatory authority if there is any unclear important measures for viral safety is how to remove or inac- point. tivate the virus, which exists in a raw material, etc. and can Followings are the raw material/substrate used for not be detected, or the virus, which is contingently contami- manufacturing biotechnological/biological products in nated in a manufacturing process. The purpose of viral Japan. clearance study is to experimentally evaluate the viral (1) Biological products derived from human removal/inactivation capability of a step that mounted in a Blood plasma, placenta, urine, etc. derived from human manufacturing process. So, it is necessary to conduct an ex- are used as the sources of raw material of biotechnological/ perimental scale spike test using an appropriate virus that is biological product. As for these raw materials, there are 2 selected by taking account the properties, such as particle cases: 1) Appropriateness can be confirmed by interview or size, shape, with or without envelope, type of nucleic acid by examination of the individual who supplies each raw ma- (DNA type, RNA type), heat and chemical treatment toler- 2172 Biotechnological/Biological Products / General Information JP XVI terial, and 2) Such sufficient interview or examination of the can be used as reference information, in addition to the individual can not be made due to type of raw material. In other information, such as; source of animal, health condi- case that sufficient examination of individual level is not tion, health and breeding control, conformity to the meat possible, it is necessary to perform test to deny virus con- standard for food, etc., to elaborate to which virus what tamination at an appropriate manufacturing stage, for ex- kind of test has to be performed, and for which virus it is not ample, the stage to decide it as a substrate for drug produc- always necessary to test for, etc. It is important to clarify tion. and record the basis of choosing the virus and the test con- (2) Biological products derived from animal besides human ducted thereof. Heparin, gonadotropin, etc. are manufactured from blood (3) Protein drug derived from cell line of human or animal plasma or from various organs of bovine, swine and equine. origin (3) Protein drug derived from cell line of human or animal It is important to conduct thorough investigation on latent origin endogenous and non-endogenous virus contamination in a In the case of protein drugs derived from cell line of hu- master cell bank (MCB), which is the cell substrate for drug man or animal origin, a cell line of human or animal is the production, in accordance with the Notice Iyakushin No. raw material per se, and the substrate for drug production is 329 entitled ``Viral safety evaluation of biotechnology prod- a cell bank prepared from cloned cell line (master cell bank ucts derived from cell lines of human or animal origin''. Fur- or working cell bank). Examination at cell bank level is con- ther, it is necessary to conduct an appropriate adventitious sidered enough for viral safety qualification, but it goes virus test (e.g., in vitro and in vivo test) and a latent without saying that the more appropriate and rational endogenous virus test on the cell at the limit of in vitro cell qualification evaluation test of cell bank can be realized age (CAL) for drug production. Each WCB as a starting cell when more information is available on the virus of the substrate for drug production should be tested for adventiti- source animal or on prehistory of driving the cell line, the ous virus either by direct testing or by analysis of cells at the base of cell bank. CAL, initiated from the WCB. When appropriate non-en- 4.2. Qualification evaluation test on human or animal as a dogenous virus tests have been performed on the MCB and source of raw material/substrate for drug production cells cultured up to or beyond the CAL have been derived (1) Biological products derived from human from the WCB and used for testing for the presence of ad- Body fluid etc. obtained from healthy human must be ventitious viruses, similar tests need not be performed on the used for biological products production. Further, in case initial WCB. that interview or examination of the individual, who supplies 5. Points of concern with respect to manufacturing and the raw material, can be possible and is necessary, interview virus testing under an appropriate protocol and a serologic test well eval- To ensure viral safety of a biological product derived from uated in aspects of specificity, sensitivity and accuracy have tissue, body fluid etc. of human or animal origin, it is neces- to be performed, so that only the raw material, which is de- sary to exclude any possibility of virus contamination from a nied latent HBV, HCV and HIV, will be used. In addition to raw material, such as tissue and body fluid, or a substrate, the above, it is necessary to test for gene of HBV, HCV and paying attention to the source of virus contamination as HIV by a nucleic amplification test (NAT) well evaluated in mentioned in above 3.5, and to adopt appropriate manufac- aspects of specificity, sensitivity and accuracy. turing conditions and technologies in addition to enhance- In case of the raw material (e.g., urine), which can not be ment of manufacturing environment, so that virus contami- tested over the general medical examination of the individual nation in the course of process and handling and from oper- who supplies the material, or of the raw material which is ators, facilities and environment can be minimized. irrational to conduct individual test, the pooled raw mate- In addition to the above, effective virus test and viral inac- rial, as the substrate for drug production, has to be conduct- tivation/removal technology, which are reflected by rapid ed at least to deny existence of HBV, HCV and HIV, using a progress of science, have to be introduced. Adoption of two method well evaluated in aspects of specificity, sensitivity or more steps with different principles is recommended for and accuracy, such as the antigen test or NAT. virus inactivation/removal process. Further, it is important (2) Biological products derived from animal besides human to minimize any possible virus derivation by using a reagent, The animal used for manufacturing biological product has which quality is equivalent to that of a drug. Examples of to be under appropriate health control, and has to be con- virus inactivation/removal processes are  heating (It is firmed of its health by various tests. Further, it is necessary reported that almost viruses are inactivated by heating at that the population, to which the animal belongs, has been 55–609C for 30 minutes with exceptions of hepatitis virus, under an appropriate breeding condition, and that no abnor- etc. and that dry heating at 609C for 10 – 24 hours is effec- mal individual has been observed in the population. Further, tive in case of the products of blood or urine origin.),  it is necessary to demonstrate information or scientific basis treatment with organic solvent/surfactant (S/D treatment), which can deny known causes infection or disease to human,  membrane filtration (15 – 50 nm),  acid treatment,  or to deny such animal inherent latent virus by serologic test irradiation (g-irradiation, etc.),  treatment with column or by nucleic amplification test (NAT). The infectious virus chromatograph (e.g. affinity chromatography, ion-exchange that is known to be common between human and animal, chromatography),  fractionation (e.g. organic solvent or and known to cause infection in each animal are tentatively ammonium sulfate fractionation),  extraction. listed in Table 2. It is necessary that the table is completed 5.1. Virus test conducted in advance of purification under careful examination, and denial of all of them, by process means of tests on individual animal, tissue, body fluid, etc. (1) Biological products derived from human as a raw material, or on pooled raw material (as a direct sub- In many cases, samples for virus test before purification strate for drug production), is not always necessary. Table 2 JP XVI General Information / Biotechnological/Biological Products 2173

Table 2 Infectious viruses known to be common Parainfluenza virus Type 3 w between human and animal and known to cause infection to each animal Talfan/Teschen disease virus w bovine swine sheep goat equine Reovirus w Cowpox virus w Endogenous retrovirus w Paravaccinia virus wwww Porcine adenovirus w Murray valley encephalitis ww virus Porcine circovirus w

Louping ill virus wwww Porcine parvovirus w

Wesselsbron virus w Porcine poxvirus w

Foot-and-mouth disease Porcine cytomegalovirus w virus ww Pseudorabies virus w Japanese encephalitis w virus Russian spring-summer encephalitis virus ww Vesicular stomatitis virus w Rift Valley fever virus ww Bovine papular stomatitis w virus Crimean-Congo hemor- rhagic fever virus www Orf virus w (Nairovirus)

Borna disease virus wwTorovirus w Rabies virus wwwww

Influenza virus w process are body fluid or tissue of individual collected as a Hepatitis E virus w raw material, or its pooled material or extraction as a substrate. As mentioned in 4.2 (1), it is necessary to deny latent Encephalomyocarditis virus ww HBV, HCV and HIV by the test evaluated enough in aspects of specificity, sensitivity and accuracy. Even in a case that a Rotavirus w non-purified bulk before purification process is produced from a substrate, it is not always necessary to conduct virus Eastern equine encephalitis virus w test again at the stage before purification, so long as the presence of any latent virus can be denied at the stage of sub- Western equine strate by an appropriate virus test, with cases where the non- w encephalitis virus purified bulk is made from the substrate by adding any reagent etc. of living organisms origin are an exception. Venezuelan equine encephalitis virus w (2) Biological products derived from animal besides human Similar to (1) above, samples for virus test before purifica- Morbillivirus w tion process are, in many cases, body fluid or tissue of individual collected as a raw material, or its pooled material or Hendra virus w extraction as a substrate. In these cases, it is necessary to Nipah virus w have a data, which can deny latent virus of probable cause of human infection or disease as mentioned in the above 4.2 Transmissible gastroente- w (2), or to have a result of serologic test or nucleic amplifica- ritis virus tion test (NAT) evaluated enough in aspects of specificity, Porcine respiratory sensitivity and accuracy. The concept, which is applied to a coronavirus w case that non-purified bulk before purification process is produced from substrate, is the same as those provided in Porcine epidemic the above 4.2 (1). diarrhea virus w (3) Protein drug derived from cell line of human or animal Hemagglutinating origin encephalomyelitis virus w Generally, substrate in this case is cell bank, and the sample for testing before purification process is a harvested cell Porcine respiratory and after cell culturing or unprocessed bulk which consists of reproductive syndrome virus w single or pooled complex culture broth. The unprocessed Hog cholera virus w bulk may be sometimes culture broth without cell. Denial of latent virus, which is determined by virus test at a MCB or WCB level, does not always deny latent virus in unprocessed 2174 Biotechnological/Biological Products / General Information JP XVI bulk after culturing. Further, it is noted that the viral test at product to reach the final product) has to be defined under the CAL is meaningful as a validation but can not guarantee comprehensive consideration of the type of raw material or definite assurance of latent virus denial, since the test is gen- substrate, the result of virus test conducted on raw material/ erally performed only once. In case of using a serum or a substrate, the result of evaluation on viral removal/inactiva- component of blood origin in a culture medium, definite tion process, any possibility of virus contamination in the denial of latent virus at the level of unprocessed bulk can not manufacturing process, etc. Comprehensive viral safety as- be assured so long as the viral test has not been conducted on surance can only be achieved by appropriate selection of the each lot at the CAL, since lot renewal can be a variable fac- raw material/substrate, an appropriate virus test conducted tor on viral contamination. on the raw material/substrate/intermediate material, the vi- A representative sample of the unprocessed bulk, removed rus test conducted at an appropriate stage of manufacturing, from the production reactor prior to further processing, an appropriate viral clearance test, etc. However, there are represents one of the most suitable levels at which the pos- cases of having specific backgrounds, such as 1) use of the sibility of adventitious virus contamination can be deter- raw material derived from unspecified individual human, 2) mined with a high probability of detection. Appropriate test- possible existence of virus at window period, 3) specific de- ing for viruses should be performed at the unprocessed bulk tection limit of virus test, etc. and in these cases, virus con- level unless virus testing is made more sensitive by initial par- tamination to the final product may occur if there is any tial processing (e.g., unprocessed bulk may be toxic in test deficiency on the manufacturing process (e.g., damage of cell cultures, whereas partially processed bulk may not be membrane filter) or any mix-up of the raw materials, etc. To toxic). In certain instances it may be more appropriate to test avoid such accidental virus contamination, it may be recom- a mixture consisting of both intact and disrupted cells and mended to conduct nucleic amplification test (NAT) on the their cell culture supernatants removed from the production final product focusing on the most risky virus among those reactor prior to further processing. that may possibly to exist in the raw material. In case of unprocessed bulk, it is required to conduct virus 6. Process evaluation on viral clearance test on at least 3 lots obtained from pilot scale or commercial 6.1. Rationale, objective and general items to be concerned scale production. It is recommended that manufacturers de- with respect to viral clearance process evaluation velop programs for the ongoing assessment of adventitious Evaluation of a viral inactivation/removal process is im- viruses in production batches. The scope, extent and fre- portant for ensuring safety of a biological product derived quency of virus testing on the unprocessed bulk should be from tissue or body fluid of human or animal origin. Con- determined by taking several points into consideration in- ducting evaluation on viral clearance is to assure, even to cluding the nature of the cell lines used to produce the some extent, elimination of the virus, which may exist in a desired products, the results and extent of virus tests per- raw material, etc. or may be derived to the process due to formed during the qualification of the cell lines, the cultiva- unexpected situation. Viral clearance studies should be made tion method, raw material sources and results of viral clear- by a carefully designed appropriate method, and has to be ance studies. Screening in vitro tests, using one or several cell rationally evaluated. lines, are generally employed to test unprocessed bulk. If ap- The objective of viral clearance studies is to assess process propriate, a NAT test or other suitable methods may be step(s) that can be considered to be effective in inactivating/ used. removing viruses and to estimate quantitatively the overall Generally, harvest material in which adventitious virus has level of virus reduction obtained by the process. This should been detected should not be used to manufacture the be achieved by the deliberate addition (``spiking'') of sig- product. If any adventitious viruses are detected at this level, nificant amounts of a virus at different manufacturing/ the process should be carefully checked to determine the purification steps and demonstrating its removal or inactiva- cause of the contamination, and appropriate actions taken. tion during the subsequent steps. It is not necessary to evalu- 5.2. Virus test as an acceptance test of an intermediate ma- ate or characterize every step of a manufacturing process if terial, etc. adequate clearance is demonstrated by the use of fewer When a biological product is manufactured from tissue, steps. It should be borne in mind that other steps in the body fluid etc. of human or animal origin, there are cases process may have an indirect effect on the viral inactivation/ that an intermediate material, partially processed as a raw removal achieved. Manufacturers should explain and justify material or substrate by outside manufacturer, is purchased the approach used in studies for evaluating viral clearance. andusedformanufacturing.Insuchcase,ifanytesttomeet The reduction of virus infectivity may be achieved by this General Information has been conducted by such out- removal of virus particles or by inactivation of viral infectiv- side manufacturer, it is necessary for the manufacturer of ity. For each production step assessed, the possible mechan- the biological product, who purchased the intermediate ma- ism of loss of viral infectivity should be described with terial, to examine what sort of virus test has to be conducted regard to whether it is due to inactivation or removal. For in- as acceptance tests, and to keep record on the basis of activation steps, the study should be planned in such a way rationality including the details of the test conducted. that samples are taken at different times and an inactivation On the other hand, if no test to meet this General Infor- curve constructed. mation has been conducted by such outside manufacturer of 6.2. Selection of virus the raw material, all necessary virus free test has to be con- To obtain broad range of information of viral inactiva- ducted to meet this General Information on the intermediate tion/removal, it is desirable that a model virus used for viral material regarding it as the direct substrate for drug produc- clearance studies should be chosen from the viruses with tion. broad range of characteristics in aspects of DNA/RNA, with 5.3. Virus test on a final product or without envelope, particle size, significant resistance to Virus tests to be conducted on a final product (or on a JP XVI General Information / Biotechnological/Biological Products 2175

Table 3 Example of viruses which have been used for viral clearance studies

Virus Family Genus Natural host Genome Env Size (nm) Shape Resistance

Vesicular Stomatitis Virus Rhabdo Vesiculovirus Equine RNA yes 70×150 Bullet Low Bovine Parainfluenza Virus Paramyxo Type 1,3 Various RNA yes 100 – 200＋ Pleo-Spher Low Respirovirus Type 2,4 Rubulavirus MuLV Retro Type C Mouse RNA yes 80 – 110 Spherical Low oncovirus Sindbis Virus Toga Alphavirus Human RNA yes 60 – 70 Spherical Low BVDV Flavi Pestivirus Bovine RNA yes 50 – 70 Pleo-Spher Low Pseudorabies Virus Herpes Varicellovirus Swine DNA yes 120 – 200 Spherical Med Poliovirus Sabin Type 1 Picorna Enterovirus Human RNA no 25 – 30 Icosahedral Med Encephalomyocardititis Picorna Cardiovirus Mouse RNA no 25 – 30 Icosahedral Med Virus Reovirus Type 3 Reo Orthoreovirus Various kind RNA no 60 – 80 Spherical Med SV 40 Papova Polyomavirus Monkey DNA no 40 – 50 Icosahedral Very high Parvovirus: canine, por- Parvo Parvovirus Canine DNA no 18 – 24 Icosahedral Very high cine Porcine

physical/chemical treatment, etc. and it is necessary to com- actual production. bine about 3 model viruses to cover these characteristics. (2) Virus detection method gives great influence to viral At choice of a model virus, there are also the ways to clearance factor. Accordingly, it is advisable to gain detec- choose a virus closely related to or having the same charac- tion sensitivity of the methods available in advance, and use teristics of the virus known to exist in the raw material. In a method with a detection sensitivity as high as possible. such case, it is in principle recommendable to choose a virus Quantitative infectivity assays should have adequate sensi- which demonstrates a higher resistance to inactivation/ tivity and reproducibility in each manufacturing process, removal treatment if two or more candidate viruses are avail- and should be performed with sufficient replicates to ensure able for choice. Further, a virus which can grow at a high adequate statistical validity of the result. Quantitative assays titer is desirable for choice, although this may not always be not associated with infectivity may be used if justified. Ap- possible. In addition to the above, choosing a virus, which propriate virus controls should be included in all infectivity will provide effective and reliable assay result at each step, is assays to ensure the sensitivity of the method. Also, the necessary, since sample condition to be tested at each step of statistics of sampling virus when at low concentrations (for a production process may influence the detection sensitivity. example, number of virus is 1-1000/L) should be considered. Consideration should also be given to health hazard which (3) Viral clearance studies are performed in a miniature may pose to the personnel performing the clearance studies. size system that simulates the actual production process of For the other items taken for consideration at choice of the biotechnological/biological product used by the manu- virus, the Notice, Iyakushin No. 329 can be used as a refer- facturer. It is inappropriate to introduce any virus into a ence. Examples of the virus which have been used for viral production facility because of GMP constraints. Therefore, clearance studies are shown in Table 3 which was derived viral clearance studies should be conducted in a separate from Iyakushin No. 329. However, the Notice, Iyakushin laboratory equipped for virological work and performed by No. 329, is on viral safety of a product derived cell line of staff with virological expertise in conjunction with produc- human or animal origin, and a more appropriate model virus tion personnel involved in designing and preparing a scaled- has to be chosen taking into account the origin/raw material down version of the purification process. The viral clearance of biological products. studies should be performed under the basic concept of 6.3. Design of viral clearance studies GLP. The purpose of viral clearance studies is to quantitatively (4) Each factor on a viral clearance study of a process, evaluate removal or inactivation capability of a process, in which is performed in miniature size, should reflect that of which a virus is intentionally spiked to a specific step of a actual manufacturing as far as possible, and its rationality manufacturing process. should be clarified. In case of chromatograph process, Following are the precautions to be taken at planning viral length of column bed, linear velocity, ratio of bed volume clearance studies. per velocity (in other words, contact time), buffer, type of (1) Care should be taken in preparing the high-titer virus column packing, pH, temperature, protein concentration, to avoid aggregation which may enhance physical removal salt concentration and concentration of the objective and decrease inactivation thus distorting the correlation with product are all correspondent to those of the actual produc- 2176 Biotechnological/Biological Products / General Information JP XVI tion. Further, similarity of elution profile should be passes through a similar manipulation, should be conducted achieved. For the other process, similar concept should be to assess infectivity variance at the manipulation. applied. If there is any factor which can not reflect the actual (11) Buffers and product (desired protein or other com- production, its effect to the result should be examined. ponent contained therein) should be evaluated independently (5) It is desirable that two or more inactivation/removal for toxicity or interference in assays used to determine the processes of different principles are selected and examined. virus titer, as these components may adversely affect the (6) As for the process which is expected to inactivate/ indicator cells. If the solutions are toxic to the indicator remove virus, each step should be evaluated in aspect of cells, dilution, adjustment of the pH, or dialysis of the clearance capability, and carefully determined if it is the buffer containing spiked virus might be necessary. If the stage of inactivation, removal or their combination for product itself has anti-viral activity, the clearance study may designing the test. Generally, in viral clearance test, a virus is need to be performed without the product in a ``mock'' run, spiked in each step which is the object of the test, and after although omitting the product or substituting a similar pro- passing through the process in question, the reduction level tein that does not have anti-viral activity could affect the be- of infectivity is evaluated. But, in some case, it is accepted haviour of the virus in some production steps. that a high potential virus is spiked at a step of the process, (12) Many purification schemes use the same or similar and virus concentration of each succeeding step is carefully buffers or columns, repetitively. The effects of this approach monitored. When removal of virus is made by separation or should be taken into account when analyzing the data. The fractionation, it is desirable to investigate how the virus is effectiveness of virus elimination by a particular process may separated or fractionated (mass balance). vary with the stage in manufacture at which it is used. (7) For assessment of viral inactivation, unprocessed (13) Overall reduction factors may be underestimated crude material or intermediate material should be spiked where production conditions or buffers are too cytotoxic or with infectious virus and the reduction factor calculated. It virucidal and should be discussed on a case-by-case basis. should be recognized that virus inactivation is not a simple, Overall reduction factors may also be overestimated due to first order reaction and is usually more complex, with a fast inherent limitations or inadequate design of viral clearance ``phase 1'' and a slow ``phase 2''. The study should, there- studies. fore, be planned in such a way that samples are taken at (14) It has to be noted that clearance capability of viral different times and an inactivation curve constructed. It is removal/inactivation process may vary depending upon the recommended that studies for inactivation include at least type of virus. The viral removal/inactivation process, which one time point less than the minimum exposure time and displays viral clearance by specific principle or mechanism, greater than zero, in addition to the minimum exposure may be quite effective to the virus, which meets such time. The reproducible clearance should be demonstrated in mechanism of action, but not effective to the other type of at least two independent studies. When there is a possibility viruses. For example, S/D treatment is generally effective to that the virus is a human pathogen, it is very important that the virus with lipid membrane, but not effective to the non- the effective inactivation process is designed and additional enveloped virus. Further, some virus is resistant to the gen- data are obtained. The initial virus load should be deter- eral heating process (55 – 609C, 30 minutes). When clearance mined from the virus which can be detected in the spiked is expected for such virus, introduction of a further severe starting material. If this is not possible, the initial virus load condition or process, which has different principle or may be calculated from the titer of the spiking virus prepara- mechanism, is necessary. Virus removal by membrane filtration. Where inactivation is too rapid to plot an inactivation tion, which is different from S/D or heat treatment in aspect curve using process conditions, appropriate controls should of principle, is effective to a broad range of virus that can be performed to demonstrate that infectivity is indeed lost by not pass through the membrane. Affinity chromatography inactivation. process, which specifically absorbs the objective protein, can (8) If antibody against virus exists in an unprocessed ma- thoroughly wash out the materials other than the objective terial, caution should be taken at clearance studies, since it protein including virus etc. and is generally effective for viral may affect the behavior of virus at viral removal or inactiva- removal. Separation/fractionation of a virus from an objec- tion process. tive protein is sometimes very difficult, but there are not so (9) Virus spiked in unprocessed material should be rare that ion exchange chromatography, ethanol fractiona- sufficient enough to evaluate viral removal or inactivation tion, etc. is effective for clearance of a virus which can not capability of the process. However, the virus ``spike'' to be be sufficiently inactivated or removed by the other process. added to the unprocessed material should be as small as (15) Effective clearance may be achieved by any of the possible in comparison with the sample volume of the un- following: multiple inactivation steps, multiple complemen- processed material so as not to cause characteristic change of tary separation steps, or combinations of inactivation and the material by addition of the virus nor to cause behavioral separation steps. Separation methods may be dependent on change of the protein in the material. the extremely specific physico-chemical properties of a virus (10) It is desirable that the virus in the sample is subject which influence its interaction with gel matrices and precipi- for quantitative determination without applying ultracen- tation properties. However, despite these potential variables, trifuge, dialysis, storage, etc. as far as possible. However, effective removal can be obtained by a combination of com- there may be a case that any handling before quantitative plementary separation steps or combinations of inactivation test, such as remove procedure of inhibitor or toxic sub- and separation steps. Well designed separation steps, such as stance, storage for a period to realize test at a time, etc., is chromatographic procedures, filtration steps and extrac- inevitable. If any manipulation, such as dilution, concentrations, can be also effective virus removal steps provided that tion, filtration, dialysis, storage, etc., is applied for prepara- they are performed under appropriately controlled condition of the sample for testing, a parallel control test, which tions. JP XVI General Information / Biotechnological/Biological Products 2177

(16) An effective virus removal step should give ration of the unprocessed material after addition of virus, reproducible reduction of virus load shown by at least two not the viral titer added to the sample preparation wherever independent studies. possible. If this is not possible, loaded virus amount is calcu- (17) Over time and after repeated use, the ability of lated from virus titer of the solution used for spike. chromatography columns and other devices used in the 6.4.3. Interpretation of results and items to be concerned purification scheme to clear virus may vary. Some estimate at evaluation of the stability of the viral clearance after several uses may At the interpretation and the evaluation of the data on ef- provide support for repeated use of such columns. fectiveness of viral inactivation/removal process, there are (18) The Notice, Iyakushin No. 329, would be used as a various factors to be comprehensively taken into account, reference when viral clearance studies on biological products such as  appropriateness of the virus used for the test,  are designed. design of the viral clearance studies,  virus reduction ratio 6.4. Interpretation of viral clearance studies shown in logarithm,  time dependence of inactivation,  6.4.1. Evaluation on viral clearance factor factors/items which give influence to the inactivation/ Viral clearance factor is a logarithm of reduction ratio of removal process,  sensitivity limit of virus assay method, viral amount (infectious titer) between each step applied for  possible effect of the inactivation/removal process which viral clearance of a manufacturing process. Total viral clear- is specific to certain class of viruses. ance factor throughout the process is sum of the viral clear- Additional items to be concerned at appropriate interpre- ance factor of each step appropriately evaluated. tation and evaluation of the viral clearance data are as Whether each and total viral clearance factor obtained are follows: acceptable or should not be evaluated in aspects of every vi- (1) Behavior of virus used to the test rus that can be realistically anticipated to derive into the raw At interpretation of the vial clearance results, it is neces- material or the manufacturing process, and its rationality sary to recognize that clearance mechanism may differ de- should be recorded. pending upon the virus used for the test. Virus used for a test In case that existence of any viral particle is recognized in is generally produced in tissue culture, but behavior of the a substrate for drug production, e.g., a substrate of rodent virus prepared in the tissue culture may be different from origin for biodrug production, it is important not only to that of the native virus. Examples are possible differences of demonstrate removal or inactivation of such virus, but also purity and degree of aggregation between the native and the to demonstrate that the purification process has enough cultured viruses. Further, change of surface properties of a capability over the required level to assure safety of the final virus, e.g., addition of a sucrose chain which is ascribed to product at an appropriate level. The virus amount removed specific nature of a separation process, may give effect to the or inactivated in a manufacturing process should be com- separation. These matters should be also considered at inter- pared with the virus amount assumed to exist in the substrate pretation of the results. etc. used for manufacturing drug, and for this purpose, it is (2) Design of test necessary to obtain the virus amount in the raw materials/ Viral clearance test should have been designed taking into substrate, etc. Such figure can be obtained by measuring account variation factors of the manufacturing process and infectious titer or by the other method such as transmission scaling down, but there still remain some variance from ac- electron microscope (TEM). For evaluation of overall tual production scale. It is necessary to consider such vari- process, a virus amount, far larger than that assumed to exist ance at the interpretation of the data and limitation of the in the amount of the raw materials/substrate which is test. equivalent to single administration of the final product, has (3) Acceptability of viral reduction data to be removed. It is quite rare that existence of virus can be Total viral clearance factor is expressed as a sum of assumed in a substrate for drug production, with the excep- logarithm of reduction ratio obtained at each step. The sum- tion of the substrate of rodent origin, and such suspicious mation of the reduction factor of multiple steps, particularly raw material/substrate should not be used for manufactur- of steps with little reduction (e.g., below 1 log10), may over- ing drug with a special exceptional case that the drug in ques- estimate viral removal/inactivation capability of the overall tion is not available from the other process and is clinically process. Therefore, virus titer of the order of 1 log10 or less indispensable, and that the information including infectious has to be ignored unless justified. Further, viral clearance properties of the virus particle assumed to exist has been factor achieved by repeated use of the same or similar clarified. method should be ignored for calculation unless justified. 6.4.2. Calculation of viral clearance index (4) Time dependence of inactivation Viral clearance factor, ``R'', for viral removal/inactiva- Inactivation of virus infectivity frequently shows biphasic tion process can be calculated by the following formula. curve, which consists of a rapid initial phase and subsequent slow phase. It is possible that a virus not inactivated in a step R ＝ log[(V × T )/(V × T )] 1 1 2 2 may be more resistant to the subsequent step. For example, In which if an inactivated virus forms coagulation, it may be resistant R: Logarithm of reduction ratio to any chemical treatment and heating.

V1: Sample volume of the unprocessed material (5) Evaluation of viral reduction ratio shown logarithm T1: Virus amount (titer) of the unprocessed material Viral clearance factor shown in logarithm of reduction V2: Sample volume of the processed material ratio of virus titer can demonstrate drastic reduction of T2: Virus amount (titer) of the processed material residual infectious virus, but there is a limit that infectious titer can never be reduced to zero. For example, reduction in At the calculation of viral clearance factor, it is recom- infectivity of a preparation containing 8 log infectious unit mendable to use the virus titer detected in the sample prepa- 10 permLbyafactorof8log10 leaves zero log10 per mL or one 2178 Biotechnological/Biological Products / General Information JP XVI infectious unit per mL, taking into account the detection mate of whose potency should be within approximately 0.5 limit of assay. log10 of the mean estimate established in the laboratory for (6) Variable factor of manufacturing process the assay to be acceptable. Assays with lower precision may Minor variance of a variation factor of a manufacturing be acceptable with appropriate justification. process, e.g., contact time of a spiked sample to a buffer or 7.2. Reproducibility and confidence limit of viral clearance a column, will sometimes give influence to viral removal or studies inactivation effect. In such case, it may be necessary to in- An effective virus inactivation/removal step should give vestigate to what extent such variance of the factor has given reproducible reduction of virus load shown by at least two influence to the process concerned in aspect of viral inactiva- independent studies. The 95z confidence limits for the tion. reduction factor observed should be calculated wherever (7) Existence of anti-viral antiserum possible in studies of viral clearance. If the 95z confidence Anti-viral antiserum that exists in the sample preparation limits for the viral assays of the starting material are ±s, and used for a test may affect sensitivity of distribution or inacti- for the viral assays of the material after the step are ±a, the vation of a virus, which may result in not only defusing the 95z confidence limits for the reduction factor are virus titer but complicating interpretation of the test result. ± s2 ＋ a2. So, existence of anti-viral antiserum is one of the important 8. Re-evaluation of viral clearance variable factors. Whenever significant changes in the production or purifi- (8) Introduction of a new process for removal/inactivation cation process are made, the effect of that change, both Viral clearance is an important factor for securing safety direct and indirect, on viral clearance should be re-evaluated of drug. In case that an achievement level of infective clear- as needed. Changes in process steps may also change the ance of a process is considered insufficient, a process which extent of viral clearance. is characterized by inactivation/removal mechanism to meet the purpose or an inactivation/removal process which can 9. Measurement for viral clearance studies mutually complement to the existence process has to be in- 9.1. Measurement of virus infective titer troduced. Assay methods may be either semiquantitative or quan- (9) Limit of viral clearance studies titative. Semiquantitative methods include infectivity assays Viral clearance studies are useful for contributing to the in animals or in cultured cell infections dose (CCID) assays, assurance that an acceptable level of safety in the final in which the animal or cell culture is scored as either infected product is achieved but do not by themselves establish safe- or not. Infectivity titers are then measured by the proportion ty. However, a number of factors in the design and execu- of animals or culture infected. In quantitative methods, the tion of viral clearance studies may lead to an incorrect esti- infectivity measured varies continuously with the virus input. mate of the ability of the process to remove virus infectivity, Quantitative methods include plaque assays where each as described above. plaque counted corresponds to a single infectious unit. Both quantal and quantitative assays are amenable to statistical 7. Statistics evaluation. The viral clearance studies should include the use of 9.2. Testing by nucleic-acid-amplification test (NAT) statistical analysis of the data to evaluate the results. The NAT can detect individual or pooled raw material/cell study results should be statistically valid to support the con- substrate or virus genome at a high sensitivity even in a stage clusions reached. thatserumtestoneachvirusisnegative.Further,itcande- 7.1. Statistical considerations for assessing virus assays tect HBV or HCV gene, which can not be measured in cul- Virus titrations suffer the problems of variation common ture system. Window period can be drastically shortened at to all biological assay systems. Assessment of the accuracy the test on HBV, HCV and HIV, and the method is expected of the virus titrations and reduction factors derived from to contribute as an effective measure for ensuring viral safe- them and the validity of the assays should be performed to ty. However, depending upon a choice of primer, there may define the reliability of a study. The objective of statistical be a case that not all the subtype of objective virus can be de- evaluation is to establish that the study has been carried out tected by this method, and, therefore, it is recommendable to an acceptable level of virological competence. to evaluate, in advance, if subtype of a broad range can be Assay detected. 1. Assay methods may be either semiquantitative or NAT will be an effective evaluation method for virus quantitative. Both semiquantitative and quantitative assays removal capability for viral clearance. However, in case of are amenable to statistical evaluation. viral inactivation process, viral inactivation obtained by this 2. Variation can arise within an assay as a result of dilu- method may be underrated, since there is a case that inacti- tion errors, statistical effects and differences within the assay vated virus still shows positive on nucleic acid. Further, at system which are either unknown or difficult to control. introduction of NAT, cautions should be taken on These effects are likely to be greater when different assay rationality of detection sensitivity, choice of a standard runs are compared (between-assay variation) than when which is used as run-control, quality assurance and main- results within a single assay run are compared (within-assay tenance of a reagent used for primer, interpretation of posi- variation). tive and negative results, etc. 3. The 95z confidence limits for results of within-assay variation normally should be on the order of ±0.5 log10 of 10. Reporting and preservation the mean. Within-assay variation can be assessed by stand- All the items relating to virus test and viral clearance stu- ard textbook methods. Between-assay variation can be moni- dies should be reported and preserved. tored by the inclusion of a reference preparation, the esti- JP XVI General Information / Biotechnological/Biological Products 2179

11. Others (meo) which in turn depends on the charge density on the The Notice, Iyakushin No. 329, should be used as a refer- capillary internal wall and the buffer characteristics. The ence at virus test and viral clearance studies. electroosmotic velocity (neo) is given by the equation: Conclusion ez V n ＝ m E ＝ As mentioned at the Introduction, assurance of quality/ eo eo Ø h »Ø L » safety etc. of JP listed drugs should be achieved by state-of- e: dielectric constant of the buffer, the-art methods and concepts reflecting the progress of z: zeta potential of the capillary surface. science and accumulation of experiences. The basis for ensuring viral safety of JP listed biotechno- The velocity of the solute (n)isgivenby: logical/biological products is detailed in this General Infor- n ＝ n ＋ n mation. What is discussed here is that an almost equal level ep eo of measures are required for both development of new drugs The electrophoretic mobility of the analyte and the elec- and for existing products as well, which means that similar troosmotic mobility may act in the same direction or in op- level of concerns should be paid on both existing and new posite directions, depending on the charge of the solute. In products in aspect of viral safety. This document is intended normal capillary electrophoresis, anions will migrate in the to introduce a basic concept that quality and safety assur- opposite direction to the electroosmotic flow and their veloc- ance of JP listed product should be based upon the most ad- ities will be smaller than the electroosmotic velocity. Cations vanced methods and concepts. This document has been writ- will migrate in the same direction as the electroosmotic flow ten to cover all conceivable cases, which can be applied to all and their velocities will be greater than the electroosmotic biotechnological/biological products. Therefore, there may velocity. Under conditions in which there is a fast electroos- be cases that it is not so rational to pursue virus tests and motic velocity with respect to the electrophoretic velocity of viral clearance studies in accordance with this document on the solutes, both cations and anions can be separated in the each product, which has been used for a long time without same run. any safety issue. So, it will be necessary to elaborate the Thetime(t) taken by the solute to migrate the distance (l) most rational ways under a case-by-case principle taking into from the injection end of the capillary to the detection point due consideration source, origin, type, manufacturing (capillary effective length) is given by the expression: process, characteristics, usages at clinical stage, accumula- l l × L tion of the past usage record, etc. relating to such biotechno- t ＝＝ n ＋ n (m ＋ m )V logical/biological products. ep eo ep eo In general, uncoated fused-silica capillaries above pH 3 have negative charge due to ionized silanol groups in the inner wall. Consequently, the electroosmotic flow is from Capillary Electrophoresis anode to cathode. The electroosmotic flow must remain constant from run to run if good reproducibility is to be ob- This test is harmonized with the European Pharmacopoeia tained in the migration velocity of the solutes. For some ap- and the U.S. Pharmacopeia. plications, it may be necessary to reduce or suppress the elec- Capillary electrophoresis is a physical method of analysis troosmotic flow by modifying the inner wall of the capillary based on the migration, inside a capillary, of charged ana- or by changing the concentration, composition and/or pH of lytes dissolved in an electrolyte solution, under the influence the buffer solution. of a direct-current electric field. After the introduction of the sample into the capillary, The migration velocity of an analyte under an electric field each analyte ion of the sample migrates within the back- of intensity E, is determined by the electrophoretic mobility ground electrolyte as an independent zone, according to its of the analyte and the electroosmotic mobility of the buffer electrophoretic mobility. Zone dispersion, that is the spread- inside the capillary. The electrophoretic mobility of a solute ing of each solute band, results from different phenomena. (mep) depends on the characteristics of the solute (electric Under ideal conditions the sole contribution to the solute- charge, molecular size and shape) and those of the buffer in zone broadening is molecular diffusion of the solute along which the migration takes place (type and ionic strength of the capillary (longitudinal diffusion). In this ideal case the the electrolyte, pH, viscosity and additives). The elec- efficiency of the zone, expressed as the number of theoretical trophoretic velocity (nep) of a solute, assuming a spherical plates (N), is given by: shape, is given by the equation: (mep ＋ meo) × V × l q V N ＝ n ＝ m E ＝ 2 × D × L ep ep Ø6phr»Ø L » D: molecular diffusion coefficient of the solute in the q: effective charge of the solute, buffer. h: viscosity of the electrolyte solution, In practice, other phenomena such as heat dissipation, r: Stoke's radius of the solute, sample adsorption onto the capillary wall, mismatched con- V: applied voltage, ductivity between sample and buffer, length of the injection L: total length of the capillary. plug, detector cell size and unlevelled buffer reservoirs can When an electric field is applied through the capillary also significantly contribute to band dispersion. filled with buffer, a flow of solvent is generated inside the Separation between two bands (expressed as the resolu- capillary, called electroosmotic flow. The velocity of the tion, RS) can be obtained by modifying the electrophoretic electroosmotic flow depends on the electroosmotic mobility mobility of the analytes, the electroosmotic mobility induced 2180 Biotechnological/Biological Products / General Information JP XVI in the capillary and by increasing the efficiency for the band electroosmotic flow in the capillary (see General Principles). of each analyte, according to the equation: Coated capillaries can be used to increase the separation capacity of those substances adsorbing on fused-silica sur- N(m － m ) R ＝ epb epa faces. S 4(mš ＋ m ) ep eo Using this mode of capillary electrophoresis, the analysis

mepa and mepb: electrophoretic mobilities of the two analytes of both small (Mr ＜ 2000) and large molecules (2000 ＜ Mr separated, ＜ 100,000) can be accomplished. Due to the high efficiency

mš ep: mean electrophoretic mobility of the two analytes achieved in capillary zone electrophoresis, separation of 1 molecules having only minute differences in their charge-to- mš ＝ (m ＋ m ). ep 2 epb epa mass ratio can be effected. This separation mode also allows Apparatus the separation of chiral compounds by addition of chiral An apparatus for capillary electrophoresis is composed of: selectors to the separation buffer. (1) a high-voltage, controllable direct-current power supp- Optimization ly, Optimization of the separation is a complex process where (2) two buffer reservoirs, held at the same level, containing several separation parameters can play a major role. The the prescribed anodic and cathodic solutions, main factors to be considered in the development of separa- (3) two electrode assemblies (the cathode and the anode), tions are instrumental and electrolytic solution parameters. immersed in the buffer reservoirs and connected to the pow- Instrumental parameters er supply, (1) Voltage: A Joule heating plot is useful in optimizing (4) a separation capillary (usually made of fused-silica) the applied voltage and column temperature. Separation which, when used with some specific types of detectors, has time is inversely proportional to applied voltage. However, an optical viewing window aligned with the detector. The an increase in the voltage used can cause excessive heat ends of the capillary are placed in the buffer reservoirs. The production, giving rise to temperature and, as a result there- capillary is filled with the solution prescribed in the mono- of, viscosity gradients in the buffer inside the capillary. This graph, effect causes band broadening and decreases resolution. (5) a suitable injection system, (2) Polarity: Electrode polarity can be normal (anode at (6) a detector able to monitor the amount of substances of the inlet and cathode at the outlet) and the electroosmotic interest passing through a segment of the separation capil- flow will move toward the cathode. If the electrode polarity lary at a given time. It is usually based on absorption spec- is reversed, the electroosmotic flow is away from the outlet trophotometry (UV and visible) or fluorometry, but conduc- and only charged analytes with electroosmotic mobilities timetric, amperometric or mass spectrometric detection can greater than the electroosmotic flow will pass to the outlet. be useful for specific applications. Indirect detection is an al- (3) Temperature: The main effect of temperature is ternative method used to detect non-UV-absorbing and non- observed on buffer viscosity and electrical conductivity, and fluorescent compounds, therefore on migration velocity. In some cases, an increase in (7) a thermostatic system able to maintain a constant tem- capillary temperature can cause a conformational change in perature inside the capillary is recommended to obtain a proteins, modifying their migration time and the efficiency good separation reproducibility, of the separation. (8) a recorder and a suitable integrator or a computer. (4) Capillary: The dimensions of the capillary (length and The definition of the injection process and its automation internal diameter) contribute to analysis time, efficiency of are critical for precise quantitative analysis. Modes of injec- separations and load capacity. Increasing total length can tion include gravity, pressure or vacuum injection and elec- decrease the electric fields (working at constant voltage), and trokinetic injection. The amount of each sample component increasing both effective length and total length increase introduced electrokinetically depends on its electrophoretic migration time. For a given buffer and electric field, heat mobility, leading to possible discrimination using this injec- dissipation, and hence sample band-broadening, depend on tion mode. the internal diameter of the capillary. The latter also affects Use the capillary, the buffer solutions, the preconditioning the detection limit, depending on the sample volume injected method, the sample solution and the migration conditions and the detection system employed. prescribed in the monograph of the considered substance. Since the adsorption of the sample components on the The employed electrolytic solution is filtered to remove par- capillary wall limits efficiency, methods to avoid these inter- ticles and degassed to avoid bubble formation that could in- actions should be considered in the development of a separa- terfere with the detection system or interrupt the electrical tion method. In the specific case of proteins, several strate- contact in the capillary during the separation run. A rigorous gies have been devised to avoid adsorption on the capillary rinsing procedure should be developed for each analytical wall. Some of these strategies (use of extreme pH and ad- method to achieve reproducible migration times of the so- sorption of positively charged buffer additives) only require lutes. modification of the buffer composition to prevent protein adsorption. In other strategies, the internal wall of the capil- 1. Capillary Zone Electrophoresis lary is coated with a polymer, covalently bonded to the In capillary zone electrophoresis, analytes are separated in silica, that prevents interaction between the proteins and the a capillary containing only buffer without any anticonvective negatively charged silica surface. For this purpose, ready-to- medium. With this technique, separation takes place because use capillaries with coatings consisting of neutral-hydro- the different components of the sample migrate as discrete philic, cationic and anionic polymers are available. bands with different velocities. The velocity of each band de- Electrolytic solution parameters pends on the electrophoretic mobility of the solute and the (1) Buffer type and concentration: Suitable buffers for JP XVI General Information / Biotechnological/Biological Products 2181 capillary electrophoresis have an appropriate buffer capacity the gels are used for protein analysis under reducing condi- in the pH range of choice and low mobility to minimize cur- tions, the separation buffer usually contains sodium dodecyl rent generation. sulfate and the samples are denatured by heating a mixture Matching buffer-ion mobility to solute mobility, whenever of sodium dodecyl sulfate and 2-mercaptoethanol or possible, is important for minimizing band distortion. The dithiothreitol before injection. When non-reducing condi- type of sample solvent used is also important to achieve on- tions are used (for example, analysis of an intact antibody), column sample focusing, which increases separation efficien- 2-mercaptoethanol and dithiothreitol are not used. Separa- cy and improves detection. tion in cross-linked gels can be optimized by modifying the An increase in buffer concentration (for a given pH) separation buffer (as indicated in the capillary zone elec- decreases electroosmotic flow and solute velocity. trophoresis section) and controlling the gel porosity during (2) Buffer pH: The pH of the buffer can affect separa- the gel preparation. For cross-linked polyacrylamide gels, tion by modifying the charge of the analyte or additives, and the porosity can be modified by changing the concentration by changing the electroosmotic flow. In protein and peptide of acrylamide and/or the proportion of cross-linker. As a separation, changing the pH of the buffer from above to rule, a decrease in the porosity of the gel leads to a decrease below the isoelectric point (pI) changes the net charge of the in the mobility of the solutes. Due to the rigidity of these solute from negative to positive. An increase in the buffer gels, only electrokinetic injection can be used. pH generally increases the electroosmotic flow. Dynamically coated gels are hydrophilic polymers, such as (3) Organic solvents: Organic modifiers (methanol, linear polyacrylamide, cellulose derivatives, dextran, etc., acetonitrile, etc.) may be added to the aqueous buffer to in- which can be dissolved in aqueous separation buffers giving crease the solubility of the solute or other additives and/or rise to a separation medium that also acts as a molecular to affect the degree of ionization of the sample components. sieve. These separation media are easier to prepare than The addition of these organic modifiers to the buffer gener- cross-linked polymers. They can be prepared in a vial and ally causes a decrease in the electroosmotic flow. filled by pressure in a wall-coated capillary (with no electro- (4) Additives for chiral separations:Fortheseparationof osmotic flow). Replacing the gel before every injection gen- optical isomers, a chiral selector is added to the separation erally improves the separation reproducibility. The porosity buffer. The most commonly used chiral selectors are cy- of the gels can be increased by using polymers of higher mo- clodextrins, but crown ethers, polysaccharides and proteins lecular mass (at a given polymer concentration) or by may also be used. Since chiral recognition is governed by the decreasing the polymer concentration (for a given polymer different interactions between the chiral selector and each of molecular mass). A reduction in the gel porosity leads to a the enantiomers, the resolution achieved for the chiral com- decrease in the mobility of the solute for the same buffer. pounds depends largely on the type of chiral selector used. In Since the dissolution of these polymers in the buffer gives this regard, for the development of a given separation it may low viscosity solutions, both hydrodynamic and electrokinet- be useful to test cyclodextrins having a different cavity size ic injection techniques can be used. (a-, b-, or g-cyclodextrin) or modified cyclodextrins with 3. Capillary Isoelectric Focusing neutral (methyl, ethyl, hydroxyalkyl, etc.) or ionizable In isoelectric focusing, the molecules migrate under the (aminomethyl, carboxymethyl, sulfobutyl ether, etc.) influence of the electric field, so long as they are charged, in groups. When using modified cyclodextrins, batch-to-batch a pH gradient generated by ampholytes having pI values in a variations in the degree of substitution of the cyclodextrins wide range (poly-aminocarboxylic acids), dissolved in the must be taken into account since it will influence the selectiv- separation buffer. ity. Other factors controlling the resolution in chiral separa- The three basic steps of isoelectric focusing are loading, tions are concentration of chiral selector, composition and focusing and mobilization. pH of the buffer and temperature. The use of organic addi- (1) Loading step: Two methods may be employed: tives, such as methanol or urea can also modify the resolu- ( i ) loading in one step: the sample is mixed with ampho- tion achieved. lytes and introduced into the capillary either by pres- 2. Capillary Gel Electrophoresis sure or vacuum; In capillary gel electrophoresis, separation takes place in- (ii) sequential loading: a leading buffer, then the ampho- side a capillary filled with a gel that acts as a molecular sieve. lytes, then the sample mixed with ampholytes, again Molecules with similar charge-to-mass ratios are separated ampholytes alone and finally the terminating buffer according to molecular size since smaller molecules move are introduced into the capillary. The volume of the more freely through the network of the gel and therefore mi- sample must be small enough not to modify the pH grate faster than larger molecules. Different biological mac- gradient. romolecules (for example, proteins and DNA fragments), (2) Focusing step: When the voltage is applied, ampho- which often have similar charge-to-mass ratios, can thus be lytes migrate toward the cathode or the anode, according to separated according to their molecular mass by capillary gel their net charge, thus creating a pH gradient from anode electrophoresis. (lower pH) to cathode (higher pH). During this step the com- Characteristics of Gels ponents to be separated migrate until they reach a pH cor- Two types of gels are used in capillary electrophoresis: responding to their isoelectric point (pI) and the current permanently coated gels and dynamically coated gels. Per- drops to very low values. manently coated gels, such as cross-linked polyacrylamide, (3) Mobilization step: If mobilization is required for de- are prepared inside the capillary by polymerization of the tection, use one of the following methods. Three methods monomers. They are usually bonded to the fused-silica wall are available: and cannot be removed without destroying the capillary. If ( i ) in the first method, mobilization is accomplished 2182 Biotechnological/Biological Products / General Information JP XVI

during the focusing step under the effect of the elec- nique that can be used for the separation of both neutral and troosmotic flow; the electroosmotic flow must be charged solutes, maintaining the efficiency, speed and in- small enough to allow the focusing of the compo- strumental suitability of capillary electrophoresis. One of the nents; most widely used surfactants in MEKC is the anionic sur- ( ii ) in the second method, mobilization is accomplished factant sodium dodecyl sulfate, although other surfactants, by applying positive pressure after the focusing step; for example cationic surfactants such as cetyltrimethylam- (iii) in the third method, mobilization is achieved after monium salts, are also used. the focusing step by adding salts to the cathode The separation mechanism is as follows. At neutral and reservoir or the anode reservoir (depending on the alkaline pH, a strong electroosmotic flow is generated and direction chosen for mobilization) in order to alter moves the separation buffer ions in the direction of the the pH in the capillary when the voltage is applied. cathode. If sodium dodecyl sulfate is employed as the sur- As the pH is changed, the proteins and ampholytes factant, the electrophoretic migration of the anionic micelle are mobilized in the direction of the reservoir which is in the opposite direction, towards the anode. As a result, contains the added salts and pass the detector. the overall micelle migration velocity is slowed down com- The separation achieved, expressed as DpI, depends on the pared to the bulk flow of the electrolytic solution. In the case pH gradient (dpH/dx), the number of ampholytes having of neutral solutes, since the analyte can partition between the different pI values, the molecular diffusion coefficient (D), micelle and the aqueous buffer, and has no electrophoretic the intensity of the electric field (E) and the variation of the mobility, the analyte migration velocity will depend only on electrophoretic mobility of the analyte with the pH (－dm/ the partition coefficient between the micelle and the aqueous dpH): buffer. In the electropherogram, the peaks corresponding to each uncharged solute are always between that of the electro- D(dpH/dx) DpI ＝ 3 osmotic flow marker and that of the micelle (the time E(－dm/dpH) elapsed between these two peaks is called the separation window). For electrically charged solutes, the migration velocity Optimization depends on both the partition coefficient of the solute be- The main parameters to be considered in the development tween the micelle and the aqueous buffer, and on the elec- of separations are: trophoretic mobility of the solute in the absence of micelle. (1) Voltage: Capillary isoelectric focusing utilises very Since the mechanism in MEKC of neutral and weakly high electric fields, 300 V/cm to 1000 V/cm in the focusing ionized solutes is essentially chromatographic, migration of step. the solute and resolution can be rationalized in terms of the (2) Capillary: The electroosmotic flow must be reduced retention factor of the solute (k?), also referred to as mass or suppressed depending on the mobilization strategy (see distribution ratio (D ), which is the ratio of the number of above). Coated capillaries tend to reduce the electroosmotic m moles of solute in the micelle to those in the mobile phase. flow. For a neutral compound, k? is given by: (3) Solutions: The anode buffer reservoir is filled with a solution with a pH lower than the pI of the most acidic am- t － t V k?＝ R 0 ＝ K S pholyte and the cathode reservoir is filled with a solution t V t 1 － R M with a pH higher than the pI of the most basic ampholyte. 0 Ø » tmc Phosphoric acid for the anode and sodium hydroxide for the cathode are frequently used. tR: migration time of the solute, Addition of a polymer, such as methylcellulose, in the am- t0: analysis time of an unretained solute (determined by in- pholyte solution tends to suppress convective forces (if any) jecting an electroosmotic flow marker which does not and electroosmotic flow by increasing the viscosity. Com- enter the micelle, for instance methanol), mercial ampholytes are available covering many pH ranges tmc: micelle migration time (measured by injecting a andmaybemixedifnecessarytoobtainanexpandedpH micelle marker, such as Sudan III, which migrates range. Broad pH ranges are used to estimate the isoelectric while continuously associated in the micelle), point whereas narrower ranges are employed to improve ac- K: partition coefficient of the solute, curacy. Calibration can be done by correlating migration VS: volume of the micellar phase, time with isoelectric point for a series of protein markers. VM: volume of the mobile phase. During the focusing step precipitation of proteins at their Likewise, the resolution between two closely-migrating so- isoelectric point can be prevented, if necessary, using buffer lutes (RS)isgivenby: additives such as glycerol, surfactants, urea or zwitterionic t buffers. However, depending on the concentration, urea 1 － Ø 0 » N a － 1 k? tmc denatures proteins. R ＝ × × b × S 4 a k? ＋ 1 t b 0 ? 4. Micellar Electrokinetic Chromatography (MEKC) 1 ＋ Ø » k a tmc In micellar electrokinetic chromatography, separation takes place in an electrolyte solution which contains a sur- N: number of theoretical plates for one of the solutes, factant at a concentration above the critical micellar concen- a: selectivity, ? ? tration (cmc). The solute molecules are distributed between k a and k b: retention factors for both solutes, respectively ? ? the aqueous buffer and the pseudo-stationary phase com- (k b ＞ k a). posed of micelles, according to the partition coefficient of Similar, but not identical, equations give k? and RS values the solute. The technique can therefore be considered as a for electrically charged solutes. hybrid of electrophoresis and chromatography. It is a tech- JP XVI General Information / Biotechnological/Biological Products 2183

Optimization micellar system, either covalently bound to the surfactant or The main parameters to be considered in the development added to the micellar separation electrolyte. Micelles that of separations by MEKC are instrumental and electrolytic have a moiety with chiral discrimination properties include solution parameters. salts of N-dodecanoyl-L-amino acids, bile salts, etc. Chiral Instrumental parameters resolution can also be achieved using chiral discriminators, (1) Voltage: Separation time is inversely proportional to such as cyclodextrins, added to the electrolytic solutions applied voltage. However, an increase in voltage can cause whichcontainmicellizedachiralsurfactants. excessive heat production that gives rise to temperature (5) Other additives: Several strategies can be carried out gradients and viscosity gradients of the buffer in the cross- to modify selectivity, by adding chemicals to the buffer. The section of the capillary. This effect can be significant with addition of several types of cyclodextrins to the buffer can high conductivity buffers such as those containing micelles. also be used to reduce the interaction of hydrophobic solutes Poor heat dissipation causes band broadening and decreases with the micelle, thus increasing the selectivity for this type resolution. of compound. (2) Temperature: Variations in capillary temperature af- The addition of substances able to modify solute-micelle fect the partition coefficient of the solute between the buffer interactions by adsorption on the latter, is used to improve and the micelles, the critical micellar concentration and the the selectivity of the separations in MEKC. These additives viscosity of the buffer. These parameters contribute to the may be a second surfactant (ionic or non-ionic) which gives migration time of the solutes. The use of a good cooling sys- rise to mixed micelles or metallic cations which dissolve in tem improves the reproducibility of the migration time for the micelle and form co-ordination complexes with the so- the solutes. lutes. (3) Capillary: As in capillary zone electrophoresis, the Quantification dimensions of the capillary (length and internal diameter) Peak areas must be divided by the corresponding migra- contribute to analysis time and efficiency of separations. In- tion time to give the corrected area in order to: creasing both effective length and total length can decrease (1) compensate for the shift in migration time from run to the electric fields (working at constant voltage), increase run, thus reducing the variation of the response, migration time and improve the separation efficiency. The (2) compensate for the different responses of sample con- internal diameter controls heat dissipation (for a given stituents with different migration times. buffer and electric field) and consequently the sample band Where an internal standard is used, verify that no peak of broadening. the substance to be examined is masked by that of the inter- Electrolytic solution parameters nal standard. (1) Surfactant type and concentration: The type of sur- Calculations factant, in the same way as the stationary phase in chroma- From the values obtained, calculate the content of the tography, affects the resolution since it modifies separation component or components being examined. When selectivity. Also, the log k? of a neutral compound increases prescribed, the percentage content of one or more compo- linearly with the concentration of surfactant in the mobile nents of the sample to be examined is calculated by deter- phase. Since resolution in MEKC reaches a maximum when mining the corrected area(s) of the peak(s) as a percentage of k? approaches the value of t /t , modifying the concentra- m 0 the total of the corrected areas of all peaks, excluding those tion of surfactant in the mobile phase changes the resolution due to solvents or any added reagents (normalization proce- obtained. dure). The use of an automatic integration system (integrator (2) Buffer pH: Although pH does not modify the parti- or data acquisition and processing system) is recommended. tion coefficient of non-ionized solutes, it can modify the electroosmotic flow in uncoated capillaries. A decrease in the System Suitability buffer pH decreases the electroosmotic flow and therefore In order to check the behavior of the capillary electropho- increases the resolution of the neutral solutes in MEKC, resis system, system suitability parameters are used. The resulting in a longer analysis time. choice of these parameters depends on the mode of capillary (3) Organic solvents: To improve MEKC separation of electrophoresis used. They are: retention factor (k?) (only for hydrophobic compounds, organic modifiers (methanol, micellar electrokinetic chromatography), apparent number propanol, acetonitrile, etc.) can be added to the electrolytic of theoretical plates (N), symmetry factor (AS) and resolu- solution. The addition of these modifiers usually decreases tion (RS). In previous sections, the theoretical expressions migration time and the selectivity of the separation. Since for N and RS have been described, but more practical equa- the addition of organic modifiers affects the critical micellar tions that allow these parameters to be calculated from the concentration, a given surfactant concentration can be used electropherograms are given below. only within a certain percentage of organic modifier before Apparent Number of Theoretical Plates the micellization is inhibited or adversely affected, resulting The apparent number of theoretical plates (N)maybecal- in the absence of micelles and, therefore, in the absence of culated using the expression: partition. The dissociation of micelles in the presence of a t 2 high content of organic solvent does not always mean that N ＝ 5.54 R Øw » the separation will no longer be possible; in some cases the h hydrophobic interaction between the ionic surfactant t : migration time or distance along the baseline from the monomer and the neutral solutes forms solvophobic com- R point of injection to the perpendicular dropped from plexes that can be separated electrophoretically. the maximum of the peak corresponding to the compo- (4) Additives for chiral separations:Fortheseparationof nent, enantiomers using MEKC, a chiral selector is included in the 2184 Biotechnological/Biological Products / General Information JP XVI

wh: width of the peak at half-height. in the electropherogram obtained with the prescribed reference solution and, if possible, situated equally around the Resolution place where this peak would be found. The resolution (RS) between peaks of similar height of two components may be calculated using the expression:

1.18(tR2 － tR1) RS ＝ Ø » Isoelectric Focusing wh1 ＋ wh2

tR2 ＞ tR1 This test is harmonized with the European Pharmacopoeia and the U.S. Pharmacopeia. The parts of the text that are t and t :migrationtimesordistances along the baseline R1 R2 not harmonized are marked with symbols ( ). from the point of injection to the perpendicu-  lars dropped from the maxima of two adjacent General Principles peaks, Isoelectric focusing (IEF) is a method of electrophoresis

wh1 and wh2: peak widths at half-height. that separates proteins according to their isoelectric point. Separation is carried out in a slab of polyacrylamide or When appropriate, the resolution may be calculated by agarose gel that contains a mixture of amphoteric electro- measuring the height of the valley (H ) between two partly v lytes (ampholytes). When subjected to an electric field, the resolved peaks in a standard preparation and the height of ampholytes migrate in the gel to create a pH gradient. In the smaller peak (H ) and calculating the peak-to-valley p some cases gels containing an immobilized pH gradient, pre- ratio: pared by incorporating weak acids and bases to specific H regions of the gel network during the preparation of the gel, p/n ＝ p Hv are used. When the applied proteins reach the gel fraction that has a pH that is the same as their isoelectric point (pI), Symmetry Factor their charge is neutralized and migration ceases. Gradients

The symmetry factor (AS) of a peak may be calculated can be made over various ranges of pH, according to the using the expression: mixture of ampholytes chosen. w Theoretical Aspects A ＝ 0.05 S 2d When a protein is at the position of its isoelectric point, it has no net charge and cannot be moved in a gel matrix by the w : width of the peak at one-twentieth of the peak 0.05 electric field. It may, however, move from that position by height, diffusion. The pH gradient forces a protein to remain in its d: distance between the perpendicular dropped from the isoelectric point position, thus concentrating it; this concen- peak maximum and the leading edge of the peak at one- trating effect is called ``focusing''. Increasing the applied twentieth of the peak height. voltage or reducing the sample load result in improved sepa- Tests for area repeatability (standard deviation of areas or ration of bands. The applied voltage is limited by the heat of the area/migration-time ratio) and for migration time generated, which must be dissipated. The use of thin gels and repeatability (standard deviation of migration time) are in- an efficient cooling plate controlled by a thermostatic circu- troduced as suitability parameters. Migration time repeata- lator prevents the burning of the gel whilst allowing sharp bility provides a test for the suitability of the capillary wash- focusing. The separation R is estimated by determining the ing procedures. An alternative practice to avoid the lack of minimum pI difference (DpI), which is necessary to separate repeatability of the migration time is to use migration time 2 neighboring bands: relative to an internal standard. D(dpH/dx) A test for the verification of the signal-to-noise ratio for a R: DpI ＝ 3 E(－dm/dpH) standard preparation (or the determination of the limit of quantification) may also be useful for the determination of D: Diffusion coefficient of the protein related substances. dpH/dx:pHgradient Signal-to-noise Ratio E: Intensity of the electric field, in volts per centimeter The detection limit and quantification limit correspond to －dm/dpH: Variation of the solute mobility with the pH in signal-to-noise ratios of 3 and 10 respectively. The signal-to- the region close to the pI noise ratio (S/N) is calculated using the expression: Since D and －dm/dpH for a given protein cannot be al- 2H tered, the separation can be improved by using a narrower S/N ＝ h pH range and by increasing the intensity of the electric field. Resolution between protein bands on an IEF gel prepared H: height of the peak corresponding to the component with carrier ampholytes can be quite good. Improvements in concerned, in the electropherogram obtained with the resolution may be achieved by using immobilized pH prescribed reference solution, measured from the maximum gradients where the buffering species, which are analogous of the peak to the extrapolated baseline of the signal ob- to carrier ampholytes, are copolymerized within the gel served over a distance equal totwentytimesthewidthat matrix. Proteins exhibiting pIs differing by as little as 0.02 half-height, pH units may be resolved using a gel prepared with carrier h: range of the background in an electropherogram ob- ampholytes while immobilized pH gradients can resolve pro- tained after injection of a blank, observed over a distance teins differing by approximately 0.001 pH units. equal to twenty times the width at the half-height of the peak JP XVI General Information / Biotechnological/Biological Products 2185

Practical Aspects Special attention must be paid to sample characteristics and/or preparation. Having salt in the sample can be problematic and it is best to prepare the sample, if possible, in deionized water or 2 per cent ampholytes, using dialysis or gel filtration if necessary. The time required for completion of focusing in thin-layer polyacrylamide gels is determined by placing a colored protein (e.g. hemoglobin) at different positions on the gel surface and by applying the electric field: the steady state is reached when all applications give an identical band pattern. In some protocols the completion of the focusing is indicated bythetimeelapsedafterthe sample application. Figure. Mould The IEF gel can be used as an identity test when the migration pattern on the gel is compared to a suitable standard preparation and IEF calibration proteins, the IEF gel can be few millilitres of a suitable liquid, taking care to avoid form- used as a limit test when the density of a band on IEF is com- ing air bubbles. Prepare the test solutions and reference solu- pared subjectively with the density of bands appearing in a tions as specified in the monograph. Place strips of paper for standard preparation, or it can be used as a quantitative test sample application, about 10 mm × 5 mm in size, on the gel when the density is measured using a densitometer or similar and impregnate each with the prescribed amount of the test instrumentation to determine the relative concentration of and reference solutions. Also apply the prescribed quantity protein in the bands subject to validation. of a solution of proteins with known isoelectric points as pH markers to calibrate the gel. In some protocols the gel has Apparatus pre-cast slots where a solution of the sample is applied in- An apparatus for IEF consists of: stead of using impregnated paper strips. Cut 2 strips of —a controllable generator for constant potential, current paper to the length of the gel and impregnate them with the and power. Potentials of 2500 V have been used and are electrolyte solutions: acid for the anode and alkaline for the considered optimal under a given set of operating condi- cathode. The compositions of the anode and cathode solutions. Supply of up to 30 W of constant power is recom- tions are given in the monograph. Apply these paper wicks mended, to each side of the gel several millimetres from the edge. Fit —a rigid plastic IEF chamber that contains a cooled plate, the cover so that the electrodes are in contact with the wicks of suitable material, to support the gel, (respecting the anodic and cathodic poles). Proceed with the —a plastic cover with platinum electrodes that are con- isoelectric focusing by applying the electrical parameters nected to the gel by means of paper wicks of suitable described in the monograph. Switch off the current when the width, length and thickness, impregnated with solutions migration of the mixture of standard proteins has stabilized. of anodic and cathodic electrolytes. Using forceps, remove the sample application strips and the Isoelectric Focusing in Polyacrylamide Gels: Detailed Proce- 2 electrode wicks. Immerse the gel in fixing solution for isoe- dure lectric focusing in polyacrylamide gel. Incubate with gentle The following method is a detailed description of an IEF shaking at room temperature for 30 minutes. Drain off the procedure in thick polyacrylamide slab gels, which is used solution and add 200 mL of destaining solution. Incubate unless otherwise stated in the monograph. with shaking for 1 hour. Drain the gel, add coomassie staining TS. Incubate for 30 minutes. Destain the gel by passive Preparation of the Gels diffusion with destaining solution until the bands are well Mould The mould (see Figure) is composed of a glass visualized against a clear background. Locate the position plate (A) on which a polyester film (B) is placed to facilitate and intensity of the bands in the electropherogram as handling of the gel, one or more spacers (C), a second glass prescribed in the monograph. plate (D) and clamps to hold the structure together. 7.5z Polyacrylamide gel Dissolve 29.1 g of acrylamide Variations to the Detailed Procedure (Subject to Validation) and 0.9 g of N,N?-methylenebisacrylamide in 100 mL of Where reference to the general method on isoelectric water. To 2.5 volumes of this solution, add the mixture of focusing is made, variations in methodology or procedure ampholytes specified in the monograph and dilute to 10 may be made subject to validation. These include: volumes with water. Mix carefully and degas the solution. (1) the use of commercially available pre-cast gels and of Preparation of the mould Place the polyester film on the commercial staining and destaining kits, lower glass plate, apply the spacer, place the second glass (2) the use of immobilized pH gradients, plate and fit the clamps. Place 7.5z polyacrylamide gel pre- (3) the use of rod gels, pared before use on a magnetic stirrer, and add 0.25 volumes (4) theuseofgelcassettesofdifferent dimensions, includ- of a solution of ammonium persulfate (1 in 10) and 0.25 ing ultra-thin (0.2 mm) gels, volumes of N,N,N?,N?-tetramethylethylenediamine. Imme- (5) variations in the sample application procedure, includ- diately fill the space between the glass plates of the mould ing different sample volumes or the use of sample appli- with the solution. cation masks or wicks other than paper, (6) the use of alternate running conditions, including varia- Method tions in the electric field depending on gel dimensions Dismantle the mould and, making use of the polyester and equipment, and the use of fixed migration times film, transfer the gel onto the cooled support, wetted with a 2186 Biotechnological/Biological Products / General Information JP XVI

rather than subjective interpretation of band stability, Destaining solution A mixture of water, methanol and

(7) the inclusion of a pre-focusing step, acetic acid (100) (5:4:1). (8) the use of automated instrumentation, (9) the use of agarose gels. Validation of Iso-Electric Focusing Procedures Mass Spectrometry of Peptides Where alternative methods to the detailed procedure are employed they must be validated. The following criteria may and Proteins be used to validate the separation: Mass spectrometry (MS) is based on the ionization of (1) formation of a stable pH gradient of desired character- molecules and separation of the electrically charged ions istics, assessed for example using colored pH markers according to the m/z. The results are expressed as a mass of known isoelectric points, spectrum with m/z values of the ions on the x-axis and signal (2) comparison with the electropherogram provided with intensity of the ions on the y-axis. The mass of the molecule the chemical reference substance for the preparation to calculated from the m/z values is expressed in unified atomic be examined, mass units (u) or daltons (Da). Tandem mass spectrometry (3) any other validation criteria as prescribed in the mono- (MS/MS) is based on the selection of the precursor ion in the graph. first stage analyzer, fragmentation of the precursor ion and Specified Variations to the General Method measurement of the product ions in the second stage mass Variations to the general method required for the analysis analyzer. This technique provides useful information for of specific substances may be specified in detail in mono- structural analysis of the molecule. Information obtained in graphs. These include: MS is qualitative and is sometimes used for qualification. (1) the addition of urea in the gel (3 mol/L concentration is MS and MS/MS are useful for measuring masses of peptides often satisfactory to keep protein in solution but up to 8 and proteins and for confirming amino acid sequences and mol/L can be used): some proteins precipitate at their post-translational modifications. Both methods are therefore isoelectric point. In this case, urea is included in the gel used for identification of pharmaceutical peptides and pro- formulation to keep the protein in solution. If urea is teins. used, only fresh solutions should be used to prevent 1. Instrument carbamylation of the protein, A mass spectrometer is composed of an ion source, an (2) the use of alternative staining methods, analyzer, an ion detector, and a data system (Figure 1). A (3) the use of gel additives such as non-ionic detergents peptide or protein sample introduced into the ion source is (e.g. octylglucoside) or zwitterionic detergents (e.g., 3- ionized by soft-ionization methods, such as matrix-assisted [(3-Cholamidopropyl)dimethylammonio]-1-propanesul- laser desorption/ionization (MALDI) and electrospray ioni- fonate (CHAPS) or 3-[(3-Cholamidopropyl)dimethy zation (ESI). The charged and gas-phased ions are sorted ac- lammonio]-2-hydroxy-1-propanesulfonate (CHAPSO)), cording to the m/z under a vacuum in the analyzer, which and the addition of ampholyte to the sample, to prevent may be a quadrupole, time-of-flight, ion trap or Fourier proteins from aggregating or precipitating. transform ion cyclotron resonance analyzer. The ion flux Points to Consider collected in the detector is converted to an electric signal. Samples can be applied to any area on the gel, but to pro- Then the signal is recorded as a mass spectrum. MS/MS is tect the proteins from extreme pH environments samples carried out by using two mass spectrometers connected in should not be applied close to either electrode. During series, an ion-trap mass spectrometer and Fourier transform method development the analyst can try applying the protein ion cyclotron resonance mass spectrometer. The precursor in 3 positions on the gel (i.e. middle and both ends); the pat- ions are generally fragmented by collision-induced dissocia- tern of a protein applied at opposite ends of the gel may not tion, post-source decay, electron capture dissociation, etc. be identical. 2. Analytical mode A phenomenon known as cathodic drift, where the pH 2.1. MS gradient decays over time, may occur if a gel is focused too There are two useful modes for MS: long. Although not well understood, electroendoosmosis and (1) Full scan mode absorption of carbon dioxide may be factors that lead to The signals of the entire ion are acquired over the chosen cathodic drift. Cathodic drift is observed as focused protein range of m/z. This mode provides information on the migrating off the cathode end of the gel. Immobilized pH masses of the molecule of interest and different species. gradients may be used to address this problem. (2) Selected ion monitoring Efficient cooling (approximately 49C) of the bed that the The signals of the ion at chosen m/z are acquired. This gel lies on during focusing is important. High field strengths mode is useful for the sensitive measurement of the chosen used during isoelectric focusing can lead to overheating and molecule. affect the quality of the focused gel. 2.2. MS/MS Reagents and Solutions— There are four essential modes for MS/MS: Fixing solution for isoelectric focusing in polyacrylamide (1) Product ion analysis gel Dissolve 35 g of 5-sulfosalicylic acid dihydrate and The signals of all the product ions produced from the 100 g of trichloroacetic acid in water to make 1000 mL. precursor at chosen m/z are acquired. This mode provides Coomassie staining TS Dissolve 125 mg of coomassie structural information on the analytes and various co- brilliant blue R-250 in 100 mL of a mixture of water, metha- existing species. nol and acetic acid (100) (5:4:1), and filter. JP XVI General Information / Biotechnological/Biological Products 2187

Fig‚ 1 Concept of MS and MS/MS

(2) Precursor ion scan mode (2) Electrospray ionization (ESI) The precursor that yields the product ion at chosen m/z A desalted peptide or protein sample is dissolved in a suit- are monitored. This mode is used for sorting the molecules able solvent, such as an aqueous solution containing acetic containing a component of interest. acid and methanol or acetonitrile. The sample solution is (3) Constant neutral loss scan mode sprayed through a capillary and held at a potential of several The precursor that loses the fragment at chosen m/z are kilovolts. The sample is introduced by using a syringe or monitored. This mode is useful to sort the molecules con- HPLC. taining a component of interest. 3.2. MS/MS (4) Selected reaction monitoring System suitability is tested by MS/MS of the test sample The product ions at chosen m/z that are produced from specified in the monograph. The detectability and system the precursor at chosen m/z are monitored. This mode performance should be confirmed based on the detection of allows for sensitive and selective measurement and is used the product ions specified in the monograph. The sample is for quantification of a molecule in a complex mixture. ionized in the same way as for MS, and the chosen precursor is fragmented by the suitable conditions specified in the 3. Analytical procedure monograph. The signals are recorded as a mass spectrum. A 3.1. MS peptide containing disulfide bonds is generally reduced by System suitability is tested by measuring the mass of a test dithiothreitol, 2-mercaptethanol and tris (2-carboxyethyl) sample specified in the monograph. The system performance phosphine. The reduced peptides are derivatized with should be confirmed based on the difference between the cal- monoiodoacetic acid, iodoacetamide, and 4-binylpyridine. culated mass and observed mass of the test sample. If the detectability and system performance do not meet the criteria, 4. Identification test the system should be optimized by adjustment of the ion 4.1. Mass of the molecule source, analyzer and detector, as well as by calibration using The monoisotopic mass of the peptide is preferably ac- appropriate molecules. MS is performed according to the quired. If the monoisotopic peak is not detectable, the sample preparation and operating conditions indicated in the average mass is calculated using the weighed average of monograph. The general procedure is described as follows. isotopic masses. Deconvolution is effective for calculating (1) Matrix-assisted laser desorption/ionization (MALDI) the average mass of multiply-charged proteins. The mass A desalted peptide or protein sample is dissolved in an should meet the criteria specified in the monograph. appropriate solvent, e.g., an aqueous solution of trifluoroa- 4.2. Amino acid sequence cetic acid. A suitable matrix, such as a-ciano-4-hydroxycin- After measuring the mass of the sample peptide, the namic acid, 2,5-dihydroxybenzoic acid, or sinapic acid, is presence of the specified product ions that arise from the dissolved in an aqueous solution containing acetonitrile and selected precursor is confirmed according to the conditions trifluoroacetic acid. A mixture of sample solution and indicated in the monograph. Digestion of sample proteins matrix solution is deposited on a sample plate and dried. The with a suitable enzyme followed by MS/MS is sometimes ef- sample on the plate is set in the ion source, and ionized by a fective for sequencing of the high-molecular weight proteins laser beam at suitable intensity. which provide insufficient product ions. Details of the diges- 2188 Biotechnological/Biological Products / General Information JP XVI tion procedure are provided in the section on peptide map- Matrix-assisted laser desorption/ionization (MALDI) ping. The sample, which is mixed with a suitable matrix and deposited on a target plate, is ionized by irradiation with 5. Glossary nanosecond laser pulses. Proteins, carbohydrates, Ion-trap (IT) oligonucleotides, and lipids can be ionized without any dis- Ion-trap refers to the quadrupole ion trap mass analyzer in sociation. Singly-charged ions are mainly detected. a restricted sense. Ions stored in the analyzer by applying radio frequency voltage to ring electrodes are separated by subsequent ejection of the ions from the analyzer by varying the voltage on the ring electrodes. This allows multiple stage Mycoplasma Testing for Cell MS in which a selected ion is repeatedly trapped, fragmented and ejected. Substrates used for the Production Electrospray ionization (ESI) of Biotechnological/Biological The sample in solution is sprayed through a capillary and Products held at high-voltage at atmospheric pressure. The sample is ionized by a formation of charged liquid droplets. High- This document describes the currently available methods molecular weight proteins are detected as multiply-charged of mycoplasma testing that should be performed for cell sub- ions. The analyzer can be connected with HPLC. strates that are used in the manufacture of biotechnological/ Quadrupole (Q) biological products. The analyzer is composed of four parallel electrodes which Methods suggested for detection of mycoplasma are, A. have a hyperboloidal or cylindrical cross-section. The ions culture method, B. indicator cell culture method, and C. transmitted to the analyzer are separated by varying the polymerase chain reaction (PCR) method. potential of direct and radio frequency components applied Mycoplasma testing should be performed on the master to the rods so that the filter for sorting the m/z values of cell bank (MCB) and the working cell bank (WCB), as well ions is changed. as on the cell cultures used during the manufacturing process of the product. For the assessment of these cells, mycoplas- Collision-induced dissociation (CID) ma testing should be performed using both methods A and When an ion collides with a neutral atom or molecule (He, B. Method B, however, does not detect only DNA derived Ar, N2 and so on), some of the translational energy of the from mycoplasma. Therefore, if a positive result is obtained collision is converted into internal energy, thereby causing only from method B, method C can be used to determine dissociation. The terms low-energy CID and high-energy whether mycoplasma is actually present. When method C is CID refer to those CIDs for which the translational energy used, it is necessary to demonstrate the rationale for deter- of the precursor ions is lower than 1000 eV and higher than mining a negative result. In such a case, the sensitivity and 1000 eV, respectively. specificity of the method, the appropriateness of the sample Electron capture dissociation (ECD) preparation, and the suitability of the selection of the test Multiply-charged positive ions interact with low energy method, including selection of reagents, reaction conditions electrons producing charge-reduced radical ions, which rea- and primers should be taken into account. dily dissociate. This method is primarily used for MS/MS in Prior to mycoplasma testing, the sample should be tested FT-ICR MS or IT MS. to detect the presence of any factors inhibiting the growth of mycoplasma. If such growth-inhibiting factors are detected Time-of-flight (TOF) they should be neutralized or eliminated by an appropriate The ionized sample is accelerated at high-voltage and sepa- method, such as centrifugation or cell passage. rated based on the time required for an ion to travel to the If the test will be performed within 24 hours of obtaining detector. There are two types of analyzer, a linear type in the sample, the sample should be stored at a temperature be- which ions travel linearly from the ion source to the detector, tween 29Cand89C. If more than 24 hours will elapse before and a reflectron type where ions are inverted by a reflectron. the test is performed, the sample should be stored at －609C Thelattertypeallowshigh-resolution measurement by cor- or lower. rection of the variation in the initial energy of ions. If mycoplasma is detected, additional testing to identify Fourier transform ion cyclotron resonance (FT-ICR) the species may be helpful in determining the source of con- The analyzer is based on the principle that the cyclotron tamination. frequency of the ions in a magnetic field is inversely propor- A. Culture Method tional to its m/z value. Ions are excited to a larger radius or- 1. Culture Medium bit using radio frequency energy and their image current is Both agar plates and broth are used. Each lot of agar and detected on receiver plates. The resulting data are devolved broth medium should be free of antibiotics except for by applying a Fourier transform to give a mass spectrum. penicillin. Refer to the Minimum Requirements for Biologi- Post-source decay (PSD) cal Products regarding selection of the culture media. Other Metastable ion decay occurs by excess internal energy and culture media may be used if they fulfill the requirements collision with residual gas during ion acceleration out of the described in the following section 2. MALDI ion source and prior to reaching the detector. This 2. Suitability of Culture Medium method is used for MS/MS by using MALDI-TOF MS with Each lot of medium should be examined for mycoplasma a reflectron mode. growth-promoting properties. To demonstrate the capacity of the media to detect known mycoplasma, each test should JP XVI General Information / Biotechnological/Biological Products 2189 include control cultures of at least two known species or The test should include a negative (non-infected) control strains of mycoplasma, one of which should be a dextrose and two positive mycoplasma controls, such as M. hyorhinis fermenter (i.e., M. pneumoniae ATCC15531 or equivalent (ATCC29052, ATCC17981 or equivalent species or strains) species or strains) and one of which should be an arginine and M. orale (ATCC23714 or equivalent species or strains). hydrolyser (i.e., M. orale ATCC23714 or equivalent species Use an inoculum of 100 CFU or 100 CCU or less for the or strains). The mycoplasma strains used for the positive positive controls. control tests should be those with a low number of passages Incubate the cell cultures for 3 to 6 days at 36 ± 19Cinan obtained from an official or suitably accredited agency, and atmosphere of air containing 5 percent carbon dioxide. handled appropriately. Inoculate the culture medium with Examine the cell cultures after fixation for the presence of 100 colony-forming units (CFU) or 100 color-changing units mycoplasma by epifluorescence microscopy (400 to 600 (CCU) or less. times magnification or greater) using a DNA-binding 3. Culture and Observation fluorochrome, such as bisbenzimidazole or an equivalent 1) Inoculate no less than 0.2 mL of test sample (cell sus- stain. Compare the microscopical appearance of the test cul- pension) in evenly distributed amounts over the surface of tures with that of the negative and positive controls. each of two or more agar plates. After the surfaces of the in- Procedure oculated plates are dried, the plates should be incubated 1) Aseptically place a sterilized glass cover slip into each under microaerophilic conditions in an atmosphere of nitro- cell culture dish (35 mm diameter). gen containing 5 to 10 percent carbon dioxide and adequate 2) Prepare Vero cell suspension in Eagle's minimum humidity at 36 ± 19C for no less than 14 days. essential medium containing 10 percent bovine calf serum at 2) Inoculate no less than 10 mL of the test sample (cell a concentration of 1 × 104 cells per 1 mL. The bovine calf suspension) into each of one or more vessels containing 100 serum should be tested and confirmed to be free from mL of broth medium, and incubate at 36 ± 19C. mycoplasma prior to use. If the culture medium for the sample cells contains any 3) Inoculate aliquots of 2 mL of the Vero cell suspension growth-inhibiting factors, such as antibiotics, these factors into each culture dish. Ensure that the cover slips are com- must be removed. A method such as centrifugation is recom- pletely submerged, and not floating on the surface of the cul- mended for this purpose. Refer to the Validation tests for ture medium. Incubate the cultures at 36 ± 19Cinanat- growth-inhibiting factors described in the Minimum Re- mosphere of air containing 5 percent carbon dioxide for one quirements for Biological Products for the detection of day, so that the cells are attached to the glass cover slip. growth-inhibiting factors. 4) Replace 2 mL of the culture medium with fresh me- 3) Subculture 0.2 mL of broth culture from each vessel dium, then add 0.5 mL of the test sample (cell culture super- on the 3rd,7th,and14th days of incubation onto two or more natant) to each of two or more culture dishes. Perform the agar plates. The plates should be inoculated microaerophili- same procedure for the positive (2 types of mycoplasma, cally at 36 ± 19C for no less than 14 days. such as M. hyorhinis (ATCC29052, ATCC17981 or equiva- 4) Examination of all plates for mycoplasma colonies lent species or strains) and M. orale (ATCC23714 or equiva- should be done microscopically on the 7th and 14th day at 100 lent species or strains) and negative controls. times magnification or greater. 5) Incubate the cultures for 3 to 6 days at 36 ± 19Cinan atmosphere of air containing 5 percent carbon dioxide. B. Indicator Cell Culture Method 6) Remove the culture medium from the culture dishes, Using Vero cell culture substrate, pretest the suitability of and add 2 mL of a mixture of acetic acid (100) and methanol the method using an inoculum of 100 CFU or 100 CCU or (1:3)(fixative)toeachdish;then,allowthemtostandfor5 less of M. hyorhinis (ATCC29052, ATCC17981 or equiva- minutes. lent species or strains) and M. orale (ATCC23714 or equiva- 7) Remove the fixative from each dish, then add the lent species or strains). same amount of fixative again, and leave the dishes to stand An equivalent indicator cell substrate and suitable for 10 minutes. mycoplasma strains may be acceptable if data demonstrate 8) Remove the fixative and then completely air-dry all at least equal sensitivity for the detection of known the dishes. mycoplasma contaminants. The mycoplasma strains should 9) Add 2 mL of bisbenzamide fluorochrome staining so- be those with a low number of passages obtained from an lution to each culture dish. Cover the dishes and let them official or suitably accredited agency, and handled appropri- stand at room temperature for 30 minutes. ately, and the unit of inoculation should be determined be- 10) Aspirate the staining solution and rinse each dish fore use. The cell substrate used should be obtained from a with 2 mL of distilled water 3 times. Take out the glass cover qualified cell bank and certified to be mycoplasma free. The slips and dry them. acquired cells should be carefully cultured and propagated, 11) Mount each cover slip with a drop of a mounting and sufficient volumes of seed stock should be prepared with fluid. Blot off surplus mounting fluid from the edges of the the proper precautions to avoid mycoplasma contamination. cover slips. The stock should be tested for mycoplasma contamination 12) Examine by epifluorescence microscopy at 400 to 600 using at least one of the methods described in this document, times magnification or greater. then frozen for storage. For each test a new container from 13) Compare the microscopic appearance of the test the stock should be thawed and used within 6 passages. sample with that of the negative and positive controls. Indicator cell cultures should be grown on cover slips sub- 14) The test result is judged to be positive if there are merged in culture dishes or equivalent containers for one more than 5 cells per 1000 (0.5z) that have minute fluores- day. Inoculate no less than 1 mL of the test sample (cell cul- cent spots that appear to surround, but are outside, the cell ture supernatant) into two or more of the culture dishes. nucleus. 2190 Biotechnological/Biological Products / General Information JP XVI

C. Polymerase Chain Reaction (PCR) Detection Method or twice with 200 to 300 mLof80z ethanol. Remove the The PCR method is a highly specific method that enables rinse solution using a pipette. Centrifuge at 15,000 min－1 for the detection of trace amounts of mycoplasma DNA, and 10 minutes at 49C, then remove the supernatant thoroughly hascometobewidelyusedinrecentyearsasameansofde- and dry up the precipitate. tecting mycoplasma contamination. However, the sensitivity 6) Dissolve the precipitate in 40 mL of distilled water. and specificity depend on the procedure employed, and a 2. Perform the same procedure for the positive and nega- positive result from PCR does not always indicate the tive controls presence of viable mycoplasma. 3. First stage of a two-step PCR The PCR method is based on amplifying DNA extracted 1) Make a mixture of the heat-resistant DNA poly- from the cell culture with specific primers so that the merase, dNTP solution, outer primer, and reaction buffer presence of the target DNA is detected. A two-step PCR solution (including Mg ions), and place 90 mL in each tube. (nestedPCR)isrecommendedinorder to increase sensitivity 2) Add 10 mL of the template prepared as above to each and specificity. The tests should include both a positive tube containing the first stage PCR solution (90 mL). control (such as M. hyorhinis (ATCC29052, ATCC17981 or 3) Perform the DNA amplification by repeating 30 cy- equivalent species or strains) of 100 CFU or 100 CCU or cles of denaturation at 949C for 30 seconds, annealing at an less) and a negative control. appropriate temperature for the primer (559Cfortheprimer Mycoplasma DNA from the sample of cells or cell cultures in this example), and elongation at 729C for 2 minutes. is amplified using primers which should be able to amplify 4. Second stage of a two-step PCR some common conserved mycoplasma DNA sequence. The 1) Make a mixture of the heat-resistant DNA poly- amplification should be performed using an appropriate merase, dNTP solution, inner primer, and reaction buffer heat-resistant DNA polymerase, and suitable conditions. solution (including Mg ions), and place 99 mL in each tube. The amplified DNA can be identified after agarose gel elec- 2) Add 1 mL of the first stage PCR product from each trophoresis, followed by ethidium bromide staining and UV tube to a tube containing the second stage PCR solution (99 irradiation of the gel. mL). For this method, it is important to use primers that are 3) Perform the DNA amplification by repeating 30 cy- specific to mycoplasma by choosing base sequences that are cles of denaturation at 949C for 30 seconds, annealing at an well-conserved for a wide range of mycoplasma species, for appropriate temperature for the primer (559Cfortheprimer example, the spacer region between the 16S-23S ribosome in this example), and elongation at 729C for 2 minutes. genes. 5. Agarose gel electrophoresis It is recommended that a two-step PCR using nested 1) Mix 10 mL of each of the first stage and second stage primers should be performed to increase the sensitivity and PCR products with 2 mL of an appropriate dye as a migra- specificity, if the one-step PCR is negative. tion marker, and perform 1z agarose gel electrophoresis. The primers to be selected for the second stage of a two- 2) Stain the gel with ethidium bromide and take a photo- step PCR are nested primers from the inner portion of the graph under UV irradiation. sequence. The outer and inner primers should have proven 3) The test is judged to be positive if a DNA band is de- effectiveness and specificity as described in publications or tected. be validated experimentally. It is possible to increase the accuracy of the detection of [An Example of Primer] mycoplasma DNA by performing PCR tests after cultivation For mycoplasma detection of mycoplasma that may be present in samples using Vero Outer primer cells. F1 : 5?-ACACCATGGGAG(C/T)TGGTAAT-3? The following is an example of a two-step PCR procedure. R1 : 5?-CTTC(A/T)TCGACTT(C/T)CAGACCCAAGG- The reagents and reaction conditions in this example are not CAT-3? exclusive. If the suitability of other reagents and conditions Inner primer is verified, they may be used. If another procedure is used, F2 : 5?-GTG(G/C)GG(A/C)TGGATCACCTCCT-3? the procedure should be justified and documented in detail, R2 : 5?-GCATCCACCA(A/T)A(A/T)AC(C/T)CTT-3? and the information provided should include the sensitivity ( ) indicates a mixture. and specificity of the method. Example Procedure [PCR reaction solution] 1. Preparation of template [First stage] [Second stage] 1) Place 600 mL of the test cell suspension (if necessary, dNTP solution (1.25 mmol/L) 16 mL16mL subcultured with Vero cells) in a tube and dissolve the cells Primer (10 pmol/mL) F1 2 mLF22mL with 0.1z SDS or an equivalent. Add an equal volume (600 Primer (10 pmol/mL) R1 2 mLR22mL mL) of TE (10 mmol/L tris-hydrochloric acid (pH 8.0), 1 Heat-resistant DNA poly- 2 mL2mL mmol/L EDTA) buffer-saturated phenol, and mix. merase (1 U/mL) 2) Centrifuge at 15,000 min－1 for 5 minutes at room Reaction buffer solution temperature. 25 mmol/L magnesium 8 mL8mL 3) Transfer 400 mL of the supernatant to another tube, chloride hexahydrate and add 10 mL of 3 mol/L sodium acetate. 10-fold buffer solution* 10 mL10mL 4) Add 1 mL (2.5 volumes) of ethanol (95) and stir thor- Sterile distilled water 50 mL59mL oughly. Ice the mixture for 15 minutes, then centrifuge at *Composition of 10-fold buffer solution 15,000 min－1 for 10 minutes at 49C. 2-amino-2-hydroxymethyl-1,3- 5) Discard the supernatant and rinse the precipitate once JP XVI General Information / Biotechnological/Biological Products 2191

propanediol-hydrochloric acid that are generally accepted. Variations of these methods will (pH 8.4) 100 mmol/L be indicated, when appropriate, in specific monographs. Potassium chloride 500 mmol/L A peptide map may be viewed as a fingerprint of a protein Gelatin 0.1 g/L and is the end product of several chemical processes that provide a comprehensive understanding of the protein being [Method of cultivating mycoplasma within Vero cells] analyzed. Four major steps are necessary for the develop- 1) Use at least two cell culture dishes for each of the test ment of the procedure: isolation and purification of the pro- sample, positive control and negative control. tein, if the protein is part of a formulation; selective cleavage 2) Into each cell culture dish (35 mm diameter), inoculate of the peptide bonds; chromatographic separation of the 2 mL of the Vero cell suspension (1 × 104 cells per 1 mL) in peptides; and analysis and identification of the peptides. A Eagle's minimum essential medium containing 10 percent test sample is digested and assayed in parallel with a refer- bovine calf serum (tested in advance using the PCR method ence standard or a reference material. Complete cleavage of to verify that it does not contain any detectable mycoplasma peptide bonds is more likely to occur when enzymes such as DNA). Incubate the cultures at 36 ± 19C in an atmosphere endoproteases (e.g., trypsin) are used, instead of chemical of air containing 5 percent carbon dioxide for one day. cleavage reagents. A map should contain enough peptides to 3) Replace the culture media with fresh media, and add be meaningful. On the other hand, if there are too many 0.5 mL of the test sample (cell culture supernatant) to each fragments, the map might lose its specificity because many of two or more Vero cell culture dishes. Perform the same proteins will then have the same profiles. procedure for the positive (such as 100 CFU or 100 CCU or 2. Isolation and Purification less of M. hyorhinis (ATCC29052, ATCC17981 or equiva- Isolation and purification are necessary for analysis of lent species or strains)) and negative controls. bulk drugs or dosage forms containing interfering excipients 4) Incubate the Vero cell culture dishes for the test and carrier proteins and, when required, will be specified in sample, positive and negative controls for 3 to 6 days at the monograph. Quantitative recovery of protein from the 36 ± 19C in an atmosphere of air containing 5 percent dosage form should be validated. carbon dioxide. 3. Selective Cleavage of Peptide Bonds The selection of the approach used for the cleavage of peptide bonds will depend on the protein under test. This Peptide Mapping selection process involves determination of the type of cleavage to be employed —enzymatic or chemical— and the This test is harmonized with the European Pharmacopoeia type of cleavage agent within the chosen category. Several and the U.S. Pharmacopeia. cleavage agents and their specificity are shown in Table 1. Peptide mapping is an identity test for proteins, especially This list is not all-inclusive and will be expanded as other those obtained by r-DNA technology. It involves the chemi- cleavage agents are identified. cal or enzymatic treatment of a protein, resulting in the for- 3.1. Pretreatment of Sample mation of peptide fragments, followed by separation and Depending on the size or the configuration of the protein, identification of the fragments in a reproducible manner. It different approaches in the pretreatment of samples can be is a powerful test that is capable of identifying single amino used. For monoclonal antibodies, the heavy and light chains acid changes resulting from events such as errors in the read- will need to be separated before mapping. If trypsin is used ing of complementary DNA (cDNA) sequences or point mu- as a cleavage agent for proteins with a molecular mass great- tations. Peptide mapping is a comparative procedure be- er than 100,000 Da, lysine residues must be protected by cause the information obtained, compared to a reference citraconylation or maleylation; otherwise, too many peptides standard or reference material similarly treated, confirms the will be generated. primary structure of the protein, is capable of detecting 3.2. Pretreatment of the Cleavage Agent whether alterations in structure have occurred, and demon- Pretreatment of cleavage agents —especially enzymatic strates process consistency and genetic stability. Each pro- agents— might be necessary for purification purposes to tein presents unique characteristics which must be well ensure reproducibility of the map. For example, trypsin used understood so that the scientific and analytical approaches as a cleavage agent will have to be treated with tosyl-L- permit validated development of a peptide map that provides phenylalanine chloromethyl ketone to inactivate chymotryp- sufficient specificity. sin. Other methods, such as purification of trypsin by HPLC This chapter provides detailed assistance in the application or immobilization of enzyme on a gel support, have been of peptide mapping and its validation to characterize the successfully used when only a small amount of protein is desired protein product, to evaluate the stability of the available. expression construct of cells used for recombinant DNA 3.3. Pretreatment of the Protein products, to evaluate the consistency of the overall process Under certain conditions, it might be necessary to concen- and to assess product stability as well as to ensure the identi- trate the sample or to separate the protein from added sub- ty of the protein product, or to detect the presence of protein stances and stabilizers used in formulation of the product, if variant. these interfere with the mapping procedure. Physical procedures used for pretreatment can include ultrafiltration, 1. The Peptide Map column chromatography, and lyophilization. Other pretreat- Peptide mapping is not a general method, but involves de- ments, such as the addition of chaotropic agents (e.g., urea) veloping specific maps for each unique protein. Although can be used to unfold the protein prior to mapping. To allow the technology is evolving rapidly, there are certain methods the enzyme to have full access to cleavage sites and permit 2192 Biotechnological/Biological Products / General Information JP XVI

Table 1 Examples of Cleavage Agents fragmentation reaction. (ii) Temperature: A temperature between 259Cand Type Agent Specificity 379C is adequate for most digestions. The temperature used Enzymatic Trypsin (EC 3.4.21.4) C-terminal side of is intended to minimize chemical side reactions. The type of Arg and Lys protein under test will dictate the temperature of the reaction Chymotrypsin C-terminal side of milieu, because some proteins are more susceptible to (EC 3.4.21.1) hydrophobic denaturation as the temperature of the reaction increases. residues (e.g., For example, digestion of recombinant bovine somatropin is Leu, Met, Ala, conducted at 49C, because at higher temperatures it will pre- aromatics) cipitate during digestion. Pepsin (EC 3.4.23.1 & 2) Nonspecific digest (iii) Time: If sufficient sample is available, a time Lysyl endopeptidase C-terminal side of course study is considered in order to determine the opti- (Lys-C Endopeptidase) Lys mum time to obtain a reproducible map and avoid incom- (EC 3.4.21.50) plete digestion. Time of digestion varies from 2 to 30 hours. Glutamyl endopeptidase C-terminal side of The reaction is stopped by the addition of an acid which (from S. aureus strain V8) Glu and Asp does not interfere in the tryptic map or by freezing. (EC 3.4.21.19) (iv) Amount of Cleavage Agent: Although excessive Peptidyl-Asp metallo N-terminal side of amounts of cleavage agent are used to accomplish a reasona- endopeptidase Asp bly rapid digestion time (i.e., 6 to 20 hours), the amount of (Endoproteinase Asp-N) cleavage agent is minimized to avoid its contribution to the (EC 3.24.33) chromatographic map pattern. A protein to protease ratio Clostripain (EC 3.4.22.8) C-terminal side of between 20:1 and 200:1 is generally used. It is recommended Arg that the cleavage agent can be added in two or more stages to optimize cleavage. Nonetheless, the final reaction volume Chemical Cyanogen bromide C-terminal side of remains small enough to facilitate the next step in peptide Met mapping —the separation step. To sort out digestion ar- 2-Nitro-5-thio-cyanobenzoic tifacts that might be interfering with the subsequent analysis, acid N-terminal side of a blank determination is performed, using a digestion con- Cys trol with all the reagents, except the test protein. o-Iodosobenzoic acid C-terminal side of 4. Chromatographic Separation Trp and Tyr Dilute acid Asp and Pro Many techniques are used to separate peptides for mapping. The selection of a technique depends on the protein BNPS-skatole Trp being mapped. Techniques that have been successfully used for separation of peptides are shown in Table 2. In this section, a most widely used reverse-phase High Performance Liquid Chromatographic (RP-HPLC) method is described as some unfolding of the protein, it is often necessary to reduce one of the procedures of chromatographic separation. and alkylate the disulfide bonds prior to digestion. The purity of solvents and mobile phases is a critical fac- Digestion with trypsin can introduce ambiguities in the tor in HPLC separation. HPLC-grade solvents and water tryptic map due to side reactions occurring during the diges- that are commercially available are recommended for RP- tion reaction, such as nonspecific cleavage, deamidation, HPLC. Dissolved gases present a problem in gradient sys- disulfide isomerization, oxidation of methionine residues, or tems where the solubility of the gas in a solvent may be less formation of pyroglutamic groups created from the deami- in a mixture than in a single solvent. Vacuum degassing and dation of glutamine at the N-terminal side of a peptide. agitation by sonication are often used as useful degassing Furthermore, peaks may be produced by autohydrolysis of procedures. When solid particles in the solvents are drawn trypsin. Their intensities depend on the ratio of trypsin to into the HPLC system, they can damage the sealing of pump protein. To avoid autohydrolysis, solutions of proteases may valves or clog the top of the chromatographic column. Both be prepared at a pH that is not optimal (e.g., at pH 5 for pre- and post-pump filtration is also recommended. trypsin), which would mean that the enzyme would not 4.1. Chromatographic Column become active until diluted with the digest buffer. The selection of a chromatographic column is empirically 3.4. Establishment of Optimal Digestion Conditions determined for each protein. Columns with 100 Å or 300 Å Factors that affect the completeness and effectiveness of pore size with silica support can give optimal separation. For digestion of proteins are those that could affect any chemical smaller peptides, octylsilane chemically bonded to totally or enzymatic reactions. porous silica articles, 3 to 10 mm in diameter (L7) and oc- (i) pH: The pH of the digestion mixture is empirically tadecylsilane chemically bonded to porous silica or ceramic determined to ensure the optimal performance of the given micro-particles, 3 to 10 mm in diameter (L1) column pack- cleavage agent. For example, when using cyanogen bromide ings are more efficient than the butyl silane chemically bond- as a cleavage agent, a highly acidic environment (e.g., pH 2, ed to totally porous silica particles, 5 to 10 mmindiameter formic acid) is necessary; however, when using trypsin as a (L26) packing. cleavage agent, a slightly alkaline environment (pH 8) is op- 4.2. Solvent timal. As a general rule, the pH of the reaction milieu should The most commonly used solvent is water with acetonitrile not alter the chemical integrity of the protein during the as the organic modifier to which less than 0.1z trifluoroa- digestion and should not change during the course of the JP XVI General Information / Biotechnological/Biological Products 2193

Table 2 Techniques Used for the Separation of Peptides parallel with the protein under test is critical in the development and establishment of system suitability limits. In addi- Reverse-Phase High Performance Liquid Chromatography tion a specimen chromatogram should be included with the (RP-HPLC) reference standard or reference material for additional com- Ion-Exchange Chromatography (IEC) parison purposes. Other indicators may include visual in- Hydrophobic Interaction Chromatography (HIC) spection of protein or peptide solubility, the absence of in- Polyacrylamide Gel Electrophoresis (PAGE), nondenaturat- tact protein, or measurement of responses of a digestion- ing dependent peptide. The critical system suitability parameters SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) for peptide analysis will depend on the particular mode of Capillary Electrophoresis (CE) peptide separation and detection and on the data analysis Paper Chromatography-High Voltage (PCHV) requirements. High-Voltage Paper Electrophoresis (HVPE) When peptide mapping is used as an identification test, the system suitability requirements for the identified peptides covers selectivity and precision. In this case, as well as when identification of variant protein is done, the identification of cetic acid is added. If necessary, add isopropyl alcohol or n- the primary structure of the peptide fragments in the peptide propyl alcohol to solubilize the digest components, provided map provides both a verification of the known primary that the addition does not unduly increase the viscosity of structure and the identification of protein variants by com- the components. parison with the peptide map of the reference standard/ 4.3. Mobile Phase reference material for the specified protein. The use of a Buffered mobile phases containing phosphate are used to digested reference standard or reference material for a given provide some flexibility in the selection of pH conditions, protein in the determination of peptide resolution is the since shifts of pH in the 3.0 to 5.0 range enhance the separa- method of choice. For an analysis of a variant protein, a tion of peptides containing acidic residues (e.g., glutamic characterized mixture of a variant and a reference standard and aspartic acids). Sodium or potassium phosphates, am- or reference material can be used, especially if the variant monium acetate, phosphoric acid, and a pH between 2 and 7 peptide is located in a less-resolved region of the map. The (or higher for polymer-based supports) have also been used index of pattern consistency can be simply the number of with acetonitrile gradients. Acetonitrile containing major peptides detected. Peptide pattern consistency can be trifluoroacetic acid is used quite often. best defined by the resolution of peptide peaks. Chromato- 4.4. Gradient Selection graphic parameters —such as peak-to-peak resolution, maxi- Gradients can be linear, nonlinear, or include step func- mum peak width, peak area, peak tailing factors, and tions. A shallow gradient is recommended in order to sepa- column efficiency— may be used to define peptide resolu- rate complex mixtures. Gradients are optimized to provide tion. Depending on the protein under test and the method of clear resolution of one or two peaks that will become ``mar- separation used, single peptide or multiple peptide resolution ker'' peaks for the test. requirements may be necessary. 4.5. Isocratic Selection The replicate analysis of the digest of the reference stand- Isocratic HPLC systems using a single mobile phase are ard or reference material for the protein under test yields used on the basis of their convenience of use and improved measures of precision and quantitative recovery. Recovery detector responses. Optimal composition of a mobile phase of the identified peptides is generally ascertained by the use to obtain clear resolution of each peak is sometimes difficult of internal or external peptide standards. The precision is ex- to establish. Mobile phases for which slight changes in compressed as the relative standard deviation (RSD). Differences ponent ratios or in pH significantly affect retention times of in the recovery and precision of the identified peptides are peaks in peptide maps should not be used in isocratic HPLC expected; therefore, the system suitability limits will have to systems. be established for both the recovery and the precision of the 4.6. Other Parameters identified peptides. These limits are unique for a given pro- Temperature control of the column is usually necessary to tein and will be specified in the individual monograph. achieve good reproducibility. The flow rates for the mobile Visual comparison of the relative retention times, the peak phases range from 0.1 to 2.0 mL per minute, and the detec- responses (the peak area or the peak height), the number of tion of peptides is performed with a UV detector at 200 to peaks, and the overall elution pattern is completed initially. 230 nm. Other methods of detection have been used (e.g., It is then complemented and supported by mathematical postcolumn derivatization), but they are not as robust or analysis of the peak response ratios and by the chromato- versatile as UV detection. graphic profile of a 1:1 (v/v) mixture of sample and refer- 4.7 Validation ence standard or reference material digest. If all peaks in the This section provides an experimental means for measur- sample digest and in the reference standard or reference ma- ing the overall performance of the test method. The accep- terial digest have the same relative retention times and peaks tance criteria for system suitability depend on the identifica- response ratios, then the identity of the sample under test is tion of critical test parameters that affect data interpretation confirmed. and acceptance. These critical parameters are also criteria If peaks that initially eluted with significantly different that monitor peptide digestion and peptide analysis. An indi- relative retention times are then observed as single peaks in cator that the desired digestion endpoint was achieved is by the 1:1 mixture, the initial difference would be an indication the comparison with a reference standard or reference of system variability. However, if separate peaks are ob- material, which is treated exactly as the article under test. served in the 1:1 mixture, this would be evidence of the The use of a reference standard or reference material in nonequivalence of the peptides in each peak. If a peak in the 2194 Biotechnological/Biological Products / General Information JP XVI

1:1 mixture is significantly broader than the corresponding andMALDI-TOFMScanalsobeusedforcharacterization peak in the sample and reference standard or reference mate- purposes. rial digest, it may indicate the presence of different peptides. The use of MS for characterization of peptide fragments is The use of computer-aided pattern recognition software for by direct infusion of isolated peptides or by the use of on- the analysis of peptide mapping data has been proposed and line LC-MS for structure analysis. In general, it includes applied, but issues related to the validation of the computer electrospray and MALDI-TOF MS analyzer as well as fast software preclude its use in a compendial test in the near atom bombardment (FAB). Tandem MS has also been used future. Other automated approaches have been used that to sequence a modified protein and to determine the type of employ mathematical formulas, models, and pattern recog- amino acid modification that has occurred. The comparison nition. Such approaches are, for example, the automated of mass spectra of the digests before and after reduction pro- identification of compounds by IR spectroscopy and the vides a method to assign the disulfide bonds to the various application of diode-array UV spectral analysis for identifi- sulfhydryl-containing peptides. cation of peptides. These methods have limitations due to If regions of the primary structure are not clearly demon- inadequate resolutions, co-elution of fragments, or absolute strated by the peptide map, it might be necessary to develop peak response differences between reference standard or a secondary peptide map. The goal of a validated method of reference material and sample fragments. characterization of a protein through peptide mapping is to The numerical comparison of the retention times and peak reconcile and account for at least 95z of the theoretical areas or peak heights can be done for a selected group of composition of the protein structure. relevant peaks that have been correctly identified in the peptide maps. Peak areas can be calculated using one peak showing relatively small variation as an internal reference, keeping in mind that peak area integration is sensitive to Qualification of Animals as Origin baseline variation and likely to introduce error in the analy- of Animal-derived Medicinal sis. Alternatively, the percentage of each peptide peak height relative to the sum of all peak heights can be calculated for Products provided in the General the sample under test. The percentage is then compared to Notices of Japanese Pharmacopoeia that of the corresponding peak of the reference standard/ reference material. The possibility of auto-hydrolysis of and Other Standards trypsin is monitored by producing a blank peptide map, that is, the peptide map obtained when a blank solution is treated Introduction with trypsin. The Official Gazette issued on March 29, 2002 announced The minimum requirement for the qualification of peptide that General Notices of the Japanese Pharmacopoeia and mapping is an approved test procedure that includes system other standards were amended to add a provision that suitability as a test control. In general, early in the regulatory ``When a drug product or a drug substance which is used to process, qualification of peptide mapping for a protein is manufacture a drug product, is manufactured from a raw sufficient. As the regulatory approval process for the protein material of animal origin, the animal in question should be progresses, additional qualifications of the test can include a in principle a healthy subject, if not otherwise provided.''. partial validation of the analytical procedure to provide The Notice Iyaku-hatsu No. 0329001, which was issued on assurance that the method will perform as intended in the the same date, provided that ``Healthy subject herein provid- development of a peptide map for the specified protein. ed is the animal which does not cause any disease or any infection to human being at an appropriate production process 5. Analysis and Identification of Peptides and use of the drug product, and as for the oral or external This section gives guidance on the use of peptide mapping drug for example, the animal, as its raw material of animal during development in support of regulatory applications. origin, should be confirmed at this stage to meet the Food The use of a peptide map as a qualitative tool does not re- Standard. It has to be noted that this standard of healthy quire the complete characterization of the individual peptide subject has to be revised timely taking into account the up- peaks. However, validation of peptide mapping in support to-date information with respect to the amphixenosis infec- of regulatory applications requires rigorous characterization tions common between human beings and animals.''. of each of the individual peaks in the peptide map. Methods This General Information describes safety assurance to characterize peaks range from N-terminal sequencing of against infection associated with the use of drugs, which are each peak followed by amino acid analysis to the use of mass manufactured from raw materials of animal origin, to follow spectroscopy (MS). up the Notice as mentioned above. For characterization purposes, when N-terminal sequencing and amino acids analysis are used, the analytical separa- 1. Basic concept tion is scaled up. Since scale-up might affect the resolution When drugs derived from raw materials of animal origin of peptide peaks, it is necessary, using empirical data, to including human are used, it is important to take into ac- assure that there is no loss of resolution due to scale-up. count any possibility that communicable disease agents such Eluates corresponding to specific peptide peaks are collect- as virus may cause infectious disease or any possible hazards ed, vacuum-concentrated, and chromatographed again, if to patients. In such case, it goes without saying that the pri- necessary. Amino acid analysis of fragments may be limited mary subject that has to be considered is the absence of any by the peptide size. If the N-terminus is blocked, it may infectious agents such as virus in the raw materials of animal need to be cleared before sequencing. C-terminal sequencing origin including human as the source of the drug. More im- of proteins in combination with carboxypeptidase digestion portant points are whether the drugs derived from such raw JP XVI General Information / Biotechnological/Biological Products 2195 materials are free of such infectious agents and whether er'', ``hepatic disease'' and ``dementia (transmissible spon- there is any possibility of transmission of infectious agents giform encephalopathies and its suspicious case)'' should be when the drugs are administered to patient. The eligibility of checked on the case history or by the interview, etc. and the animals including human, as the source of raw materials of experience of being transfused or/and transplanted should drugs, in other words ``the subject which is free from any be checked to confirm eligibility as a donor. The most ap- disease or transmission of infectious agents that is infectious propriate check items and test methods then available are to to human being at an appropriate production process and be used, which need to be reconsidered at appropriate timing use of the drug product'' is that ``The drug should be taking into account the updated knowledge and the progress entirely free from any risk of infections by means of whole of the science and the technologies. At screening of a donor, procedures which include evaluation of appropriateness of reexaminations has to be made at appropriate timing using the animals including human as the source of their raw mate- the eligible check items and the test methods taking into rials, establishment of appropriate production processes and account the window period (Initial period after infection, their appropriate control, and strict adherence to the clinical in which antibody against bacteria, fungi or virus is not indications of the final product.'' detected.) In the case of plasma derivatives produced from the do- 2. Animals including human as the source of raw materials nated blood in Japan, the donor should be checked by means of drugs of self-assessed report about health conditions, and a sero- What is the most clear and appropriate preventive meas- logic check and a nucleic acid amplification test (NAT) on ures against infection to human being due to administration mini pooled plasma should be performed at the stage of do- of drugs which are derived from animals including human is nated blood. Further, the plasma material (i.e., critical raw to assure the absence of any infectious agents such as virus in material ) for fractionation should be stored 4 months in its raw materials or an appropriate critical raw material by minimum so that the arrangement could be taken based on each of the followings: (1) the use of raw materials of the information available after collection of the blood and healthy animal origin, which are proved to be free from the blood infusion to exclude the possibility of using any communicable disease agents to human, or (2) the use of ap- critical raw material which might cause infection to patients. propriate critical raw materials (e.g., cell substrate, blood On the other hand, as for the materials such as urine plasma, pooled urine after some treatments) for drug which are taken from the unspecified number of the donors production, which are proved to be free from communicable and come to be critical raw materials for drug production disease agents after certain appropriate processing on raw after some treatments, it is unrealistic and not practical to materials of animal origin. conduct the tests of virus infection, etc. on the individual As for raw materials of drugs of human origin, cell, tissue, donor. Consequently, appropriate tests such as virus test has blood, placenta, urine, etc. are used. Whenever it is to be performed on such critical raw materials for drug sufficient and possible each donor, as the origin of such raw production. materials, should be asked his (her) health condition and In the case of the animals besides human, the wild ones undergoes his (her) medical examination at this stage, so that should be excluded. Only the animals, which are raised the appropriateness as a donor can be confirmed from the under well sanitarily controlled conditions taken to prevent standpoint of safety concerning communicable disease bacterial contamination or under the effective bacterial pol- agents such as virus. lution monitoring systems, have to be used, and it is recom- For example, ``Basic concept on handling and use of a mended that the animals from a colony appropriately con- drug product, etc. which is derived from cell/tissue'' trolled under specific pathogen-free (SPF) environment are (Attachment 1 of the Notice Iyaku-Hatsu No. 1314 dated to be used as far as possible. Further, for the animals regu- December 26, 2000) issued by the Director-General of the lated under the Food Standard, only the animals that met Medicinal Safety Bureau, Ministry of Health and Welfare, this standard should be used. It should be confirmed by ap- states that since the cell/tissue supplied by a human donor propriate tests that the animals were free from pathogen, if comes to be applied to patients without processing through necessary. any sufficient inactivation or removal of communicable dis- The concrete measures to avoid transmittance or spread of ease agents, the selection and qualification criteria on such infectivity of prion, which is considered to be the pathogen donor has to be established. These criteria are to be com- of transmissible spongiform encephalopathies (TSEs), as far posed with the respect to the check items on the case history as possible are the followings:  avoidance of use of and the physical conditions as well as the test items on the animals, which are raised in the areas where high incidence various transmission of infectious agents through cell/tissue, or high risk of TSEs (Scrapie in sheep and goat, bovine and that the appropriateness of these criteria has to be clari- spongiform encephalopathies (BSE) in cattle, chronic wast- fied. Hepatitis Type-B (HBV), Hepatitis Type-C (HCV), ing disease (CWD) in deer, new type of Creutzfeldt-Jacob- Human Immune Deficiency Viral infections (HIV), Adult Disease (CJD) in human, etc.) is reported, and humans, who T-Cell Leukemia and Parvovirus B19 Infections should be have stayed long time (more than 6 months) in such areas, as denied through the interview to the donor and the tests raw materials or related substances of drugs;  avoidance of (serologic test, nucleic-acid amplification test, etc.). Further, use of any substances that are derived from the individual in- if necessary, Cytomegalovirus infection and EB Virus infec- fected with scrapie, BSE, CJD, etc.;  avoidance of using a tion should be denied by tests. ``Infections caused by bacter- material derived from organ, tissue and cell, etc. of high risk ia such as Treponema pallidum, Chlamydia, Gonococci, of TSEs; and  taking appropriate measures basing on the Tubercule bacillus, etc.'', ``septicemia and its suspicious information collected, which includes incidence of TSEs, the case'', ``vicious tumor'', ``serious metabolic or endocrine- results of epidemiological investigation and the experimental related disorders'', ``collagenosis and haematological disord- research on prion, and incidence of tardive infection on 2196 Biotechnological/Biological Products / General Information JP XVI donors after collecting raw materials, etc. the production of the drugs are known to have endogenous retrovirus-like particles sometimes. The reason why such 3. Human or animal cells which are used as critical raw cells can be used for the production of the drugs is that mul- materials for drug production tiple measures are applied for safety in the purification Cell substrates derived from humans or animals are used stages which include appropriate inactivation or removal for drug production. In such case, it is desirable that the processes. There are cases in which the production procedure humans or the animals, which are the origins of the cell sub- involves intentional use of a virus or a microorganism. In strates, are healthy subjects. However, it is considered prac- this case, relevant measures capable of removing or inac- tical that viral safety of the drugs derived from the cell sub- tivating of such virus or microorganism are appropriately strates are evaluated on the cells, which are so called critical incorporated in the purification process, so that the risk of raw materials for production of such drugs. In such case, the infection to human can be fully denied and its safety can be safety should be confirmed through the test and analysis on assured when it is used as a drug. Further, even in the case established cell bank thoroughly with respect to virus etc., as that it is difficult to clarify the risk of contamination of the far as possible. The items and the methods of the tests that infectious agents or that the raw material are contaminated have been followed in this case are described in detail in the by viruses etc., the raw material in question may be used for Notice of Japanese version on the internationally accepted the production of drugs so long as appropriate inactivation ICH Guideline entitled ``Viral safety evaluation of or removal processes are introduced, their effectiveness can biotechnology products derived from cell lines of human or be confirmed and the safety can be assured by appropriate animal origin'' (Iyakushin No. 329 issued on February 22, control of the manufacturing processes under GMP, etc. 2000 by Director, Evaluation and Licensing Division, Phar- maceutical and Medical Safety Bureau, Ministry of Health 5. Conclusion and Welfare). In the meantime, it is important how to han- The qualification of animals including human, as the dle the cell in case that any virus has been detected under the source of raw materials of drugs, in other words ``the subject cell level tests. This Notice describes how to cope with this which does not cause any infectious diseases to human being situation as follows: ``It is recognised that some cell lines at an appropriate production process and use of the drug used for the manufacture of product will contain en- product'' is that ``the drug has to be entirely free from any dogenous retroviruses, other viruses or viral sequences. In risk of infections by means of whole procedures which in- such circumstances, the action plan recommended for manu- clude evaluation of appropriateness of the animal including facturer is described in Section V (Rationale and action plan human as the source of their raw materials, establishment of for viral clearance studies and virus tests on purified bulk) of appropriate production processes and their appropriate the Notice. The acceptability of cell lines containing viruses control, and strict adherence to the clinical indication of the other than endogenous retroviruses will be considered on an final product.'' individual basis by the regulatory authorities, by taking into To cope with this subject, the advanced scientific meas- account a risk/benefit analysis based on the benefit of the ures, which actually reflect the updated knowledge and product and its intended clinical use, the nature of the con- progress of the science and the technology about infectious taminating viruses, their potential for infecting humans or diseases in human and infection of animal origin, have to be for causing disease in humans, the purification process for taken into account timely. the product (e.g., viral clearance evaluation data), and the extent of the virus tests conducted on the purified bulk.'' For example, it is well known that Type A-, R- and C- endogenous particles like retrovirus are observed in the cells SDS-Polyacrylamide Gel of the rodents used most often for drug production. It is also Electrophoresis known that they are not infectious to human and is not dan- gerous, and CHO cells are generally used for drug produc- This test is harmonized with the European Pharmacopoeia tion. The established cell lines (e.g., NAMALWA Cell, and the U.S. Pharmacopeia. BALL-1 Cell, etc.) derived from cancer patients are some- The SDS-Polyacrylamide Gel Electrophoresis is used for times used, but through the thorough virus tests, etc., their the characterization of proteins in biotechnological and biosafety are confirmed. The established cell lines are assumed logical products and for control of purity and quantitative to be safer than the primary cultured cells which are hard to determinations. conduct the thorough virus test. This technique is a suitable analytical method with which 4. Establishment and control of appropriate production to identify and to assess the homogeneity of proteins in process and adherence to the clinical indication of final biotechnological and biological products. The method is also product for safety assurance routinely used for the estimation of protein subunit molecu- Safety assurance against potential infections at only the lar masses and for determining the subunit compositions of level of animals that are source of raw materials of drugs is purified proteins. limited. Further, ``health of animal'' can not be defined Ready-to-use gels and reagents are widely available on the univocally, and the various factors have to be taken into market and can be used instead of those described in this account. The final goal of this subject is to protect human text, provided that they give equivalent results and that they from any infectious disease caused by drugs. Achieving this meet the validity requirements given below under Validation goal, the establishment and control of appropriate produc- of the Test. tion processes of each drug and the adherence to the clinical 1. Characteristics of Polyacrylamide Gels indications of the final product are important. The sieving properties of polyacrylamide gels are afforded As mentioned above, the rodent cells used most often for JP XVI General Information / Biotechnological/Biological Products 2197 by the three-dimensional network of fibers and pores which be estimated from its relative mobility calibrated in SDS- is formed as the bifunctional bisacrylamide cross-links Polyacrylamide Gel Electrophoresis and the occurrence of a adjacent polyacrylamide chains. Polymerization is catalyzed single band in such a gel is a criterion of purity. by a free radical-generating system composed of ammonium However, modifications to the polypeptide backbone, persulfate and N,N,N?,N?-tetramethylethylenediamine such as N- or O-1inked glycosylation, have a significant im- (TEMED). pact on the apparent molecular mass of a protein, since SDS As the acrylamide concentration of a gel increases, its does not bind to a carbohydrate moiety in a manner similar effective pore size decreases. The effective pore size of a gel to a polypeptide. Thus, a consistent charge-to-mass ratio is is operationally defined by its sieving properties; that is, by not maintained. The apparent molecular masses of proteins the resistance it imparts to the migration of macromolecules. that have undergone post-translational modifications do not There are limits on the acrylamide concentration that can be truly reflect the masses of the polypeptides. used. At high acrylamide concentrations, gels break much 2.1. Reducing conditions more easily and are difficult to handle. As the pore size of a Polypeptide subunits and three-dimensional structure of gel decreases, the migration rate of a protein through the gel proteins are often fixed, at least in part, by the presence of decreases. By adjusting the pore size of a gel, through disulfide bonds. A goal of SDS-Polyacrylamide Gel Elec- manipulating the acrylamide concentration, the resolution of trophoresis under reducing conditions is to disrupt this struc- the method can be optimized for a given protein product. ture by reducing the disulfide bonds. Complete denaturation Thus, the physical characteristics of a given gel are deter- and dissociation of proteins by treatment with 2-mercap- mined by the relative concentrations of acrylamide and toethanol or dithiothreitol (DTT) will result in unfolding of bisacrylamide, used in its preparation. the polypeptide backbone and subsequent complexation with In addition to the composition of the gel, the state of the SDS. Under these conditions, the molecular masses of the protein is an important determinant of the electrophoretic polypeptide subunits can be calculated by linear regression in mobility. In the case of proteins, the electrophoretic mobility the presence of suitable molecular-mass standards. is dependent on the pK values of the charged groups and the 2.2. Non-reducing conditions size of the molecule. It is also influenced by the type, con- For some analyses, complete dissociation of the protein of centration and pH of the buffer, the temperature and the interest into subunit peptides is not desirable. In the absence field strength, as well as by the nature of the support mate- of treatment with reducing agents such as 2-mercaptoethanol rial. or DTT, disulfide covalent bonds remain intact, preserving the oligomeric form of the protein. Oligomeric SDS-protein 2. Polyacrylamide Gel Electrophoresis under Denaturing complexes migrate more slowly than their SDS-polypeptide Conditions subunits. In addition, non-reduced proteins may not be com- Themethodcitedasanexampleislimitedtotheanalysis pletely saturated with SDS and, hence, may not bind the of monomeric polypeptides with a mass range of 14,000 to detergent in the expected mass ratio. This makes molecular- 100,000 daltons. It is possible to extend this mass range by mass determinations of these molecules by SDS- various techniques (e.g., by using gradient gels, particular Polyacrylamide Gel Electrophoresis less straightforward buffer systems, etc.), but those techniques are not discussed than analyses of fully denatured polypeptides, since it is in this chapter. necessary that both standards and unknown proteins be in Analysis by electrophoresis on sodium dodecyl sulfate similar configurations for valid comparisons. However, the (SDS) polyacrylamide gel (SDS-Polyacrylamide Gel Elec- staining of a single band in such a gel is a criterion of purity. trophoresis) under denaturing conditions is the most common mode of electrophoresis used in assessing the quality of 3. Characteristics of Discontinuous Buffer System Gel proteins in biotechnological and biological products, and Electrophoresis will be the focus of the example described here. Typically, The most widely used electrophoretic method for the analytical electrophoresis of proteins is carried out in poly- analysis of complex mixtures of proteins involves the use of acrylamide gels under conditions that ensure dissociation of a discontinuous buffer system consisting of two contiguous, the proteins into their individual polypeptide subunits and but distinct gels: a resolving (lower) gel and a stacking that minimize aggregation. Most commonly, the strongly (upper) gel. The two gels are cast with different porosities, anionic detergent SDS is used in combination with heat to pH, and ionic strengths. In addition, different mobile ions dissociate the proteins before they are loaded on the gel. The are used in the gel and electrode buffers. The buffer discon- denatured polypeptides bindtoSDS,becomenegatively tinuity acts to concentrate large-volume samples in the stack- charged and exhibit a consistent charge-to-mass ratio regarding gel, resulting in improved resolution. When power is ap- less of protein type. Because the amount of SDS bound is plied, a voltage drop develops across the sample solution almost always proportional to the molecular mass of the which drives the proteins into the stacking gel. Glycinate polypeptide and is independent of its amino acid sequence, ions from the electrode buffer follow the proteins into the SDS-polypeptide complexes migrate through polyacrylamide stacking gel. A moving boundary region is rapidly formed gels with mobilities that are dependent on the size of the with the highly mobile chloride ions in the front and the rela- polypeptides. tively slow glycinate ions in the rear. A localized high-vol- The electrophoretic mobilities of the resultant SDS- tage gradient is formed between the leading and trailing ion polypeptide complexes all assume the same functional fronts, causing the SDS-protein complex to form into a very relationship to their molecular masses. Migration of SDS- thin zone (called the stack) andtomigratebetweenthechlo- complexes occurs toward the anode in a predictable manner, ride and glycinate phases. Regardless of the height of the ap- with low-molecular-mass complexes migrating faster than plied sample solution in the wells, all SDS-protein complexes larger ones. The molecular mass of a protein can therefore condense within a very narrow range and enter the resolving 2198 Biotechnological/Biological Products / General Information JP XVI gel as a well-defined, thin zone of high protein density. The tion at room temperature to allow polymerization to occur. large-pore stacking gel does not retard the migration of most 4.2.2. Preparation of the stacking gel proteins and serves mainly as an anticonvective medium. At After polymerization is complete (about 30 minutes), pour the interface of the stacking and resolving gels, the proteins off the isobutanol and wash the top of the gel several times experience a sharp increase in retardation due to the smaller with water to remove the isobutanol overlay and any un- pore size of the resolving gel. Once the proteins are in the polymerized acrylamide. Drain as much fluid as possible resolving gel, their mobility continues to be slowed down by from the top of the gel, and then remove any remaining the molecular sieving effect of the matrix. The glycinate water with the edge of a paper towel. ions overtake the proteins, which then move in a space In a conical flask, prepare the appropriate volume of solu- of uniform pH formed by the tris(hydroxy- tion containing the desired concentration of acrylamide, methyl)aminomethane and glycine. Molecular sieving causes using the values given in Table 2. Mix the components in the the SDS-polypeptide complexes to separate on the basis of order shown. Where appropriate, before adding the ammo- their molecular masses. nium persulfate solution and the TEMED, filter the solution if necessary under vacuum through a cellulose acetate mem- 4. Preparing Vertical Discontinuous Buffer SDS- brane (pore size: 0.45 mm); keep the solution under vacuum Polyacrylamide Gels by swirling the filtration unit until no more bubbles are 4.1. Assembling of the gel moulding cassette formed in the solution. Add appropriate amounts of ammo- Clean the two glass plates (size: e.g. 10 cm × 8cm),the nium persulfate solution and TEMED as indicated in Table sample comb made of polytetrafluoroethylene, the two 2, swirl and pour immediately into the gap between the two spacers and the silicone rubber tubing (diameter, e.g. 0.6 glass plates of the mould directly onto the surface of the mm × 35 cm) with mild detergent and rinse extensively with polymerized resolving gel. Immediately insert a clean sample water. Dry all the items with a paper towel or tissue. Lubri- comb into the stacking gel solution, taking care to avoid cate the spacers and the silicone rubber tubing with non- trapping air bubbles. Add more stacking gel solution to fill silicone grease. Apply the spacers along each of the two completely the spaces of the sample comb. Leave the gel in a short sides of the glass plate 2 mm away from the edges and vertical position and allow to polymerize at room tempera- 2 mm away from the long side corresponding to the bottom ture. of the gel. Begin to lay the silicone rubber tubing on the glass 4.3. Mounting the gel in the electrophoresis apparatus and plate by using one spacer as a guide. Carefully twist the sili- electrophoretic separation cone rubber tubing at the bottom of the spacer and follow After polymerization is complete (about 30 minutes), re- the long side of the glass plate. While holding the silicone move the sample comb carefully. Rinse the wells immedi- rubber tubing with one finger along the long side again twist ately with water or with the running buffer for SDS- the tubing and lay it on the second short side of the glass Polyacrylamide Gel Electrophoresis to remove any un- plate, using the spacer as a guide. Place the second glass polymerized acrylamide. If necessary, straighten the teeth of plate in perfect alignment and hold the mould together by the sample comb of the stacking gel with a blunt hypodermic hand pressure. Apply two clamps on each of the two short needle attached to a syringe. Remove the clamps on one sides of the mould. Carefully apply four clamps on the lon- short side, carefully pull out the silicone rubber tubing and ger side of the gel mould, thus forming the bottom of the gel replace the clamps. Proceed similarly on the other short side. mould. Verify that the silicone rubber tubing is running Remove the silicone rubber tubing from the bottom part of along the edge of the glass plates and has not been extruded the gel. Mount the gel in the electrophoresis apparatus. Add whileplacingtheclamps. the electrophoresis buffers to the top and bottom reservoirs. 4.2. Preparation of the gel Remove any bubbles that become trapped at the bottom of In a discontinuous buffer SDS polyacrylamide gel, it is the gel between the glass plates. This is best done with a bent recommended to pour the resolving gel, let the gel set, and hypodermic needle attached to a syringe. Never pre-run the then pour the stacking gel, since the compositions of the two gel before loading solutions, such as samples, since this will gels in acrylamide-bisacrylamide, buffer and pH are differ- destroy the discontinuity of the buffer systems. Before load- ent. ing solutions, such as samples, carefully rinse the stacking 4.2.1. Preparation of the resolving gel gel wells with the running buffer for SDS-Polyacrylamide In a conical flask, prepare the appropriate volume of solu- Gel Electrophoresis. Prepare the test and reference solutions tion containing the desired concentration of acrylamide for in the recommended sample buffer and treat as specified in the resolving gel, using the values given in Table 1. Mix the the individual monograph. Apply the appropriate volume of components in the order shown. Where appropriate, before each solution to the stacking gel wells. Start the electropho- adding the ammonium persulfate solution and the resis using suitable operating conditions for the electropho- tetramethylethylenediamine (TEMED), filter the solution if resis equipment to be used. There are commercially available necessary under vacuum through a cellulose acetate mem- gels of different surface area and thickness that are ap- brane (pore size: 0.45 mm); keep the solution under vacuum propriate for various types of electrophoresis equipment. by swirling the filtration unit until no more bubbles are Electrophoresis running time and current/voltage may need formed in the solution. Add appropriate amounts of ammo- to be altered depending on the type of apparatus used, in nium persulfate solution and TEMED as indicated in Table order to achieve optimum separation. Check that the dye 1, swirl and pour immediately into the gap between the two front is moving into the resolving gel. When the dye is reach- glass plates of the mould. Leave sufficient space for the ing the bottom of the gel, stop the electrophoresis. Remove stacking gel (the length of the teeth of the sample comb plus the gel assembly from the apparatus and separate the glass 1 cm). Using a pipette, carefully overlay the solution with plates. Remove the spacers, cut off and discard the stacking water-saturated isobutanol. Leave the gel in a vertical posi- gel and immediately proceed with staining. JP XVI General Information / Biotechnological/Biological Products 2199

5. Detection of Proteins in Gels commercially available for calibrating gels. They are ob- Coomassie staining is the most common protein staining tainable in various molecular mass ranges. Concentrated method, with a detection level of the order of 1 mgto10mg stock solutions of proteins of known molecular mass are of protein per band. Silver staining is the most sensitive diluted in the appropriate sample buffer and loaded on the method for staining proteins in gels and a band containing l0 same gel as the protein sample to be studied. ng to 100 ng can be detected. All of the steps in gel staining Immediately after the gel has been run, the position of the are done at room temperature with gentle shaking in any bromophenol blue tracking dye is marked to identify the convenient container. Gloves must be worn when staining leading edge of the electrophoretic ion front. This can be gels, since fingerprints will stain. done by cutting notches in the edges of the gel or by inserting 5.1. Coomassie staining a needle soaked in India ink into the gel at the dye front. Immerse the gel in a large excess of Coomassie staining TS After staining, measure the migration distances of each pro- and allow to stand for at least 1 hour. Remove the staining tein band (markers and unknowns) from the top of the solution. resolving gel. Divide the migration distance of each protein Destain the gel with a large excess of destaining TS. by the distance traveled by the tracking dye. The normalized Change the destaining solution several times, until the migration distances so obtained are called the relative mobil- stained protein bands are clearly distinguishable on a clear ities of the proteins (relative to the dye front) and conven- background. The more thoroughly the gel is destained, the tionally denoted as Rf. Construct a plot of the logarithm of smaller is the amount of protein that can be detected by the therelativemolecularmasses(Mr) of the protein standards method. Destaining can be speeded up by including 2 to 3 g as a function of the Rf values. Note that the graphs are of anion-exchange resin or a small sponge in the destaining slightly sigmoid. Unknown molecular masses can be estimat- TS. ed by linear regression analysis or interpolation from the

NOTE: the acid-alcohol solutions used in this procedure curves of log Mr against Rf as long as the values obtained for do not completely fix proteins in the gel. This can lead to the unknown samples are positioned along the linear part of losses of some low-molecular-mass proteins during the stain- the graph. ing and destaining of the gel. Permanent fixation is ob- 8. Suitability of the Test (Validation) tainable by allowing the gel to stand in trichloroacetic acid The test is not valid unless the proteins of the molecular TS for fixing for 1 hour before it is immersed in Coomassie mass marker are distributed along 80z of the length of the staining TS. gel and over the required separation range (e.g., the range 5.2. Silver staining covering the product and its dimer or the product and its Immerse the gel in a large excess of fixing TS and allow to related impurities) the separation obtained for the relevant stand for 1 hour. Remove the fixing solution, add fresh fix- protein bands shows a linear relationship between the ing solution and incubate either for at least 1 hour or over- logarithmofthemolecularmassandtheRf as described in night, if convenient. Discard the fixing solution and wash 7. Additional requirements with respect to the solution under the gel in water for 1 hour. Soak the gel for 15 minutes in a 1 test may be specified in individual monographs. volz glutaraldehyde solution. Wash the gel twice for 15 minutes in water. Soak the gel in fresh silver nitrate TS for 9. Quantification of Impurities silver staining for 15 minutes, in darkness. Wash the gel Where the impurity limit is specified in the individual three times for 5 minutes in water. Immerse the gel for about monograph, a reference solution corresponding to that level 1 minute in developer TS until satisfactory staining has been of impurity should be prepared by diluting the test solution. obtained. Stop the development by incubation in blocking For example, where the limit is 5z, a reference solution TS for 15 minutes. Rinse the gel with water. would be a 1:20 dilution of the test solution. No impurity (any band other than the main band) in the electrophoreto- 6. Drying of Stained SDS-Polyacrylamide Gels gram obtained with the test solution may be more intense Depending on the staining method used, gels are pretreat- than the main band obtained with the reference solution. ed in a slightly different way. For Coomassie staining, after Under validated conditions, impurities may be quantified the destaining step, allow the gel to stand in a diluted solu- by normalization to the main band, using an integrating den- tion of concentrated glycerin (1 in 10) for at least 2 hours sitometer. In this case, the responses must be validated for (overnight incubation is possible). For silver staining, after linearity. the final rinse, allow the gel to stand in a diluted solution of concentrated glycerin (1 in 50) for 5 minutes. 10. Test solutions: Immerse two sheets of porous cellulose film in water and (i) Coomassie staining TS: Dissolve 125 mg of coomassie incubate for 5 to 10 minutes. Place one of the sheets on a brilliant blue R-250 in 100 mL of a mixture of water, metha- drying frame. Carefully lift the gel and place it on the cellu- nol and acetic acid (100) (5:4:1), and filter. lose film. Remove any trapped air bubbles and pour 2 to 3 (ii) Developer TS: Dissolve 2 g of citric acid monohydrate mL of water around the edges of the gel. Place the second in water to make 100 mL. To 2.5 mL of this solution add sheet on top and remove any trapped air bubbles. Complete 0.27 mL of formaldehyde solution and water to make 500 the assembly of the drying frame. Place in an oven or leave mL. at room temperature until dry. (iii) Fixing TS: To 250 mL of methanol add 0.27 mL of formaldehyde solution and water to make 500 mL. 7. Molecular-Mass Determination (iv) Silver nitrate TS for silver staining: To 40 mL of so- Molecular masses of proteins are determined by compari- dium hydroxide TS add 3 mL of ammonia solution (28), son of their mobilities with those of several marker proteins then add dropwise 8 mL of a solution of silver nitrate (1 in 5) of known molecular mass. Mixtures of proteins with precise- while stirring, and add water to make 200 mL. ly known molecular masses blended for uniform staining are 2200 Biotechnological/Biological Products / General Information JP XVI

Table 1 Preparation of resolving gel

Component volumes (mL) per gel mould volume of Solution components 5mL10mL15mL20mL25mL30mL40mL50mL

6z Acrylamide Water 2.6 5.3 7.9 10.6 13.2 15.9 21.2 26.5 Acrylamide solution(1) 1.0 2.0 3.0 4.0 5.0 6.0 8.0 10.0 1.5 mol/L Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5 100 g/L SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 g/L APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED(5) 0.004 0.008 0.012 0.016 0.02 0.024 0.032 0.04 8z Acrylamide Water 2.3 4.6 6.9 9.3 11.5 13.9 18.5 23.2 Acrylamide solution(1) 1.3 2.7 4.0 5.3 6.7 8.0 10.7 13.3 1.5 mol/L Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5 100 g/L SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 gL APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED(5) 0.003 0.006 0.009 0.012 0.015 0.018 0.024 0.03 10z Acrylamide Water 1.9 4.0 5.9 7.9 9.9 11.9 15.9 19.8 Acrylamide solution(1) 1.7 3.3 5.0 6.7 8.3 10.0 13.3 16.7 1.5 mol/L Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5 100 g/L SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 g/L APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED(5) 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02 12z Acrylamide Water 1.6 3.3 4.9 6.6 8.2 9.9 13.2 16.5 Acrylamide solution(1) 2.0 4.0 6.0 8.0 10.0 12.0 16.0 20.0 1.5 mol/L Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5 100 g/L SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 g/L APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED(5) 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02 14z Acrylamide Water 1.4 2.7 3.9 5.3 6.6 8.0 10.6 13.8 Acrylamide solution(1) 2.3 4.6 7.0 9.3 11.6 13.9 18.6 23.2 1.5 mol/L Tris solution (pH 8.8)(2) 1.2 2.5 3.6 5.0 6.3 7.5 10.0 12.5 100 g/L SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 g/L APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED(5) 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02 15z Acrylamide Water 1.1 2.3 3.4 4.6 5.7 6.9 9.2 11.5 Acrylamide solution(1) 2.5 5.0 7.5 10.0 12.5 15.0 20.0 25.0 1.5 mol/L Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5 100 g/L SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 g/L APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 TEMED(5) 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02

(1) Acrylamide solution: 30z acrylamide/bisacrylamide (29:1) solution (2) 1.5 mol/L Tris solution (pH 8.8): 1.5 mol/L tris-hydrochloride buffer solution, pH 8.8 (3) 100 g/L SDS: 100 g/L solution of sodium dodecyl sulfate (4) 100 g/L APS: 100 g/L solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solutions must be prepared before use. (5) TEMED: N,N,N?,N?-tetramethylethylenediamine

(v) Destaining TS: A mixture of water, methanol and trichloroacetic acid in a mixture of water and methanol (5:4) acetic acid (100) (5:4:1). to make 100 mL. (vi) Blocking TS: To 10 mL of acetic acid (100) add water to make 100 mL. (vii) Trichloroacetic acid TS for fixing: Dissolve 10 g of JP XVI General Information / Biotechnological/Biological Products 2201

Table 2 Preparation of stacking gel

Component volumes (mL) per gel mould volume of Solution components 1mL 2mL 3mL 4mL 5mL 6mL 8mL 10mL

Water 0.68 1.4 2.1 2.7 3.4 4.1 5.5 6.8 Acrylamide solution(1) 0.17 0.33 0.5 0.67 0.83 1.0 1.3 1.7 1.0 mol/L Tris solution (pH 6.8)(2) 0.13 0.25 0.38 0.5 0.63 0.75 1.0 1.25 100 g/L SDS(3) 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1 100 g/L APS(4) 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1 TEMED(5) 0.001 0.002 0.003 0.004 0.005 0.006 0.008 0.01

(1) Acrylamide solution: 30z acrylamide/bisacrylamide (29:1) solution (2) 1.0 mol/L Tris solution (pH 6.8): 1 mol/L tris-hydrochloride buffer solution, pH 6.8 (3) 100 g/L SDS: 100 g/L solution of sodium dodecyl sulfate (4) 100 g/L APS: 100 g/L solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solutions must be prepared before use. (5) TEMED: N,N,N?,N?-tetramethylethylenediamine

the Standard Solution and the Test Solution in quartz cells at Total Protein Assay a wavelength of 280 nm, with a suitable spectrophotometer, using the buffer as the blank. To obtain accurate results, the This test is harminized with the European Pharmacopoeia response should be linear in the range of protein concentra- and the U.S. Pharmacopeia. The parts of the text that are tions to be assayed.  not harmonized are marked with symbols ( ). Light-Scattering The accuracy of the UV spectroscopic determination of protein can be decreased by the scattering of The following procedures are provided as illustrations of light by the test specimen. If the proteins in solution exist as the determination of total protein content in pharmacopoeial particles comparable in size to the wavelength of the measur- preparations. Other techniques, such as HPLC, are also ac- ing light (250 to 300 nm), scattering of the light beam results ceptable if total protein recovery is demonstrated. Many of in an apparent increase in absorbance of the test specimen. the total protein assay methods described below can be per- To calculate the absorbance at 280 nm due to light-scatter- formed successfully using kits from commercial sources. ing, determine the absorbances of the Test Solution at wave- Note: Where water is required, use distilled water. lengths of 320, 325, 330, 335, 340, 345, and 350 nm. Using Method 1 (UV method) the linear regression method, plot the log of the observed ab- Protein in solution absorbs UV light at a wavelength of sorbance versus the log of the wavelength, and determine the 280 nm, due to the presence of aromatic amino acids, mainly standard curve best fitting the plotted points. From the tyrosine and tryptophan. This property is the basis of this graph so obtained, extrapolate the absorbance value due to method. Protein determination at 280 nm is mainly a func- light-scattering at 280 nm. Subtract the absorbance from tion of the tyrosine and tryptophan content of the protein. If light-scattering from the total absorbance at 280 nm to the buffer used to dissolve the protein has a high absorbance obtain the absorbance value of the protein in solution. relative to that of water, there is an interfering substance in Filtration with a filter having a 0.2-mm porosity or clarifica- the buffer. This interference can be compensated for when tion by centrifugation may be performed to reduce the effect the spectrophotometer is adjusted to zero buffer absorbance. of light-scattering, especially if the solution is noticeably If the interference results in a large absorbance that turbid. challenges the limit of sensitivity of the spectrophotometer, Calculations Calculate the concentration, CU,ofproteinin the results may be compromised. Furthermore, at low con- the test specimen by the formula: centrations protein can be absorbed onto the cuvette, there- C ＝ C (A /A ), by reducing the content in solution. This can be prevented by U S U S preparing samples at higher concentrations or by using a in which CS is the concentration of the Standard Solution; nonionic detergent in the preparation. and AU and AS are the corrected absorbances of the Test Note: Keep the Test Solution, the Standard Solution, and Solution and the Standard Solution, respectively. the buffer at the same temperature during testing. Method 2 (Lowry method) Standard Solution Unless otherwise specified in the in- This method, commonly referred to as the Lowry assay, is dividual monograph, prepare a solution of the reference based on the reduction by protein of the phosphomolybdic- standard or reference material for the protein under test in tungstic mixed acid chromogen in the Folin-Ciocalteu's the same buffer and at the same concentration as the Test phenol reagent, resulting in an absorbance maximum at 750 Solution. nm. The Folin-Ciocalteu's phenol reagent (Folin's TS) reacts Test Solution Dissolve a suitable quantity of the protein primarily with tyrosine residues in the protein, which can under test in the appropriate buffer to obtain a solution hav- lead to variation in the response of the assay to different ingaconcentrationof0.2to2mgpermL. proteins. Because the method is sensitive to interfering sub- Procedure Concomitantly determine the absorbances of 2202 Biotechnological/Biological Products / General Information JP XVI stances, a procedure for precipitation of the protein from the move interfering substances by precipitation of proteins be- test specimen may be used. Where separation of interfering fore testing. This technique also can be used to concentrate substances from the protein in the test specimen is necessary, proteins from a dilute solution. proceed as directed below for Interfering Substances prior to Sodium Deoxycholate Reagent Prepare a solution of so- preparation of the Test Solution. The effect of interfering dium deoxycholate in water having a concentration of 150 substances can be minimized by dilution provided the con- mg in 100 mL. centration of the protein under test remains sufficient for ac- Trichloroacetic Acid Reagent Prepare a solution of curate measurement. Variations of the Lowry test that are trichloroacetic acid in water having a concentration of 72 g indicated in national regulatory documents1) can be substit- in 100 mL. uted for the method described below. Procedure Add 0.1 mL of Sodium Deoxycholate Reagent Standard Solutions Unless otherwise specified in the in- to 1 mL of a solution of the protein under test. Mix on a vor- dividual monograph, dissolve the reference standard or ref- tex mixer, and allow to stand at room temperature for 10 erence material for the protein under test in the buffer used minutes. Add 0.1 mL of Trichloroacetic Acid Reagent, and to prepare the Test Solution. Dilute portions of this solution mix on a vortex mixer. Centrifuge at 3000 × gfor30 with the same buffer to obtain not fewer than five Standard minutes, decant the liquid, and remove any residual liquid Solutions having concentrations between 5 and 100 mgof with a pipet. Redissolve the protein pellet in 1 mL of Alka- protein per mL, the concentrations being evenly spaced. line Copper Reagent. Proceed as directed for the Test Solu- Test Solution Dissolve a suitable quantity of the protein tion. [Note: Color development reaches a maximum in 20 to under test in the appropriate buffer to obtain a solution hav- 30 minutes during incubation at room temperature, after ing a concentration within the range of the concentrations of which there is a gradual loss of color. Most interfering sub- the Standard Solutions. An appropriate buffer will produce stances cause a lower color yield; however, some detergents a pH in the range of 10 to 10.5. cause a slight increase in color. A high salt concentration Blank Use the buffer used for the Test Solution and the may cause a precipitate to form. Because different protein Standard Solutions. species may give different color response intensities, the Reagents and Solutions— standard protein and test protein should be the same.] Copper Sulfate Reagent Dissolve 100 mg of copper (II) Method 3 (Bradford method) sulfate pentahydrate and 200 mg of sodium tartrate dihy- This method, commonly referred to as the Bradford assay, drate in water, dilute with water to 50 mL, and mix. Dissolve is based on the absorption shift from 470 nm to 595 nm ob- 10 g of anhydrous sodium carbonate in water to a final served when Coomassie brilliant blue G-250 binds to protein. volume of 50 mL, and mix. Slowly pour the sodium carbon- The Coomassie brilliant blue G-250 binds most readily to ate solution into the copper sulfate solution with mixing. arginyl and lysyl residues in the protein, which can lead to Prepare this solution fresh daily. variation in the response of the assay to different proteins. 5z SDS TS Dissolve 5 g of sodium dodecyl sulfate in Standard Solutions Unless otherwise specified in the in- water, and dilute with water to 100 mL. dividual monograph, dissolve the reference standard or the Alkaline Copper Reagent Prepare a mixture of 5z SDS reference material for the protein under test in the buffer TS, Copper Sulfate Reagent, and Sodium Hydroxide Solu- used to prepare the Test Solution. Dilute portions of this so- tion (4 in 125) (2:1:1). This reagent may be stored at room lution with the same buffer to obtain not fewer than five temperature for up to 2 weeks. Standard Solutions having concentrations between 100 mg Diluted Folin's TS Mix 10 mL of Folin's TS with 50 mL and 1 mg of protein per mL, the concentrations being evenly of water. Store in an amber bottle, at room temperature. spaced. Procedure To 1 mL of each Standard Solution, the Test Test Solution Dissolve a suitable quantity of the protein Solution, and the Blank, add 1 mL of Alkaline Copper Rea- under test in the appropriate buffer to obtain a solution hav- gent, and mix. Allow to stand at room temperature for 10 ing a concentration within the range of the concentrations of minutes. Add 0.5 mL of the Diluted Folin's TS to each solu- the Standard Solutions. tion, and mix each tube immediately after the addition, and Blank Use the buffer used to prepare the Test Solution and allow to stand at room temperature for 30 minutes. Deter- the Standard Solutions. mine the absorbances of the solutions from the Standard So- Coomassie Reagent Dissolve 100 mg of Coomassie brilliant lutions and the Test Solution at the wavelength of maximum blue G-2502) in 50 mL of ethanol (95). [Note: Not all dyes absorbance at 750 nm, with a suitable spectrophotometer, have the same brilliant blue G content, and different prod- using the solution from the Blank to set the instrument to ucts may give different results.] Add 100 mL of phosphoric zero. acid, dilute with water to 1000 mL, and mix. Filter the solu- Calculations [Note: The relationship of absorbance to pro- tion through filter paper (Whatman No.1 or equivalent), and tein concentration is nonlinear; however, if the standard store the filtered reagent in an amber bottle at room temper- curve concentration range is sufficiently small, it will ap- ature. [Note: Slow precipitation of the dye will occur during proach linearity.] Using the linear regression method, plot storage of the reagent. Filter the reagent before use.] the absorbances of the solutions from the Standard Solu- Procedure Add 5 mL of the Coomassie Reagent to 100 mL tions versus the protein concentrations, and determine the of each Standard Solution, the Test Solution, and the Blank, standard curve best fitting the plotted points. From the and mix by inversion. Avoid foaming, which will lead to standard curve so obtained and the absorbance of the Test poor reproducibility. Determine the absorbances of the solu- Solution, determine the concentration of protein in the Test tions from the Standard Solutions and the Test Solution at Solution. 595 nm, with a suitable spectrophotometer, using the Blank Interfering Substances In the following procedure, deoxy- to set the instrument to zero. cholate-trichloroacetic acid isaddedtoatestspecimentore- JP XVI General Information / Biotechnological/Biological Products 2203

[Note: Do not use quartz (silica) spectrophotometer cells: directed for Interfering Substances under Method 2. Because the dye binds to this material. Because different protein spe- different protein species may give different color response cies may give different color response intensities, the stand- intensities, the standard protein and test protein should be ard protein and test protein should be the same.] There are the same. relatively few interfering substances, but detergents and am- Calculations [Note: The relationship of absorbance to pro- pholytes in the test specimen should be avoided. Highly alka- tein concentration is nonlinear; however, if the standard line specimens may interfere with the acidic reagent. curve concentration range is sufficiently small, it will ap- Calculations [Note: The relationship of absorbance to pro- proach linearity.] Using the linear regression method, plot tein concentration is nonlinear; however, if the standard the absorbances of the solutions from the Standard Solu- curve concentration range is sufficiently small, it will ap- tions versus the protein concentrations, and determine the proach linearity.] Using the linear regression method, plot standard curve best fitting the plotted points. From the the absorbances of the solutions from the Standard Solu- standard curve so obtained and the absorbance of the Test tions versus the protein concentrations, and determine the Solution, determine the concentration of protein in the Test standard curve best fitting the plotted points. From the Solution. standard curve so obtained and the absorbance of the Test Method 5 (Biuret method) Solution, determine the concentration of protein in the Test This method, commonly referred to as the Biuret assay, is Solution. based on the interaction of cupric (Cu2 ＋ ) ion with protein in Method 4 (Bicinchoninic acid method) an alkaline solution and the resultant development of absor- This method, commonly referred to as the bicinchoninic bance at 545 nm. acid or BCA assay, is based on reduction of the cupric Standard Solutions Unless otherwise specified in the in- (Cu2＋) ion to cuprous (Cu＋) ion by protein. The bicincho- dividual monograph, prepare a solution of Albumin Human ninic acid reagent is used to detect the cuprous ion. The for which the protein content has been previously deter- method has few interfering substances. When interfering mined by nitrogen analysis (using the nitrogen-to-protein substances are present, their effect may be minimized by di- conversion factor of 6.25) or of the reference standard or lution, provided that the concentration of the protein under reference material for the protein under test in sodium chlo- test remains sufficient for accurate measurement. ride solution (9 in 1000). Dilute portions of this solution with Standard Solutions Unless otherwise specified in the in- sodium chloride solution (9 in 1000) to obtain not fewer than dividual monograph, dissolve the reference standard or the three Standard Solutions having concentrations between 0.5 reference material for the protein under test in the buffer and 10 mg per mL, the concentrations being evenly spaced. used to prepare the Test Solution. Dilute portions of this so- [Note: Low responses may be observed if the sample under lution with the same buffer to obtain not fewer than five test has significantly different level of proline than that of Standard Solutions having concentrations between 10 and Albumin Human. A different standard protein may be 1200 mg of protein per mL, the concentrations being evenly employed in such cases.] spaced. Test Solution Prepare a solution of the test protein in so- Test Solution Dissolve a suitable quantity of the protein dium chloride solution (9 in 1000) having a concentration under test in the appropriate buffer to obtain a solution hav- within the range of the concentrations of the Standard Solu- ing a concentration within the range of the concentrations of tions. the Standard Solutions. Blank Use sodium chloride solution (9 in 1000). Blank Use the buffer used to prepare the Test Solution and Biuret Reagent Dissolve about 3.46 g of copper (II) sulfate the Standard Solutions. pentahydrate in 10 mL of water, with heating if necessary, Reagents and Solutions— and allow to cool (Solution A). Dissolve about 34.6 g of so- BCA Reagent Dissolve about 10 g of bicinchoninic acid, dium citrate dihydrate and 20.0 g of anhydrous sodium car- 20 g of sodium carbonate monohydrate, 1.6 g of sodium bonate in 80 mL of water, with heating if necessary, and tartrate dihydrate, 4 g of sodium hydroxide, and 9.5 g of allow to cool (Solution B). Mix Solutions A and B, and sodium hydrogen carbonate in water. Adjust, if necessary, dilute with water to 200 mL. This Biuret Reagent is stable at with sodium hydroxide or sodium hydrogen carbonate to a room temperature for 6 months. Do not use the reagent if it pH of 11.25. Dilute with water to 1000 mL, and mix. develops turbidity or contains any precipitate. Copper Sulfate Reagent Dissolve about 2 g of copper (II) Procedure To one volume of the Standard Solutions and a sulfate pentahydrate in water to a final volume of 50 mL. solution of the Test Solution add an equal volume of sodium Copper-BCA Reagent Mix 1 mL of Copper Sulfate Rea- hydroxide solution (6 in 100), and mix. Immediately add a gent and 50 mL of BCA Reagent. volume of Biuret Reagent equivalent to 0.4 volume of the Procedure Mix 0.1 mL of each Standard Solution, the Test Test Solution, and mix. Allow to stand at a temperature be- Solution, and the Blank with 2 mL of the Copper-BCA Rea- tween 159Cand259C for not less than 15 minutes. Within gent. Incubate the solutions at 379C for 30 minutes, note the 90 minutes after the addition of the Biuret Reagent, deter- time, and allow to come to room temperature. Within 60 mine the absorbances of the Standard Solutions and the so- minutes following the incubation time, determine the absor- lution from the Test Solution at the wavelength of maximum bances of the solutions from the Standard Solutions and the absorbance at 545 nm, with a suitable spectrophotometer, Test Solution in quartz cells at 562 nm, with a suitable spec- using the Blank to set the instrument to zero. [Note: Any so- trophotometer, using the Blank to set the instrument to zero. lution that develops turbidity or a precipitate is not accepta- After the solutions are cooled to room temperature, the ble for calculation of protein concentration.] color intensity continues to increase gradually. If substances Calculations Using the least-squares linear regression that will cause interference in the test are present, proceed as method, plot the absorbances of the Standard Solutions ver- 2204 Biotechnological/Biological Products / General Information JP XVI sus the protein concentrations, and determine the standard Dilute with water to 1000 mL, and mix. curve best fitting the plotted points, and calculate the corre- Stock OPA Reagent Dissolve about 120 mg of o- lation coefficient for the line. [Note: Within the given range phthalaldehyde in 1.5 mL of methanol, add 100 mL of of the standards, the relationship of absorbance to protein Borate Buffer, and mix. Add 0.6 mL of polyoxyethylene (23) concentration is approximately linear.] A suitable system is lauryl ether, and mix. This solution is stable at room temper- one that yields a line having a correlation coefficient of not ature for at least 3 weeks. less than 0.99. From the standard curve and the absorbance OPA Reagent To 5 mL of Stock OPA Reagent add 15 of the Test Solution, determine the concentration of protein mL of 2-mercaptoethanol. Prepare at least 30 minutes prior in the test specimen, making any necessary correction. to use. This reagent is stable for one day. Interfering Substances To minimize the effect of interfer- Procedure Adjust each of the Standard Solutions and the ing substances, the protein can be precipitated from the ini- Test Solution to a pH between 8.0 and 10.5. Mix 10 mLof tial test specimen as follows. Add 0.1 volume of 50z the Test Solution and each of the Standard Solutions with trichloroacetic acid to 1 volume of a solution of the test 100 mL of OPA Reagent, and allow to stand at room tem- specimen, withdraw the supernatant layer, and dissolve the perature for 15 minutes. Add 3 mL of 0.5 mol/L sodium precipitate in a small volume of 0.5 mol/L sodium hydroxide hydroxide TS, and mix. Using a suitable fluorometer, TS. Use the solution so obtained to prepare the Test Solu- determine the fluorescent intensities of solutions from the tion. Standard Solutions and the Test Solution at an excitation Comments This test shows minimal difference between wavelength of 340 nm and an emission wavelength between equivalent IgG and albumin samples. Addition of the so- 440 and 455 nm. [Note: The fluorescence of an individual dium hydroxide and the Biuret Reagent as a combined rea- specimen is read only once because irradiation decreases the gent, insufficient mixing after the addition of the sodium hy- fluorescent intensity.] droxide, or an extended time between the addition of the so- Calculations The relationship of fluorescence to protein dium hydroxide solution and the addition of the Biuret Rea- concentration is linear. Using the linear regression method, gent will give IgG samples a higher response than albumin plot the fluorescent intensities of the solutions from the samples. The trichloroacetic acid method used to minimize Standard Solutions versus the protein concentrations, and the effects of interfering substances also can be used to de- determine the standard curve best fitting the plotted points. termine the protein content in test specimens at concentra- From the standard curve so obtained and the fluorescent tions below 500 mgpermL. intensity of the Test Solution, determine the concentration of protein in the test specimen. Method 6 (Fluorometric method) This fluorometric method is based on the derivatization of Method 7 (Nitrogen method) the protein with o-phthalaldehyde (OPA), which reacts with This method is based on nitrogen analysis as a means of the primary amines of the protein (i.e., NH2-terminal amino protein determination. Interference caused by the presence acid and the e-amino group of the lysine residues). The sensi- of other nitrogen-containing substances in the test protein tivity of the test can be increased by hydrolyzing the protein can affect the determination of protein by this method. before testing. Hydrolysis makes the a-amino group of the Nitrogen analysis techniques destroy the protein under test constituent amino acids of the protein available for reaction but are not limited to protein presentation in an aqueous with the OPA reagent. The method requires very small quan- environment. tities of the protein. Procedure A Determine the nitrogen content of the protein Primary amines, such as tris(hydroxymethyl)amino- under test as directed elsewhere in the Pharmacopoeia. Com- methane and amino acid buffers, react with OPA and must mercial instrumentation is available for the Kjeldahl nitro- be avoided or removed. Ammonia at high concentrations gen assay. will react with OPA as well. The fluorescence obtained when Procedure B Commercial instrumentation is available for amine reacts with OPA can be unstable. The use of automat- nitrogen analysis. Most nitrogen analysis instruments use ed procedures to standardize this procedure may improve the pyrolysis (i.e., combustion of the sample in oxygen at tem- accuracy and precision of the test. peratures approaching 10009C), which produces nitric oxide

Standard Solutions Unless otherwise specified in the in- (NO) and other oxides of nitrogen (NOx) from the nitrogen dividual monograph, dissolve the reference standard or the present in the test protein. Some instruments convert the reference material for the protein under test in the buffer nitric oxides to nitrogen gas, which is quantified with a used to prepare the Test Solution. Dilute portions of this so- thermal conductivity detector. Other instruments mix nitric lution with the same buffer to obtain not fewer than five oxide (NO) with ozone (O3) to produce excited nitrogen . Standard Solutions having concentrations between 10 and dioxide (NO2 ) which emits light when it decays and can be 200 mg of protein per mL, the concentrations being evenly quantified with a chemiluminescence detector. A protein ref- spaced. erence standard or reference material that is relatively pure Test Solution Dissolve a suitable quantity of the protein and is similar in composition to the test proteins is used to under test in the appropriate buffer to obtain a solution hav- optimize the injection and pyrolysis parameters and to evalu- ing a concentration within the range of the concentrations of ate consistency in the analysis. the Standard Solutions. Calculations The protein concentration is calculated by Blank Use the buffer used to prepare the Test Solution and dividing the nitrogen content of the sample by the known the Standard Solutions. nitrogen content of the protein. The known nitrogen content Reagents and Solutions— of the protein can be determined from the chemical composi- Borate Buffer Dissolve about 61.83 g of boric acid in tion of the protein or by comparison with the nitrogen con- water, and adjust with potassium hydroxide to a pH of 10.4. tent of the appropriate reference standard or reference mate- JP XVI General Information / Microorganisms 2205 rial. estimated level of sterility assurance is greatly variable, so the conditions for disinfection and sterilization treatment 1) Example: Minimum Requirements for Biological must be chosen appropriately for each application. General- Products and individual monograph in JP.  ly, the following methods are to be used singly or in combi- 2) Purity of the reagent is important. nation after appropriate optimization of operation procedures and conditions, in accordance with the kind and the degree of the contaminating microorganisms and the nature G4 Microorganisms of the item to which the methods are applied. The validation of sterilization in accordance with Termi- nal Sterilization and Sterilization Indicators is required when the methods are applied to the manufacturing processes of Decision of Limit for Bacterial drug products. Endotoxins 1. Disinfection methods These methods are used to reduce the number of living The endotoxin limit for injections is to be decided as microorganisms, but do not always remove or kill all follows: microorganisms present. Generally, disinfection is classified Endotoxin limit ＝ K/M into chemical disinfection with the use of chemical drugs (disinfectants) and physical disinfection with the use of moist where K is a threshold pyrogenic dose of endotoxin per kg heat, ultraviolet light, and other agents. body mass (EU/kg), and M is equal to the maximum bolus 1.1. Chemical disinfection dose of product per kg body mass. When the product is to be Microorganisms are killed with chemical drugs. The kill- injected at frequent intervals or infused continuously, M is ing effect and mechanisms of a chemical drug differ depend- the maximum total dose administered in a single hour ing on the type, applied concentration, action temperature, period. and action time of the chemical drug used, the degree of con- M is expressed in mL/kg for products to be administered tamination on the object to be disinfected, and the species by volume, in mg/kg or mEq/kg for products to be ad- and state (e.g., vegetative bacteria or spore bacteria) of ministered by mass, and in Unit/kg for products to be ad- microorganisms. ministered by biological units. Depending on the administra- Therefore, in applying the method, full consideration is tion route, values for K are set as in the following table. required of the sterility and permissible storage period of the prepared chemical drug, the possibility of resistance of Intended route of administration K (EU/kg) microorganisms at the site of application, and the effect of residual chemical drug on the product. In selecting a suitable Intravenous 5.0 chemical drug, the following items should be considered in Intravenous, for relation to the intended use. radiopharmaceuticals 2.5 (i) The antimicrobial spectrum (ii) Action time for killing microorganisms Intraspinal 0.2 (iii) Action durability (iv) Effect of the presence of proteins Notes: (v) Influence on the human body 1) For products to be administered by mass or by units, (vi) Solubility in water the endotoxin limit should be decided based on the labeled (vii) Influence on the object to be disinfected amount of the principal drug. (viii) Odor 2) Sixty kg should be used as the average body mass of (ix) Convenience of use an adult when calculating the maximum adult dose per kg. (x) Easy disposability 3) The pediatric dose per kg body mass should be used (xi) Influence on the environment at disposal when this is higher than the adult dose. (xii) Frequency of occurrence of resistance 4) The K values for the intravenous route are applicable 1.2. Physical disinfection to drugs to be administered by any route other than those Microorganisms are killed without a chemical drug. shown in the table. (i) Steam flow method Microorganisms are killed by direct application of steam. This method is used for a product which may be denatured by the moist heat method. As a rule, the product is kept in Disinfection and Sterilization flowing steam at 1009C for 30 – 60 minutes. Methods (ii) Boiling method Microorganisms are killed by putting the object in boiling Disinfection and Sterilization Methods are applied to kill water. This method is used for a product which may be microorganisms in processing equipment/utensils and areas denatured by the moist heat method. As a rule, the product used for drug manufacturing, as well as to perform is put in boiling water for 15 minutes or more. microbiological tests specified in the monographs, and so (iii) Intermittent method differ from ``Terminal Sterilization'' and ``Filtration Microorganisms are killed by heating for 30 – 60 minutes Method'' described in ``Terminal Sterilization and Steriliza- repeatedly, three to five times, once a day in water at 80 – tion Indicators''. The killing effect on microorganisms or the 1009C or in steam. This method is used for a product which 2206 Microorganisms / General Information JP XVI may be denatured by the moist heat method. There is test solutions, etc. As a rule, microwave radiation with a another method called the low temperature intermittent wavelength of around 2450 ± 50 MHz is used. method with repeated heating at 60 – 809C. During the inter- 2.3. Gas methods mission periods between heating or warming, a suitable Microorganisms are killed by a sterilizing gas. Suitable temperature for the growth of microorganisms of 209Cor gases for killing microorganisms include ethylene oxide gas, higher, must be maintained. formaldehyde gas, hydrogen peroxide gas, chlorine dioxide (iv) Ultraviolet method gas, etc. Temperature, humidity, the concentration of gas, As a rule, microorganisms are killed by irradiation with and the exposure time differ in accordance with the species ultraviolet rays at a wavelength of around 254 nm. This of gas used. As sterilizing gases are generally toxic to hu- method is used for products which are resistant to ultraviolet mans, full consideration is required of the environmental rays, such as smooth-surfaced articles, facilities, and equip- control for the use of gases and the concentration of residual ment, or water and air. This method does not suffer from gas. In some of the gas methods, it may be difficult to meas- the occurrence of resistance, which is observed in chemical ure or estimate quantitatively the killing of microorganisms. disinfection, and shows a killing effect on bacteria, fungi, 2.4. Filtration method and viruses. It must be taken into consideration that direct Microorganisms are removed by filtration with a suitable ultraviolet irradiation of the human body can injure the eyes filtering device. This method is generally used for gas, water, and skin. or culture media and test solutions containing a substance that is water-soluble and unstable to heat. As a rule, a filter 2. Sterilization methods having a pore size of 0.22 mm or smaller is used for the 2.1. Heating methods sterilization. However, in this method, a filter with a pore In these methods, the heating time before the temperature size of 0.45 mm or smaller is permitted to be used. or pressure reaches the prescribed value differs according to the properties of the product, the size of the container, and the conditions. The duration of heating in conducting these methods is counted from the time when all the parts of the Media Fill Test (Process Simulation) product have reached the prescribed temperature. (i) Moist heat method The media fill test is one of the process validations em- Microorganisms are killed in saturated steam at a suitable ployed to evaluate the propriety of the aseptic processing of temperature and pressure. This method is generally used for pharmaceutical products using sterile media, etc. instead of heat-stable substances, such as glass, porcelain, metal, rub- actual products. Therefore, media fill should be conducted ber, plastics, paper, and fiber, as well as heat-stable liquids, under conditions that simulate routine manufacturing such as water, culture media, reagents, test solutions, liquid procedures, e.g. filling and sealing, operating environment, samples, etc. As a rule, one of the following conditions is processing operation, number of personnel involved, etc., used. and include permissible worst case conditions. Process simu- 115 – 1189C for 30 minutes lation can be applied to the other aseptic manufacturing 121 – 1249C for 15 minutes processes in addition to aseptic manufacturing processes for 126 – 1299C for 10 minutes finished drug products such as ``filling'' and ``sealing''. (ii) Dry-heat method 1. Frequency of media fills Microorganisms are killed in dry-heated air. This method 1.1. Initial performance qualification is generally used for heat-stable substances, such as glass, Initial performance qualification should be conducted for porcelain, and metal, as well as heat-stable products, such as each new facility, item of equipment, filling line, and con- mineral oils, fats and oils, powder samples, etc. This method tainer design (except for multiple sizes of the same container is generally conducted in the way of direct heating by gas or design), etc. As referring to Table 1, a sufficient number of electricity or circulating heated air. As a rule, one of the fol- units should be used to simulate aseptic manufacturing lowing conditions is used. process. A minimum of three consecutive separate successful 160 – 1709C for 120 minutes runs should be performed on each separate day. 170 – 1809C for 60 minutes 1.2. Periodic performance requalification 180 – 1909C for 30 minutes 1) As referring to Table 2, a sufficient number of units 2.2. Irradiation methods should be used to simulate aseptic manufacturing process. (i) Radiation method Media fill run should be conducted at least on semi-annual Microorganisms are killed by gamma-rays emitted from a base for each shift and processing line. All personnel work- radioisotope or electron beam and bremsstrahlung (X-ray) ing in the critical processing area should be trained about generated from an electron accelerator. This method is gen- aseptic processing and participate in a media fill run at least erally used for radiation-resistant substances such as glass, once a year. porcelain, metal, rubber, plastics, fiber, etc. The dose is 2) When filling lines have not been used for over six decided according to the material properties, and the degree months, conduct appropriate numbers of media fill runs in of contamination of the product to be sterilized. Special con- the same way as for the initial performance qualification sideration is necessary of the possibility of qualitative change prior to the use of the filling lines. of the product after the application of the method. 3) In cases of facility and equipment modification (inter- (ii) Microwave method changing parts may not require requalification), major Microorganisms are killed by the heat generated by direct changes in personnel working in critical aseptic processing, microwave irradiation. This method is generally used for anomalies in environmental monitoring results, or a product microwave-resistant products such as water, culture media, sterility test showing contaminated products, conduct ap- JP XVI General Information / Microorganisms 2207

Table 1 Initial performance qualification 2) Particulate monitoring data 3) Personnel monitoring data (microbial monitoring Number Contaminated Minimum data on gloves, gowns, etc. at the end of work) of units units in any of number of Action 4) Sterilization cycle data for media, commodities, eq- filled per the three simulations uipment, etc. simulation simulations 5) Calibration data of sterilization equipment Investigation, cor- 6) Storage conditions of sterile commodities 3 ＜5000 ≧1 rective measures, 7) HEPA filter evaluation (integrity tests, velocity, etc.) restart validation 8) Pre and post filter integrity test data (including filter housing assembly) Investigation, con- 9) Air flow patterns and pressures 1 sideration of repeat 10) Unusual events that occurred during the media fill run 5000 – of one media fill 11) Characterization of contaminants 3 12) Hygienic control and training programs 10000 Investigation, cor- 13) Gowning procedures and training programs ＞1 rective measures, 14) Aseptic processing technique and training programs restart validation 15) Operator's health status (especially coughing, sneez- 1 Investigation ing, etc., due to respiratory diseases) 16) Other factors that affect sterility 3 ＞10000 Investigation, cor- ＞1 rective measures, 3. Data guidance for media fills restart validation Each media fill run should be fully documented and the following information recorded: 1) Data and time of media fill 2) Identification of filling room and filling line used Table 2 Periodic performance requalification 3) Container/closure type and size Number 4) Volume filled per container Minimum of units Contaminated 5) Filling speed number of Action filled per units 6) Filter type and integrity test result (in case of filtra- simulations simulation tion) 7) Type of media filled Investigation, reval- ＜5000 1 8) Number of units filled idation 9) Number of units not incubated and reason 10) Number of units incubated Investigation, con- 11) Number of units positive 1 sideration of repeat 12) Incubation time and temperature media fill 5000 – 13) Procedures used to simulate any step of a normal Every half 10000 Investigation, cor- production fill (e.g., mock lyophilization or substitution of year ＞1 rective measures, vial headspace gas) revalidation 14) Microbiological monitoring data obtained during the media fill set-up and run 1 Investigation 15) List of personnel who took part in the media fill 16) Growth promotion results of the media (in case of ＞10000 Investigation, cor- ＞1 rective measures, powder fill, an antimicrobial activity test for the powder is revalidation necessary) 17) Identification and characterization of the microorganisms from any positive units 18) Product(s) covered by the media fill 19) Investigation of runs with a positive unit or failed propriate numbers of media fill runs in the same way as for runs the initial performance qualification prior to the scheduled 20) Management review media fills. 4. Media fill procedures 2. Acceptance criteria of media fills Methods to validate aseptic processing of liquid, powder Both in initial performance qualification and periodic per- and freeze-dried products are described. Basically, it is possi- formance qualification, the target should be zero growth ble to apply media fill procedures for liquid products to regardless of number of units filled per simulation. Where other dosage forms. contaminated units are found, action shown in Tables 1 and 4.1. Media selection and growth promotion 2 should be taken. Soybean-casein digest medium or other suitable media are 2.1. Investigation of positive units used. When strains listed in the Microbial Limit Test <4.05> Where contaminated units are found in media fill, an in- and, if necessary, one or two representative microorganisms vestigation should be conducted regarding the cause, taking which are frequently isolated in environmental monitoring into consideration the following points: are inoculated under the specified conditions, each strain 1) Microbial monitoring data should show obvious growth. 2208 Microorganisms / General Information JP XVI

4.2. Sterile medium preparation tion sterilization. The medium is sterilized according to the pre-validated B.3 Sterility of filling powders method. The powders must pass the Sterility Test. However, if the 4.3. Incubation and inspection of media filled units sterilization is fully validated, sterility testing of the powders Leaking or damaged units should be removed and record- can be omitted. ed prior to incubation of media filled units. Units should be B.4 Media fill procedures incubated at 20 – 359C for not less than 14 days. The use of Chose a suitable procedure from among the following another temperature range should be justified. If two tem- procedures. peratures are used for incubation, the units are typically in- 1) Fill sterilized liquid media into containers by suitable cubated for at least 7 days at each temperature starting with methods, and then fill actual products or sterilized placebo the lower temperature. Established temperature should be powder with the powder filling machine. If sterilized powder kept within ±2.59C. Observe the media filled units for media are used as a placebo powder, fill sterilized water in- growth of microorganisms on the last day of the incubation. steadofsterilizedliquidmedia. Microorganisms in contaminated units should be followed to 2) Distribute liquid media into containers, and then steri- identification and characterization. For the identification of lize them in an autoclave. Remove the containers to the fill- contaminants, ``Rapid Identification of Microorganisms ing area, and then fill actual products or sterilized placebo Based on Molecular Biological Method'' shown in General powder into the containers with the powder filling machine. Information or appropriate commercial kit for identification 3) Fill actual products or sterilized placebo powder into of microorganisms may be applicable. containers with the powder filling machine, and then fill sterilized liquid media into the containers by appropriate A. Liquid products methods. If sterilized powder media are used as a placebo Media fill procedure powder, fill sterilized water instead of sterilized liquid me- Media fill should include normal facility/equipment oper- dia. ations and clean-up routines. Containers, closures, parts of the filling machine, trays, etc. are washed and sterilized ac- C. Lyophilized products cording to the standard operating procedures. Media fills In the case of lyophilized products, it may be impossible to should be conducted under processing conditions that in- conduct a media fill run in the same way as used for actual clude ``worst case'' conditions, e.g., correction of line stop- processing of lyophilized products. The process of freezing page, repair or replacement of filling needles/tubes, replace- and lyophilization of the solution may kill contaminant or- ment of on-line filters, permitted interventions, duration and ganisms and change the characteristics of the media too. The size of run, number of personnel involved, etc. use of inert gas as a blanket gas may inhibit the growth of It is not necessary to put all worst-case scenarios in a aerobic bacteria and fungi. Therefore, in general, the actual single media fill run, but all worst-case scenarios should be freezing and lyophilization process should be avoided and air evaluated intentionally. While advanced processing lines are is used as the blanket gas. For products manufactured under highly automated, often operated at relatively high speeds, an anaerobic atmosphere, process simulation should be per- and are designed to limit operator intervention, there are formed with the use of anaerobic growth media and the inert processing lines showing frequent human interventions. gas such as nitrogen gas. Although the most accurate simulation model would be the Media fill procedures full batch size and duration because it most closely simulates Use the following method or other methods considered to the actual production run, other appropriate models can be be equivalent to these methods. justified. 1) After filling of the media into containers by the filling A predetermined volume of medium is filled into sterilized machine, cap the containers loosely and collect them in pre- containers at a predetermined filling speed and the contain- sterilized trays. ers are sealed. The media are contacted with all interior sur- 2) After placing the trays in the lyophilizer, close the faces in the containers by an appropriate method, and then chamber door, and conduct lyophilization according to the incubated at the predetermined temperature. procedures for production operation. Hold them without freezing and boiling-over under weak vacuum for the B. Powder products predetermined time. B.1 Powder selection and antimicrobial activity test 3) After the vacuum process, break the vacuum, and seal Actual products or placebo powder are used. In general, the stoppers. lactose monahydrate, D-mannitol, polyethylene glycol 6,000, 4) Contact the media with all product contact surfaces in carboxymethyl cellulose salts or media powder, etc. are used the containers by appropriate methods, and then cultivate as placebo powders. Prior to employing any of the powders, them at the predetermined temperature. evaluate whether the powder has antimicrobial activity. Media powders are dissolved in water and other powders in References liquid medium, and the solutions are inoculated with less ISO 13408-1 (2008): Aseptic processing of health care prod- than 100 CFU microorganisms of each kind, shown in 4.1, ucts: General requirements. for the growth promotion test. If obvious growth appears in the medium incubated at the predetermined temperature for 5 days, the powder has no antimicrobial activity and is available for the media fill test. B.2 Sterilization of powders Powders are bagged in suitable containers (e.g. double heat-sealed polyethylene bags), and are subjected to radia- JP XVI General Information / Microorganisms 2209

tus of the product batch should be established on the basis of Microbial Attributes of Non-sterile the bioburden data obtained by retrospective validation and/ or concurrent validation. In general, a mixture of samples Pharmaceutical Products randomly taken from at least different three portions, almost the same amount for each portion, is used for the This chapter is harmonized with the European Phar- tests of the product. When the sampling is difficult in a clean macopoeia and the U.S. Pharmacopeia. The parts of the text area, special care is required during sampling to avoid in- that are not harmonized are marked with symbols ( ).  troducing microbial contamination into the product or af- The presence of certain micro-organisms in non-sterile fecting the nature of the product bioburden. If it is con- preparations may have the potential to reduce or even inacti- firmed that the product bioburden is stable for a certain vate the therapeutic activity of the product and has a poten- period, as in the case of non-aqueous or dried products, it is tial to adversely affect the health of the patient. Manufac- not necessary to do the tests, immediately after the sampling. turers have therefore to ensure a low bioburden of finished 3.2. Testing frequency dosage forms by implementing current guidelines on Good The frequency of the tests should be established on the Manufacturing Practice during the manufacture, storage and basis of a variety of factors unless otherwise specified. These distribution of pharmaceutical preparations. This chapter factors include: provides guidelines for acceptable limits of viable micro- (i) Dosage forms of non-sterile pharmaceutical products organisms (bacteria and fungi) existing in raw materials and (usage); non-sterile pharmaceutical products. Microbial examina- (ii) Manufacturing processes; tion of non-sterile products is performed according to the (iii) Manufacturing frequency; methods given in the Microbial Limit Test <4.05> on (iv) Characteristics of raw materials (natural raw mate- Microbiological Examination of Non-sterile Products: I. rial, synthetic compound, etc.); Microbial Enumeration Tests and II. Tests for Specified (v) Batch sizes; Micro-organisms. When these tests are carried out, a (vi) Variations in bioburden estimates (changes in batch- microbial control program must be established as an im- es, seasonal variations, etc.); portant part of the quality management system of the (vii) Changes affecting the product bioburden (changes product. Personnel responsible for conducting the tests in manufacturing process, supplier of raw materials, batch should have specialized training in microbiology, biosafety number of raw materials, etc.); measures and in the interpretation of the testing results. (viii) Others. In general, the tests may be performed at a high frequency 1. Definitions during the initial production of a drug to get information on (i) Non-sterile pharmaceutical products: Non-sterile the microbiological attributes of the product or raw mate- drugs shown in monographs of the JP and non-sterile rials used for the production. However, this frequency may finished dosage forms. be reduced as bioburden data are accumulated through (ii) Raw materials: All materials, including raw ingredi- retrospective validation and/or concurrent validation. For ents and excipients, used for the preparation of drugs, except example, the tests may be performed at a frequency based on for water and gases. time (e.g., weekly, monthly or seasonally), or on alternate (iii) Bioburden: Number and type of viable micro-organ- batches. isms existing in non-sterile pharmaceutical products. (iv) Action levels: Established bioburden levels that re- 4. Microbial control program quire immediate follow-up and corrective action if they are When the ``Microbial Limit Test <4.05>'' is applied to a exceeded. non-sterile pharmaceutical product, the methods for the (v) Alert levels: Established bioburden levels that give recovery, cultivation and estimation of the bioburden from early warning of a potential drift from normal bioburden the product must be validated and a ``Microbial control pro- level, but which are not necessary grounds for definitive cor- gram'' covering the items listed below must be prepared. rective action, though they may require follow-up investiga- (i) Subject pharmaceutical name (product name); tion. (ii) Frequency of sampling and testing; (vi) Quality management system: The procedures, opera- (iii) Sampling methods (including responsible person, tion methods and organizational structure of a manufacturer quantity, environment, etc. for sampling); (including responsibilities, authorities and relationships be- (iv) Transfer methods of the samples to the testing area tween these) needed to implement quality management. (including storage condition until the tests); (v) Treatment of the samples (recovery methods of 2. Scope microbial contaminants); In general, the test for total viable aerobic count is not ap- (vi) Enumeration of viable micro-organisms (including plied to drugs containing viable micro-organisms as an active testing quantity, culture media, growth-supporting test of ingredient. the media, culturing methods, etc.); 3. Sampling plan and frequency of testing (vii) Detection of specified micro-organisms (including 3.1. Sampling methods testing quantity, culture media, growth-supporting test of Microbial contaminants are usually not uniformly dis- the media, culturing methods, etc.); tributed throughout the batches of non-sterile pharmaceuti- (viii) Estimation of the number of and characterization cal products or raw materials. A biased sampling plan, thereof microbial contaminants; fore, cannot be used to estimate the real bioburden in the (ix) Establishment of ``Microbial acceptance criteria'' product. A sampling plan which can properly reflect the sta- (including alert level and action level); 2210 Microorganisms / General Information JP XVI

(x) Actions to be taken when the levels exceed ``Microbi- of active ingredients or non-sterile pharmaceuticals may al acceptance criteria''; have a direct effect on the quality of the finished dosage (xi) Persons responsible for the testing and evaluation, form. This means it is necessary to keep the level of microbi- etc.; al contaminants in the water as low as possible. (xii) Other necessary items. Acceptance criteria for microbiological quality for non- sterile finished dosage forms are shown in Table 2. These 5. Microbial acceptance criteria for non-sterile pharmaceu- microbial limits are based primarily on the type of dosage tical products form, water activity, and so on. For oral liquids and phar- By establishing ``Microbial acceptance criteria'' for non- maceutical products having a high water activity, in general, sterile pharmaceutical products based upon the total aerobic low microbial acceptance criteria are given. microbial count (TAMC) and the total combined yeasts/  Table 2 includes a list of specified micro-organisms for moulds count (TYMC), it is possible to evaluate at the ini- which acceptance criteria are set. The list is not necessarily tial processing stage of the product whether the microbiological quality of the raw materials is adequate or not. Further- more, it is then possible to implement appropriate corrective Table 1 Acceptance criteria for microbiological quality of action as needed to maintain or improve the microbiological non-sterile substances for pharmaceutical use quality of the product. The target limits of microbial levels for raw materials (synthetic compounds and minerals) are Total Combined Total Aerobic showninTable1. Yeasts/Moulds Microbial Count In general, synthetic compounds have low bioburden lev- Count (CFU/g or CFU/mL) els due to the high temperatures, organic solvents, etc., used (CFU/g or CFU/mL) in their manufacturing processes. Raw materials originated from plants and animals in general have higher bioburdens Substances for 3 2 than synthetic compounds. pharmaceutical 10 10 The microbial quality of the water used in the processing use

Table 2 Acceptance criteria for microbiological quality of non-sterile dosage forms

Total Aerobic Total Combined Route of administration Microbial Count Yeasts/Moulds Count Specified Micro-organism (CFU/g or CFU/mL) (CFU/g or CFU/mL)

Non-aqueous preparations 103 102 Absence of Escherichia coli for oral use (1 g or 1 mL)

Aqueous preparations 102 101 Absence of Escherichia coli for oral use (1 g or 1 mL)

Rectal use 103 102 —

Oromucosal use 102 101 Absence of Staphylococcus aureus Gingival use (1 g or 1 mL) Cutaneous use Absence of Pseudomonas aeruginosa Nasal use (1 g or 1 mL) Auricular use

Vaginal use 102 101 Absence of Pseudomonas aeruginosa (1 g or 1 mL) Absence of Staphylococcus aureus (1 g or 1 mL) Absence of Candida albicans (1 g or 1 mL)

Transdermal patches (limits 102 101 Absence of Staphylococcus aureus for one patch including ad- (1 patch) hesive layer and backing) Absence of Pseudomonas aeruginosa (1 patch)

Inhalation use (more rigo- 102 101 Absence of Staphylococcus aureus rous requirements apply to (1 g or 1 mL) liquid preparations for Absence of Pseudomonas aeruginosa nebulization) (1 g or 1 mL) Absence of bile-tolerant gram-negative bacteria (1g or 1 mL) JP XVI General Information / Microorganisms 2211

Table 3 Acceptance criteria for crude drugs and crude directly consumed crude drug preparations containing pow- drug preparations dered crude drugs. In this guideline, enterobacteria and other gram-negative bacteria, Escherichia coli, Salmonella, Category 1 Category 2 Micro-organisms and Staphylococcus aureus are mentioned as specified (CFU/g or CFU/mL) (CFU/g or CFU/mL) micro-organisms, but other micro-organisms such as certain Aerobic bacteria 107 105 species of Bacillus cereus, Clostridia, Pseudomonas, Bur- Molds and yeasts 104 103 kholderia, Asperigillus and Enterobacter species are also Enterobacteria necessary to be tested depending on the origin of raw mate- and other rials for crude drugs or the preparation method of crude ※ 103 gram-negative drug preparations. bacteria Escherichia coli 102 Not detected Salmonella Not detected Not detected Microbiological Evaluation of Staphylococcus ※※ aureus Processing Areas for Sterile

※ : The limits are not specified. Pharmaceutical Products This chapter describes the methods for the control and evaluation of microbial contamination in areas used for the exhaustive and for a given preparation it may be necessary to test for other micro-organisms depending on the nature of processing of sterile pharmaceutical products. Such processing areas are classified into critical areas and clean areas ac- the starting materials and the manufacturing process. cording to the required levels of air-cleanliness. A critical If it has been shown that none of the prescribed tests will area is a defined space in which the airborne particulate and allow valid enumeration of micro-organisms at the level microorganism levels are controlled to meet grade A. The prescribed, a validated method with a limit of detection as close as possible to the indicated acceptance criterion is used. cleanliness requirements for such a space extend to the surfaces of the facilities and equipment which form or are locat- In addition to the micro-organisms listed in Table 2, the ed within the space, as well as to the supplied raw materials, significance of other micro-organisms recovered should be chemicals, water, etc. Environmental conditions, such as evaluated in terms of: temperature, humidity, and air pressure, are also controlled ( i ) the use of the product: hazard varies according to the route of administration (eye, nose, respiratory in this space when required. A clean area is a controlled space such that the levels of contaminants (particulates and tract); microorganisms) in air, gases and liquids are maintained ( ii ) the nature of the product: does the product support growth, does it have adequate antimicrobial preser- within specified limits, which are less stringent than those of grade A. When sterile pharmaceutical products are manufac- vation? (iii) the method of application; tured, the environment, facilities/equipment, and personnel should be routinely monitored to ensure appropriate (iv) the intended recipient: risk may differ for neonates, microbiological control in the processing areas. The detec- infants, the debilitated; ( v ) use of immunosuppressive agents, corticosteroids; tion of microorganisms should be performed under normal operational conditions, using an appropriate sampling (vi) presence of disease, wounds, organ damage. Where warranted, a risk-based assessment of the relevant device, according to an environmental control program established previously. The sampling, cultivation, counting, factors is conducted by personnel with specialized training in and evaluation methods for airborne microorganisms, as microbiology and the interpretation of microbiological data. For raw materials, the assessment takes account of well as those found on surfaces, should also be chosen appropriately, depending on the monitoring purpose, monitor- processing to which the product is subjected, the current technology of testing and the availability of materials of the ing items, and microorganisms being detected. Sampling devices, measurement methods, media, culture conditions, desired quality. Acceptance criteria are based on individual frequency of monitoring, and recommended limits for en- results or on the average of replicate counts when replicate counts are performed (e.g. direct plating methods). vironmental microorganisms shown in this chapter are for information only, and are not requirements. When an acceptance criterion for microbiological quality is prescribed it is interpreted as follows: 1. Definitions —101 CFU: maximum acceptable count ＝ 20, For the purposes of this chapter, the following definitions —102 CFU: maximum acceptable count ＝ 200, apply. —103 CFU: maximum acceptable count ＝ 2000, and so (i) Processing areas: Areas in which actions such as culti- forth. vation, extraction/purification, weighing of raw materials, washing and drying of containers and stoppers, preparation 6. Acceptance criteria for crude drugs Target limits of microbial contamination for crude drugs of solutions, filling, sealing and packaging are performed, including the gowning area. and crude drug preparations are shown in Table 3. Category (ii) Action levels: Established microbial levels (and type 1 includes crude drugs and crude drug preparations which are used for extraction by boiling water or to which boiling of microorganisms, if appropriate) that require immediate follow-up and corrective action if they are exceeded. water is added before use. Category 2 includes crude drugs which are taken directly without extraction process and (iii) Alert levels: Established microbial levels (and type 2212 Microorganisms / General Information JP XVI of microorganisms if appropriate) that give early warning of filtration, the product must be handled and filled into con- a potential drift from normal operating conditions, but tainers under grade A aseptic conditions. which are not necessarily grounds for definitive corrective 2.3. Sterile products prepared aseptically from sterile start- action, though they may require follow-up investigation. ing materials (iv) Contaminants: Particulates and microorganisms The handling of starting materials and all further process- causing contamination by adhering to surfaces or by being ing should be done under grade A conditions. incorporated into materials. 3. Microbiological environmental monitoring program (v) Cleanliness: A quantity which indicates the condition Environmental monitoring is especially important in steril- of cleanliness of a monitoreditem,expressedasmassor ity assurance for sterile pharmaceutical products that are number of contaminants contained in a certain volume or manufactured by aseptic processing. The major purpose of area. environmental monitoring is to predict potential deteriora- (vi) Contamination control: The planning, establishment tion of the processing environment before it occurs, and to of systems and implementation activities performed in order produce high-quality, sterile pharmaceutical products under to maintain the required cleanliness of a specified space or appropriate contamination control. surface. 3.1. Monitoring of environmental microorganisms (vii) Shift: Scheduled period of work or production, usu- (i) An environmental control program document is pre- ally less than 12 hours in length, during which operations are pared for each area used for the processing of sterile phar- conducted by a single defined group of workers. maceutical products. The procedures in the document in- (viii) Characterization of contaminants: Procedures for clude: 1) items to be monitored, 2) type of microorganisms classifying contaminants so that they can be differentiated. to be monitored, 3) frequency of monitoring, 4) methods of In routine control, classification to the genus level is monitoring, 5) alert and action levels, and 6) actions to be sufficient; as required, identification to the species level is taken when specified levels are exceeded. performed. (ii) The aseptic processing areas and other processing 2. Air-cleanliness of processing areas for sterile phar- areas maintained under controlled conditions are monitored maceutical products on a routine basis. The critical processing areas where sterile Airborne particulates in areas used for the processing of products are in contact with environmental air are monitored pharmaceutical products may act physically as a source of during every operational shift. The items to be monitored insoluble particles in the products, and biologically as a car- include the air, floor, walls, equipment surfaces, and the rier of microorganisms. So, it is necessary to control strictly gowns and gloves of the personnel. Table 2 shows suggested the number of particles in the air. The air-cleanliness criteria frequencies of the environmental monitoring. are shown in Table 1. (iii) The sampling devices used for monitoring environ- 2.1. Terminally sterilized products mental microorganisms, as well as the methods and culture Solutions should generally be prepared in a grade C en- media, should be suitable to detect microorganisms that may vironment. Solution preparation may be permitted in a grade be present (aerobic bacteria, anaerobic bacteria, molds, D environment if additional measures are taken to minimize yeast, etc.). The cultivation conditions, such as incubation contamination. For parenterals, filling should be done in a temperature and time, are selected to be appropriate for the grade A workstation in a grade B or C environment. The re- specific growth requirements of microorganisms to be de- quirements during the preparation and filling of other sterile tected. Table 3 shows the culture media and cultivation con- products are generally similar to those for parenterals. ditions that are generally used in testing for environmental 2.2. Sterile products prepared aseptically after filtration microorganisms. The handling of starting materials and the preparation of (iv) The number of microorganisms in the samples is solutions should be done in a grade C environment. These estimated by using the Membrane Filtration, Pour Plating, activities may be permitted in a grade D environment if addi- Spread Plating, or Serial Dilution (Most Probable Number) tional measures are taken to minimize contamination, such Methods described in the Microbial Limit Test. as the use of closed vessels prior to filtration. After sterile (v) Table 4 shows recommended limits for environmental

Table 1 Air-cleanliness requirements for processing of sterile pharmaceutical products

Air cleanliness Maximum number of airborne particulates per m3 at rest in operation Grade*1 ≧0.5 mm ≧0.5 mm A (Laminar-airflow zone) 3530 3530 B (Non laminar-airflow zone) 3530 353000 C 353000 3530000 D 3530000 —*2 *1 The maximum permitted number of particles in the ``in operation'' condition corresponds to the standards described under USP <1116> as follows. Grade A: Class 100 (M3.5); Grade B: Class 10,000 (M5.5); Grade C: Class 100,000 (M6.5); Grade D: no corresponding standard. *2 The limit for this area will depend on the nature of the operation carried out there. JP XVI General Information / Microorganisms 2213

Table 2 Suggested frequency of environmental monitoring

Processing area Frequency of monitoring Critical area (Grade A) Each shift Clean area adjacent to critical area (Grade B) Each shift Other clean areas (Grade C, D) Potential product/container contact areas Twice a week Non-product/container contact areas Once a week

Table 3 Media and culture conditions

Microorganisms *1 to be detected Media Culture conditions Soybean-casein digest agar (or fluid) medium 30–359C*2 Aerobes Brain-heart infusion agar (or fluid) medium More than 5 days*3 Nutrient agar (or fluid) medium Soybean-casein digest agar (or fluid) medium Sabouraud dextrose agar (or fluid) medium 20–259C*2 Yeast and fungi Potato-dextrose agar (or fluid) medium More than 5 days*3 Glucose peptone agar (or fluid) medium Soybean-casein digest agar medium Fluid cooked meat medium 30–359C Anaerobes*4 Reinforced clostridial agar (or fluid) medium More than 5 days*3 Thioglycolate medium I (or thioglycolate agar medium) for sterility test *1 If necessary, antibiotics may be added to media in an appropriate concentration (see Microbial Limit Test). If the existence of disinfectants that may interfere with the test on the surface of the specimen is suspected, add a substance to inactivate them. *2 When soybean-casein digest agar medium is used for the detection of aerobes, yeast and fungi, incubation at 25 to 309Cformorethan5 days is acceptable. *3 If a reliable count is obtained in a shorter incubation time than 5 days, this may be adopted. *4 Generally, anaerobes are not targets for the monitoring. For the detection of anaerobes, agar medium is incubated in an appropriate anaerobic jar.

Table 4 Recommended limits for environmental microorganisms*1

CFU on a surface Airborne 2 Minimum air sample Grade microorganisms* 3 instruments/facilities gloves (CFU/m3) (m ) (CFU/24–30 cm2)*3 A ＜10.5 ＜1 ＜1 B100.55 5 C 100 0.2 25 — D 200 0.2 50 — *1 Maximum acceptable average numbers of microorganisms under each condition. *2 These values are by using a slit sampler or equivalent. *3 Viable microbe cell number per contact plate (5.4 – 6.2 cm in diameter). When swabbing is used in sampling, the number of microorganisms is calculated per 25 cm2. For gloves, usually, put their all fingers on the plate.

microorganisms. The alert and action levels may be adjusted source of any discrepancy should be investigated immedi- if necessary after sufficient data have been accumulated. The ately and the investigation should be documented in a most important point in environmental monitoring is to con- report. After corrective action has been taken, follow-up firm that an acceptable value of each monitoring item is monitoring should be done to demonstrate that the affected maintained consistently. area is once again within specification. (vi) Microorganisms isolated are characterized if neces- (ii) The report is reviewed and approved by personnel sary. In addition, analysis of hourly or daily variation of air- responsible for quality control and distributed to all key per- borne particulate numbers will provide data to assist in the sonnel associated with the aseptic processing operation. control of the cleanliness of processing areas. 4. Sampling devices and measuring methodology 3.2. Evaluation of environmental monitoring data Various types of sampling devices and measurement (i) The data from the environmental monitoring are methods are available for the sampling and measurement of evaluated on a routine basis for each area and location. The 2214 Microorganisms / General Information JP XVI microorganisms in the air and on surfaces, and appropriate the air. The pinhole sampler resembles the slit of the slit samplers and measuring methodology are selected according sampler, but has pinholes in place of the slit. A known to the purpose of monitoring and the items to be monitored. volume of air passed through several pinholes impacts on 4.1. Evaluation of airborne microorganisms agar medium in a slowly revolving Petri dish. The cen- 4.1.1. Settle plates trifugal sampler consists of a propeller that pulls a known Petri dishes of a specified diameter containing a suitable volume of air into the device and then propels the air out- culture medium are placed at the measurement location and ward to impact on a tangentially placed nutrient agar strip. the cover is removed there. The plates are exposed for a The sampler is portable and can be used anywhere, but the given time and the microorganisms deposited from the air sampling volume of air is limited. onto the agar surface are enumerated after incubation. This See above the Measuring methods <4.1.2.1> on the charac- method is not effective for quantitative monitoring of total teristics of the filtration-type sampler. airborne microorganisms because it does not detect microor- 4.2. Measurement methods for microorganisms on sur- ganisms that do not settle onto the surface of the culture faces media, and the settling velocity of aggregates of microorgan- 4.2.1. Contact plates isms is affected by air currents and disturbances in airflow. Use a contact plate with an appropriate contact surface. Although the results obtained by the settle plate method are The culture medium surface should be brought into contact only qualitative or semi-quantitative, this method is suitable with the sampling site for several seconds by applying for long-term evaluation of possible contamination of prod- uniform pressure without circular or linear movement. After ucts or devices by airborne microorganisms. contact and removal, the plates are covered and, as soon as 4.1.2. Active microbial sampling methods possible, incubated using appropriate culture conditions. 4.1.2.1. Measuring methods After a contact plate has been used, the site to which the Methods in which a fixed volume of air is aspirated in- plate was applied must be wiped aseptically to remove any clude filtration-type sampling devices and impact-type adherent culture medium. sampling devices. With the filter-type sampling devices the 4.2.2. Swabs desired volume of air can be collected by appropriately A piece of sterilized gauze, absorbent cotton, cotton swab, changing the air intake rate or the filter size. However, care or other suitable material premoistened with an appropriate must be taken to ensure that sterility is maintained while the rinse fluid is stroked in closely parallel sweeps or slowly ro- filter is placed in and removed from the holder. When air tated over the defined sampling area. After sampling, the sampling devices are used in critical areas, care must be swab is agitated in a specified amount of an appropriate taken to avoid disturbance of the airflow around the prod- sterilized rinse fluid, and the rinse fluid is assayed for viable ucts. There are two types of filters; wet-type used gelatin organisms. filters and dry-type used membrane filters. With the dry-type 5. Test methods for collection performance of a sampling filters, static electricity effects can make it impossible to col- device for airborne microorganisms lect quantitatively microorganisms on the filter. When an The testing of the collection performance of sampling impact-type sampling device is used, the following points are devices for airborne microorganisms is performed in accor- important: 1) The speed at which the collected air strikes the dance with JIS K 3836 (Testing methods for collection ef- culture medium surface must be sufficient to capture the ficiency of airborne microbe samplers) or ISO 14698 – 1 microorganisms, but must not have an adverse effect on the (Cleanrooms and associated controlled environments. collected microorganisms. 2) A sufficient volume of air must Biocontamination control. General principles). besampledsothatevenextremely low levels of microbiological contaminants are detected, but the procedure must not 6. Growth-promotion test of media and confirmation of cause a significant change in the physical or chemical proper- antimicrobial substances ties of the culture medium. 3) When the device is used in crit- This test and confirmation are performed according to ical areas, care must be exercised to ensure that the process- ``Effectiveness of culture media and confirmation of antimi- ing of the sterile pharmaceutical products is not adversely af- crobial substances'' in the Microbial Limit Test <4.05>. fected by the air disturbance. 7. Media 4.1.2.2. Sampling devices (i) Soybean-casein digest agar medium or Fluid soybean- The most commonly used samplers are as follows: Slit casein digest medium sampler, Andersen sampler, pinhole sampler, centrifugal See Microbial Limit Test. sampler and filtration-type sampler. Each sampler has specific characteristics. The slit sampler is a device to trap (ii) Sabouraud dextrose agar medium or Fluid sabouraud microorganisms in a known volume of air passed through a dextrose medium standardized slit. The air is impacted on a slowly revolving See Microbial Limit Test. Antibiotic is added if necessary. Petri dish containing a nutrient agar. The rotation rate of the Petri dish and the distance from the slit to the agar surface (iii) Potato-dextrose agar medium or Fluid potato-dex- are adjustable and it is possible to estimate the number of trose medium microorganisms in the air passed through the device for a See Microbial Limit Test. Antibiotic is added if necessary. period of up to 1 hr. The Andersen sampler consists of a perforated cover and several pieces of Petri dishes containing a (iv) Glucose peptone agar medium or Fluid glucose pep- nutrient agar, and a known volume of air passed through the tone medium perforated cover impacts on the agar medium in the Petri Glucose 20.0 g dishes. The sampler is suitable for the determination of the Yeast extract 2.0 g distribution of size ranges of microorganism particulates in JP XVI General Information / Microorganisms 2215

Magnesium sulfate heptahydrate 0.5 g Water 1000 mL Peptone 5.0 g For fluid medium, add 0.5 g of agar. Sterilize by heating Potassium dihydrogen phosphate 1.0 g in an autoclave at 1219C for 15 to 20 min. pH after Agar 15.0 g sterilization: 6.7 – 6.9. Water 1000 mL Mix all the components, and sterilize by heating in an *1 Bovine brain extract powder Dried extract of bovine autoclave at 1219C for 15 to 20 minutes. pH after fresh brain. A yellow-brown powder having a characteristic sterilization: 5.6 – 5.8. Just prior to use, add 0.10 g of odor. benzylpenicillin potassium and 0.10 g of tetracyclin per Loss on drying: not more than 5z. liter of medium as sterile solutions or, alternatively, add *2 Bovine heart extract powder Dried extract of bovine 50 mg of chloramphenicol per liter of medium. Anti- fresh heart. A yellow-brown powder having a characteristic biotics are added, as appropriate. odor. Loss on drying: not more than 5z. (v) Thioglycolate agar medium or thioglycolate medium 8. Rinsing Liquids I for sterility test (i) Phosphate buffer (pH 7.2) See Sterility Test. The agar concentration of Thioglycolate See Microbial Limit Test. agar medium is about 1.5z. (ii) Buffered sodium chloride-peptone solution (pH 7.0) (vi) Brain-heart infusion agar medium or Fluid brain- See Microbial Limit Test. heart infusion medium Bovine brain extract powder*1 (iii) Ringer's solution, 1/4 concentration An amount equivalent to 200 g of calf brain Sodium chloride 2.25 g Bovine heart extract powder*2 Potassium chloride 0.105 g An amount equivalent to 250 g of the material Calcium chloride dihydrate 0.16 g Peptone 10.0 g Water 1000 mL Glucose 2.0 g Sterilize by heating in an autoclave at 1219C for 15 to 20 Sodium chloride 5.0 g min. pH after sterilization: 7.0. Disodium hydrogenphosphate dodecahydrate 2.5 g (iv) Thiosulfate-Ringer's solution Agar 15.0 g Sodium thiosulfate pentahydrate 0.8 g Water 1000 mL Ringer's solution, 1/4 concentration 1000 mL Sterilize by heating in an autoclave at 1219C for 15 to 20 Sterilize by heating in an autoclave at 1219C for 15 to 20 min. pH after sterilization: 7.2 – 7.6. min. pH after sterilization: 6.6. (vii) Nutrient agar medium or Fluid nutrient medium (v) LP liquid Meat extract 3.0 g Casein peptone 1.0 g Peptone 5.0 g Soybean lecithin 0.7 g Agar 15.0 g Polysorbate 80 1.0 – 20.0 g Water 1000 mL Water 1000 mL Sterilize by heating in an autoclave at 1219C for 15 to 20 Sterilize by heating in an autoclave at 1219C for 15 to 20 min. pH after sterilization: 6.6 – 7.0. min. pH after sterilization: 7.2. (viii) Fluid cooked meat medium Bovine heart extract powder*2 An amount equivalent to 450 g of the material Preservatives-Effectiveness Tests Peptone 20.0 g Glucose 2.0 g The purpose of the Preservatives-Effectiveness Tests is to Sodium chloride 5.0 g assess microbiologically the preservative efficacy, either due Water 1000 mL to the action of product components themselves or any ad- Sterilize by heating in an autoclave at 1219C for 15 to 20 ded preservative(s), for multi-dose containers. The efficacy min. pH after sterilization: 7.2 – 7.6. of the preservatives is assessed by direct inoculation and mixing of the test strains in the product, and titration of survival (ix) Reinforced clostridial agar medium or Fluid rein- of the test strains with time. forced clostridial medium Preservatives must not be used solely to comply with GMP Meat extract 10.0 g for drugs or to reduce viable aerobic counts. In addition, Peptone 10.0 g preservatives themselves are toxic substances. Therefore, Yeast extract 3.0 g preservatives must not be added to products in amounts Soluble starch 1.0 g which might jeopardize the safety of human beings, and con- Glucose 5.0 g sideration must be given to minimizing the amounts of L-Cystein hydrochloride monohydrate 0.5 g preservative used. These tests are commonly used to verify Sodium chloride 5.0 g that products maintain their preservative effectiveness at the Sodium acetate trihydrate 3.0 g design phase of formulation or in the case of periodic Agar 15.0 g monitoring. Although these tests are not performed for lot 2216 Microorganisms / General Information JP XVI release testing, the efficacy of the preservative present in the When strains other than the five listed above are cultured, product packaged in the final containers should be verified select a culture medium suitable for growth of the strain con- throughout the entire dating period. cerned. The cell suspension may also be prepared by a method suitable for that strain. Use the inoculum suspen- 1. Products and their Categories sions within 24 hours after they have been prepared from the The products have been divided into two categories for cultivations on agar plate media or in liquid media. Store the these tests. Category I products are those made with aqueous inoculum suspensions in a refrigerator if it is not possible to bases or vehicles, and Category II products are those made inoculate them into the test specimens within 2 hours. Titrate with nonaqueous bases or vehicles. Oil-in-water emulsions the viable cell count of the inocula immediately before use, are considered Category I products, and water-in-oil emul- and then calculate the theoretical viable cell count per mL (g) sions Category II products. Category I is further divided into of the product present just after inoculation. three subtypes depending on the dosage forms. Category IA: Injections and other sterile parenterals 3. Test Procedure Category IB: Non-sterile parenterals 3.1. Category I products Category IC: Oral products in liquid forms (including Inject each of the cell suspensions aseptically into five con- syrup products to be dissolved or suspended before use) tainers containing the product and mix uniformly. When it is Category II: All the dosage forms listed under Category I difficult to inject the cell suspension into the container asep- made with non-aqueous bases or vehicles. tically or the volume of the product in each container is too small to be tested, transfer aseptically a sufficient volume of 2. Test Microorganisms and Culture Media the product into each of alternative sterile containers, and The following strains or those considered to be equivalent mix the inoculum. When the product is not sterile, incubate are used as the test microorganisms. additional containers containing the uninoculated product as Escherichia coli ATCC 8739, NBRC 3972 controls and calculate their viable cell counts (the viable Pseudomonas aeruginosa ATCC 9027, NBRC 13275 counts of bacteria and those of yeasts and moulds). A sterile Staphylococcus aureus ATCC 6538, NBRC 13276 syringe, spatula or glass rod may be used to mix the cell sus- Candida albicans ATCC 10231, NBRC 1594, JCM 2085 pension uniformly in the product. The volume of the suspen- Aspergillus brasiliensis ATCC 16404, NBRC 9455 sion mixed in the product must not exceed 1/100 of the These test microorganisms are representative of those that volume of the product. Generally, the cell suspension is in- might be found in the environment in which the product is oculated and mixed so that the concentration of viable cells manufactured, used or stored, and they are also recognized is 105 to 106 cells per mL or per gram of the product. Incu- as opportunistic pathogens. In addition to these strains bate these inoculated containers at 209Cto259Cwithpro- designated as test microorganisms, it is further recommend- tection from light, and calculate the viable cell count of 1 ed to use strains that might contaminate the product and mL or 1 g of the product taken at 0, 14 and 28 days subse- grow on or in it, depending on its characteristics. For the test quent to inoculation. Record any marked changes (e.g., microorganisms received from coordinated collections of changes in color or the development of a bad odor) when ob- microorganisms, one passage is defined as the transfer of served in the mixed samples during this time. Such changes microorganisms from an established culture to fresh me- should be considered when assessing the preservative effi- dium, and microorganisms subjected to not more than five cacy of the product concerned. Express sequential changes in passages should be used for the tests. Single-strain challenges the viable counts as percentages, with the count at the start rather than mixed cultures should be used. The test strains of the test taken as 100. Titration of the viable cell counts is can be harvested by growth on solid agar or liquid media. based, in principle, on the Pour Plate Methods in ``Micro- Cultures on agar plate media: Inoculate each of the five bial Limit Tests''. In this case, confirm whether any anti- test strains on the surface of agar plates or agar slants. For microbial substance is present in the test specimen. If a growth of bacteria, use Soybean-Casein Digest Agar Medi- confirmed antimicrobial substance needs to be eliminated, um, and for yeasts and moulds, use Sabouraud Glucose incorporate an effective inactivator of the substance in the Agar, Glucose-Peptone Agar or Potato Dextrose Agar Medi- buffer solution or liquid medium to be used for dilution of um. Incubate bacterial cultures at 309Cto359C for 18 to 24 the test specimen, as well as in the agar plate count medium. hours, the culture of C. albicans at 209Cto259Cfor40to However, it is necessary to confirm that the inactivator has 48 hours and the culture of A. brasiliensis at 209Cto259C no effect on the growth of the microorganisms. When the for one week or until good sporulation is obtained. Harvest occurrence of the preservative or the product itself affects these cultured cells aseptically using a platinum loop, etc. titration of the viable cell count and there is no suitable inac- Suspend the collected cells in sterile physiological saline or in tivator available, calculate the viable cell counts by the Mem- 0.1z Peptone Water and adjust the viable cell count to brane Filtration Method in ``Microbial Limit Tests''. about 108 microorganisms per mL. In the case of A. 3.2. Category II products brasiliensis, suspend the cultured cells in sterile physiological The procedures are the same as those described for Cate- saline or 0.1z Peptone Water containing 0.05 w/vz of gory I products, but special procedures and considerations polysorbate 80 and adjust the spore count to about 108 per are required for both uniform dispersion of the test microor- mL. Use these suspensions as the inocula. ganism in the product and titration of viable cell counts in Liquid cultures: After culturing each of the four strains the samples. except for A. brasiliensis in a suitable medium, remove the For semisolid ointment bases, heat the sample to 459Cto medium by centrifugation. Wash the cells in sterile physio- 509C until it becomes oily, add the cell suspension and logical saline or 0.1z Peptone Water and resuspend them in disperse the inoculum uniformly with a sterile glass rod or the same solution with the viable cell or spore count of the spatula. Surfactants may also be added to achieve uniform inoculum adjusted to about 108 per mL. JP XVI General Information / Microorganisms 2217 dispersion, but it is necessary to confirm that the surfactant Effectiveness Tests are described below. Other media may be added has no effect on survival or growth of the test used if they have similar nutritive ingredients and selective microorganisms and that it does not potentiate the preserva- and growth-promoting properties for the microorganisms to tive efficacy of the product. For titration of the viable cell be tested. count, a surfactant or emulsifier may be added to disperse (i) Soybean Casein Digest Agar Medium the product uniformly in the buffer solution or liquid me- Casein peptone 15.0 g dium. Sorbitan monooleate, polysorbate 80 or lecithin may Soybean peptone 5.0 g be added to improve miscibility between the liquid medium Sodium chloride 5.0 g and semisolid ointments or oils in which test microorganisms Agar 15.0 g were inoculated. These agents serve to inactivate or neutral- Water 1000 mL ize many of the most commonly used preservatives. Mix all of the components and sterilize at 1219Cfor15– 20 minutes in an autoclave. pH after sterilization: 7.1 – 7.3. Table 1 Interpretation criteria by product category (ii) Sabouraud Glucose Agar Medium Peptone (animal tissue and casein) 10.0 g Product Interpretation criteria category Microorganisms Glucose 40.0 g After 14 days After 28 days Agar 15.0 g Bacteria 0.1z of inocu- Same or less Water 1000 mL lum count or than level after Mix all of the components and sterilize at 1219Cfor15– less 14 days 20 minutes in an autoclave. pH after sterilization: 5.4 – 5.8. Category IA Yeasts/moulds Same or less Same or less (iii) Glucose Peptone (GP) Agar Medium than inoculum than inoculum Glucose 20.0 g count count Yeast extract 2.0 g Bacteria 1z of inoculum Same or less Magnesium sulfate heptahydrate 0.5 g count or less than level after Peptone 5.0 g 14 days Monobasic potassium phosphate 1.0 g Category IB Agar 15.0 g Yeasts/moulds Same or less Same or less Water 1000 mL than inoculum than inoculum count count Mix all of the components and sterilize at 1219Cfor15– 20 minutes in an autoclave. pH after sterilization: 5.6 – 5.8. Bacteria 10z of inocu- Same or less lum count or than level after (iv) Potato Dextrose Agar Medium less 14 days Potato extract 4.0 g Category IC Glucose 20.0 g Yeasts/moulds Same or less Same or less Agar 15.0 g than inoculum than inoculum count count Water 1000 mL Mix all of the components and sterilize at 1219Cfor15– Bacteria Same or less Same or less 20 minutes in an autoclave. pH after sterilization: 5.4 – 5.8. than inoculum than inoculum count count (v) 0.1z Peptone Water Category II Peptone 1.0 g Yeasts/moulds Same or less Same or less Sodium chloride 8.0 g than inoculum than inoculum count count Water 1000 mL Mix all of the components and sterilize at 1219Cfor15– 20 minutes in an autoclave. pH after sterilization: 7.2 – 7.4. 4. Interpretation Interpret the preservative efficacy of the product according to Table 1. When the results described in Table 1 are obtained, the product examined is considered to be effectively Rapid Counting of Microbes using preserved. There is a strong possibility of massive microbial Fluorescent Staining contamination having occurred when microorganisms other than the inoculated ones are found in the sterile product to This chapter provides rapid methods using fluorescence be examined, and caution is required in the test procedures staining for the quantitative estimation of viable microor- and/or the control of the manufacturing process of the ganisms. Incubation on an agar medium has been widely product. When the contamination level in a nonsterile used for quantitative estimation of viable microorganisms, product to be examined exceeds the microbial enumeration but a number of environmental microorganisms of interest limit specified in ``Microbial Attributes of Nonsterile Phar- are not easy to grow in culture under usual conditions, thus maceutical Products'' in General Information, caution is new microbial detection methods based on fluorescence or also required in the test procedures and/or the control of the luminescence have been developed. In the fluorescence stain- manufacturing process of the product. ing method, microorganisms are stained with fluorescent dye, and can easily be detected and counted with various 5. Culture Media sorts of apparatus, such as a fluorescence microscope or Culture media and buffer solution used for Preservatives- flow cytometer. Methods are available to detect total 2218 Microorganisms / General Information JP XVI microorganisms, including bothdeadandviablecells,orto thatcantrapparticlesonthesurfacecanbeusedsuchas detect only cells with a specified bioactivity by choosing the polycarbonate filter, alumina filter, etc. dye reagent appropriately. Nucleic acid staining reagents, (iii) Glass slide which bind with DNA or RNA, detect all cells containing (iv) Cover glass nucleic acids, whether they are live or dead. This technique is (v) Ocular micrometer for counting (with 10 × 10 grids) the most fundamental for the fluorescence staining method. 1.3. Procedure On the other hand, fluorescent vital staining methods target An example of the procedure using fluorescence micro- the respiratory activity of the microorganism and the activity scope is described below. of esterase, which is present universally in microorganisms. 1.3.1. Preparation of samples In the microcolony method, microcolonies in the early stage Prepare samples by ensuring that microbes are dispersed of colony formation are counted. The CFDA-DAPI double evenly in the liquid (water or buffer solution). staining method and the microcolony method are described 1.3.2. Filtration below. These methods can give higher counts than the other Set a membrane filter (poresize: 0.2 mm) on the funnel of techniques, because these rapid and accurate techniques pro- the filtering equipment. Filter an appropriate amount of vide quantitative estimation of viable microorganisms based sample to trap microbes in the sample on the filter. on a very specific definition of viability, which may be 1.3.3. Staining different from that implicit in other methods. The proce- Pour sufficient amount of buffer solution for CFDA dures of these methods described here may be changed as ex- staining, mixed to provide final concentration of 150 mg/mL perience with the methods is accumulated. Therefore, other of CFDA and 1 mg/mL of DAPI, into the funnel of the reagents, instruments and apparatus than those described filtering equipment and allow staining in room temperature here may also be used if there is a valid reason for so doing. for 3 minutes, then filter the liquid by suction. Pour in sufficient amount of aseptic water, suction filter and remove 1. CFDA-DAPI double staining method excess fluorescent reagent left on the filter. Thoroughly dry Fluorescein diacetate (FDA) reagents are generally used the filter. for the detection of microorganisms possessing esterase 1.3.4. Slide preparation activity. These reagents are hydrolyzed by intracellular Put one drop of immersion oil for fluorescence micro- esterase, and the hydrolyzed dye exhibits green fluorescence scope on the glass slide. Place the air dried filter over it, with under blue excitation light (about 490 nm). Modified FDAs the filtering side on the top. Then put one drop of immersion such as carboxyfluorescein diacetate (CFDA) are used be- oil for fluorescence microscope on the surface of the filter, cause of the low stainability of gramnegative bacteria with place a cover glass. Put another drop of immersion oil for FDA. The principle of the CFDA-DAPI double staining fluorescence microscope on the cover glass when using an method, which also employs a nucleic acid staining reagent, oilimmersion objective lens. 4?,6-diamidino-2-phenylindole (DAPI), is as follows. The 1.3.5. Counting nonpolar CFDA penetrates into the cells and is hydrolyzed Observe and count under fluorescence microscope, with to fluorescent carboxyfluorescein by intracellular esterase. 1000 magnification. In case of CFDA-DAPI double staining The carboxyfluorescein is accumulated in the living cells due method, count the microorganisms (with esterase activity) to its polarity, and therefore green fluorescence due to car- exhibiting green fluorescence under the blue excitation light boxyfluorescein occurs when cells possessing esterase activity first to avoid color fading by the ultraviolet light, then count are illuminated with blue excitation light. No fluorescent car- the microorganisms (with DNA) exhibiting blue fluorescence boxyfluorescein is produced with dead cells, since they are under the ultraviolet excitation light in the same microscopic unable to hydrolyze CFDA. On the other hand, DAPI binds field. Count the organisms exhibiting fluorescence on more preferentially to the adenine and thymine of DNA after than 20 randomly selected fields among 100 grids observed penetration into both viable and dead microorganisms, and throughanocularmicrometerof the microscope, and calcu- consequently all of the organisms containing DNA exhibit late the total number of organisms using the following for- blue fluorescence under ultraviolet excitation light. There- mula. The area of the microscopic field should be previously fore, this double staining method enables to count spe- determined with the ocular and objective micrometers. The cifically only live microorganisms possessing esterase activity amount of the sample to be filtered must be adjusted so that under blue excitation light, and also to determine the total the cell number per field is between 10 and 100. It might be microbial count (viable and dead microorganisms) under ul- necessary to reprepare the sample in certain instances. (In traviolet excitation light. such case that the average count number is not more than 2 1.1. Apparatus organisms per field, or where more than 5 fields are found 1.1.1. Fluorescence microscope or fluorescence observa- whichhavenoorganismperfield, it is assumed that the tion apparatus microorganism count is below the detection limit.) Various types of apparatus for counting fluorescen- cestained microorganisms are available. Appropriate filters Number of microbes (cells/mL) are provided, depending on the fluorescent dye reagents ＝ s(average number of microbes per visual field) used. A fluorescence microscope, laser microscope, flow × (area of filtration)t/s(amount of sample cytometer, and various other types of apparatus may be used filtered) × (area of one microscopic field)t for fluorescence observation. 1.4. Reagents and test solutions 1.2. Instruments (i) Aseptic water: Filter water through a membrane filter (i) Filtering equipment (funnels, suction flasks, suction with 0.2 mm pore size to remove particles, then sterilize it by pumps) heating in an autoclave at 1219C for 15 minutes. Water for (ii) Membrane filters (poresize: 0.2 mm); A suitable filter injection may be used. JP XVI General Information / Microorganisms 2219

(ii) CFDA solution, 10 mg/mL: Dissolve 50 mg of dark place. It should be noted that the appropriate incuba- CFDA in dimethylsulfoxide to prepare a 5 mL solution. tion conditions (such as medium, incubation temperature Store at －209C in light shielded condition. and/or incubation time) are different, depending on the (iii) Buffer solution for CFDA staining: Dissolve 5 g of sample. sodium chloride with 0.5 mL of 0.1 mol/L disodium dihy- 2.3.4. Fixation drogen ethylenediamine tetraacetate TS and diluted disodi- Soak filter paper with an appropriate amount of neutral um hydrogen phosphate TS (1 in 3) to prepare 100 mL of so- buffered formaldehyde test solution, then place the filter lution. Add sodium dihydrogen phosphate dihydrate solu- that has been removed from the culture medium on top with tion (1 in 64) to adjust the pH level to 8.5. Filter the solution filtering side up, and allow to remain at room temperature through a membrane filter with a pore size of 0.2 mm. for more than 30 minutes to fix the microcolonies. (iv) DAPI solution, 10 mg/mL: Dissolve 10 mg of DAPI 2.3.5. Staining in 100 mL of aseptic water. Dilute this solution 10 times with Soak filter paper with an appropriate amount of staining aseptic water and filter through a membrane filter with a solution (such as 1 mg/mL of DAPI, 2z polyox- poresizeof0.2mm. Store at 49C in light shielded condition. yethylenesorbitan monolaurate), then place the filter on top (v) Immersion oil for fluorescence microscope with filtering side up, and then leave at room temperature, light shielded for 10 minutes to stain microcolonies. Wash 2. Microcolony method the filter by placing it with the filtering side facing up on top Microcolonies, which are in early stages of colony forma- of a filter paper soaked with aseptic water for 1 minute. tion, are fluorescently stained, then observed and counted Thoroughly air dry the filter. under fluorescence microscope or other suitable systems. 2.3.6. Slide preparation This method enables to count the number of proliferative Put one drop of immersion oil for fluorescence micro- microorganisms, with short incubation time. In this method, scope on the slide glass. Place anairdriedfilteroverit,with the organisms are trapped on a membrane filter, the filter is the filtering side on the top. Then, put one drop of immer- incubated on a medium for a short time, and the microcolo- sion oil for fluorescence microscope on top, place a cover nies are counted. By this method, even colonies which are glass. undetectable with the naked eye can be identified, so viable 2.3.7. Counting organisms can be counted rapidly and with high precision. Count the organisms exhibiting fluorescence on more than Various nucleic acid staining reagents can be used for stain- 20 randomly selected fields among the 100 grids observed ing of microcolonies. through an ocular micrometer of the microscope with 400 or 2.1. Apparatus 200 magnification, and calculate the total number of organ- 2.1.1. Fluorescence microscope or fluorescence observa- isms using the following formula. The area of the tion apparatus microscopic field should be previously determined with the Various types of apparatus for counting fluorescen- ocular and objective micrometers. In such case that the cestained microorganisms are available. Appropriate filters average count number is not more than 2 microcolonies per are provided, depending on the fluorescence dye reagents field, or where more than 5 fields are found which have no used. A fluorescence microscope, laser microscope and vari- microcolony per field, it is assumed that the microorganism ous other types of apparatus may be used for fluorescence count is below the detection limit. observation. 2.2. Instruments Number of microcolonies (cells/mL) (i) Filtering equipment (funnels, suction flasks, suction ＝ s(average number of microcolonies per visual field) pumps) × (area of filtration)t/s(amount of sample (ii) Membrane filters (pore size: 0.2 mm); A suitable filter filtered) × (area of one microscopic field)t thatcantrapparticlesonthesurfacecanbeusedsuchas 2.4. Reagents and test solutions polycarbonate filter, alumina filter, etc. (i) Aseptic water: Filter water through a membrane filter (iii) Glass slide with 0.2 mm pore size to remove particles, and sterilize it by (iv) Cover glass heating in an autoclave at 1219C for 15 minutes. Water for (v) Filter paper (No. 2) injection may be used. (vi) Ocular micrometer for counting (with 10 × 10 grids) (ii) Staining solution: Dissolve 10 mg of DAPI in 100 mL 2.3. Procedure of aseptic water. Dilute the solution 10 times with aseptic An example of the procedure using a fluorescence micro- water and filter through a membrane filter with pore size of scope is described below. 0.2 mm. Store at 49C in light shielded condition. Dissolve 2.3.1. Preparation of samples polyoxyethylene sorbitan monolaurate to the final concen- Prepare samples by ensuring that microbes are dispersed tration of 2z, when using. evenly in the liquid (water or buffer solution). (iii) Neutral buffered formaldehyde solution (4w/vz 2.3.2. Filtration formaldehyde solution; neutrally buffered). Set a membrane filter (pore size: 0.2 mm) on the funnel of (iv) Immersion oil for fluorescence microscope the filtering equipment. Filter an appropriate amount of sample to trap microbes in the sample on the filter. 2.3.3. Incubation Remove the filter from the filtering equipment and place it with filtering side facing up on a culture medium avoiding formation of airbubbles between the filter and the medium. Incubate at a suitable temperature for appropriate hours in a 2220 Microorganisms / General Information JP XVI

solution, and then heated at 1009C for 10 min. In general, Rapid Identification of PCR can be run for bacteria and yeasts heated in DNA releasing solution. For fungi, DNA extraction from culture Microorganisms Based on fluid is better because some of colony samples can disturb Molecular Biological Method PCR reaction. 2.2. PCR This chapter describes the methods for the identification Add 2 mL of template DNA in PCR reaction solution. Use or estimation of microorganisms (bacteria and fungi), found 10F/800R primers (or 800F/1500R primers in the case to in in-process control tests or lot release tests of pharmaceuti- analyze also a latter part of 16S rRNA) for bacteria and cal products, at the species or genus level based on their ITS1F/ITS1R primers for fungi, and then perform 30 am- DNA sequence homology. The identification of isolates plification cycles at 949C for 30 sec, 559Cfor60sec,and found in the sterility test or aseptic processing can be helpful 729C for 60 sec. DNA fragments are amplified about 800 bp for investigating the causes of contamination. Furthermore, in the case of bacteria and about 150 – 470 bp depending on information on microorganisms found in raw materials used the strain in the case of fungi. Include a negative control for pharmaceutical products, processing areas of phar- (water instead of the test solution) in the PCR. maceutical products, and so on is useful in designing meas- 2.3. Confirmation of PCR products ures to control the microbiological quality of drugs. For the Mix 5 mL of PCR product with 1 mL of loading buffer so- identification of microorganisms, phenotypic analysis is lution, place it in a 1.5 w/vz agarose gel well, and carry out widely used, based on morphological, physiological, and electrophoresis with TAE buffer solution (1-fold concentra- biochemical features and analysis of components. Commer- tion). Carry out the electrophoresis together with appropri- cial kits based on differences in phenotype patterns have ate DNA size markers. After the electrophoresis, observe been used for the identification of microorganisms, but are PCR products on a trans-illuminator (312 nm) and confirm not always applicable to microorganisms found in raw mate- the presence of a single band of the targeted size. If multiple rials used for pharmaceutical products and in processing bands are observed, cut the targeted band out of the gel, and areas of pharmaceutical products. In general, the identifica- extract DNA by using appropriate commercial DNA ex- tion of microorganisms based on phenotypic analysis needs traction kit. special knowledge and judgment is often subjective. It is 2.4. Purification of PCR products considered that the evolutionary history of microorganisms Remove unincorporated PCR primers and deoxynucleo- (bacteria and fungi) is memorized in their ribosomal RNAs side triphosphates (dNTP) from PCR products by using ap- (rRNAs), so that systematic classification and identification propriate purification methods. of microorganisms in recent years have been based on the 2.5. Quantification of purified DNA analysis of these sequences. This chapter presents a rapid When purified DNA is measured by spectrophotometer, method to identify or estimate microorganisms based on par- calculate 1 OD260 nm as 50 mg/mL. tial sequences of divergent regions of the 16S rRNA gene for 2.6. Labeling of PCR products with sequencing reagents bacteria and of the internal transcribed spacer 1 (ITS1) Use an appropriate fluorescence-labeled sequencing rea- region located between 18S rRNA and 5.8S rRNA for fungi, gent suitable for the available DNA sequencer or its program followed by comparison of the sequences with those in the and label the PCR products according to the instructions database. Methods described in this chapter do not take the provided with the reagent. place of usual other methods for the identification, and can 2.7. Purification of sequencing reagent-labeled PCR prod- be modified based on the examiner's experience, and on the ucts available equipment or materials. Other gene regions besides Transfer the product in 75 mL of diluted ethanol (7 in 10) those mentioned in this chapter can be used if appropriate. into a 1.5 mL centrifuge tube, keep in an ice bath for 20 min, and centrifuge at 15,000 rpm for 20 min. After removal of 1. Apparatuses supernatant, add 250 mL of diluted ethanol (7 in 10) to the (i) DNA sequencer precipitate and centrifuge at 15,000 rpm for 5 min. Remove Various types of sequencers used a gel board or capillary the supernatant and dry the precipitate. can be used. 2.8. DNA homology analysis (ii) DNA amplifier Place sequencing reagent-labeled PCR products in the To amplify target DNA and label amplified (PCR) prod- DNA sequencer and read the nucleotide sequences of the ucts with sequencing reagents. PCR products. Compare the partial nucleotide sequence 2. Procedures with those in the BLAST database. The following procedures are described as an example. 3. Judgment 2.1. Preparation of template DNA If sequencing data show over 90z identity with a se- It is important to use a pure cultivated bacterium or fun- quence in the database, in general, judgment may be made as gus for identification. In the case of colony samples, colo- follows. nies are picked up with a sterilized toothpick (in the case of ( i ) In the case of bacteria, compare the nucleotides in fungi, a small fragment of colony sample is picked up), and the product obtained with the 10F primer (the 800F suspended in 0.3 mL of DNA releasing solution in a 1.5 mL primer when 800F/1500R primers are used) with the centrifuge tube. In the case of culture fluid, a 0.5 mL por- BLAST database. Higher ranked species are judged tion of fluid is put in a 1.5 mL centrifuge tube and centri- as identified species or closely related species. fuged at 10,000 rpm for 10 min. After removal of the super- (ii) In the case of fungi, compare sequencing data for the natant, the pellet is suspended in 0.3 mL of DNA releasing product obtained with the ITS1F primer with the JP XVI General Information / Microorganisms 2221

BLAST database. Higher ranked species are judged as identified species or closely related species. For Primer 4. Reagents, Test Solutions Bacteria 10F 5?-GTTTGATCCTGGCTCA-3? (i) 0.5 mol/L Disodium dihydrogen ethylenediamine 800R 5?-TACCAGGGTATCTAATCC-3? tetraacetate TS: Dissolve 18.6 g of disodium dihydrogen 800F 5?-GGATTAGATACCCTGGTA-3? ethylenediamine tetraacetate dihydrate in water to make 100 1500R 5?-TACCTTGTTACGACTT-3? mL. Fungi ITS1F 5?-GTAACAAGGT(T/C)TCCGT-3? (ii) 1 mol/L Tris buffer solution, pH 8.0: Dissolve 24.2 g ? ? of 2-amino-2-hydroxymethyl-1,3-propanediol in a suitable ITS1R 5 -CGTTCTTCATCGATG-3 amount of water, adjust the pH to 8.0 with 0.2 mol/L hydrochloric acid TS, and add water to make 200 mL. (xii) Polyoxyethylene(10)octylphenyl ether: A pale yellow, (iii) TE buffer solution: Mix 1.0 mL of 1 mol/L tris buffer viscous liquid. solution, pH 8.0 and 0.2 mL of 0.5 mol/L disodium dihydrogen ethylenediamine tetraacetate TS, and add water to make 100 mL. (iv) DNA releasing solution: Divide TE buffer solution Sterility Assurance for Terminally containing 1 volz of polyoxyethylene (10) octylphenyl ether Sterilized Pharmaceutical Products into small amounts and store frozen until use. (v) PCR reaction solution As indicated in the ``Terminal Sterilization and Steriliza- 10-fold buffer solution* 5 mL tion Indicators'', the pharmaceuticals to which terminal dNTP mixture** 4 mL sterilization can be applied, generally must be sterilized so 10 mmol/L Sense primer 1 mL that a sterility assurance level of 10－6 or less is obtained. The 10 mmol/L Anti-sense primer 1 mL sterility assurance level of 10－6 or less can be proven by Heat-resistant DNA polymerase (1 U/mL) 1 mL using a sterilization process validation based on physical and Water 36 mL microbiological methods, but cannot be proven by sterility * Being composed of 100 mmol/L 2-amino-2-hydrox- tests of the sterilized products. This chapter deals with the ymethyl-1,3-propanediol hydrochloride, pH 8.4, 500 mmol/ necessary requirements for the appropriate management of L potassium chloride, 20 mmol/L magnesium chloride and the important control points of the sterilization process for 0.1 g/L gelatin. the parametric release of products, without performing ** A solution containing 2.5 mmol/L each of dGTP (sodi- sterility tests on products which have been subjected to um 2?-deoxyguanosine 5?-triphosphate), dATP (sodium 2?- terminal sterilization (in the case of radiation sterilization, deoxyadenosine 5?-triphosphate), dCTP (sodium 2?-deox- called dosimetric release). Parametric release is a method ycytidine 5?-triphosphate) and dTTP (sodium 2?-deoxythymi- that can be applied in cases where the sterilization system is dine 5?-triphosphate). Adequate products containing these clearly defined, important control points are clearly speci- components as described above may be used. fied, and the sterilization system process can be validated by (vi) Sequencing reagent: There are many kinds of sequenc- microbiological methods using appropriate biological indica- ing methods, such as the dye-primer method for labeling of tors. primer, the dye-terminator method for labeling of dNTP ter- 1. Definitions minator and so on. Use an appropriate sequencing reagent The definitions of the terminology used in this chapter are kit for the apparatus and program to be used. provided below. (vii) 50-Fold concentrated TAE buffer solution: Dissolve 1.1. Terminal sterilization 242 g of 2-amino-2-hydroxymethyl-1,3-propanediol in 57.1 A process whereby a product is sterilized in its final con- mL of acetic acid (100) and 100 mL of 0.5 mol/L disodium tainer or packaging, and which permits the measurement and dihydrogen ethylenediamine tetraacetate TS, and add water evaluation of quantifiable microbial lethality. to make 1000 mL. 1.2. Validation (viii) 1-Fold concentrated TAE buffer solution: Diluted 50- A documented procedure for obtaining, recording and fold concentrated TAE buffer solution (1 in 50) prepared be- interpreting the results needed to show that a process will fore use is referred to as 1-fold concentrated TAE buffer so- consistently yield a product complying with predetermined lution. specifications. (ix) Agarose gel: Mix 1.5 g of agarose, 2.0 mL of 50-fold 1.3. Periodic re-validation concentrated TAE buffer solution, 10 mL of a solution of Validation that is regularly performed to reconfirm that a ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenan- process is consistently yielding a product complying with thridinium bromide) (1 in 100) and 100 mL of water. After predetermined specifications. It should confirm that varia- dissolving the materials by heating, cool the solution to bles and the acceptable ranges are permissible to yield a about 609C, and prepare gels. product consistently of the required quality. (x) Loading buffer solution (6-fold concentrated): Dissolve 1.4. Facility/equipment qualification 0.25 g of bromophenol blue, 0.25 g of xylene cyanol FF and This is to provide evidence that the manufacturing facili- 1.63 g of disodium dihydrogen ethylenediamine tetraacetate ties/equipment, measuring equipment, and manufacturing dihydrate in 50 mL of water, and add 30 mL of glycerol and environment control facilities, etc. have been properly select- water to make 100 mL. ed, correctly installed, and are operated in conformity with (xi) PCR primers the specifications at the time of installation and during operation. 2222 Microorganisms / General Information JP XVI

1.5. Operation qualification process. This is to provide evidence to confirm physically, chemi- a) Details related to the range of duties of the persons cally and microbiologically that equipment, operated in ac- responsible for the validation, as well as the extent of their cordance with its operational instructions, operates as speci- authority fied and affords a product meeting the specifications. b) Details related to the implementation period for the 1.6. Support system for sterilization process sterilization validation This refers to the facility/equipment that is associated c) Details related to the creation, modification, and ap- with the sterilization devices, such as the preconditioning proval of the sterilization validation plan documents and aeration for ethylene oxide sterilization, the steam supp- d) Details related to the reporting, evaluation, and ap- ly equipment for moist heat sterilization, and the loading proval of the sterilization validation implementation results devices for radiation sterilization. e) Details related to the storage of documentation con- 1.7. Quality system cerning the sterilization validation The procedures, resources and organizational structure of f) Other required matters a manufacturer (responsibilities, authorities and relation- 2.2.2. The sterilization validation procedure must list the ships between these) required to implement quality manage- names of the enactors, the date of enactment, and when ment. there are revisions, must also list the revisers, date of revi- 1.8. Change control system sions, revised sections and reasons for the revisions. A system designed to evaluate all of the changes that may 2.2.3. The manufacturer must properly store and maintain affect the quality of the pharmaceutical product, in order to the sterilization validation procedure after clarifying the ensure that the process is continuously controlled. procedures related to alterations and deletions of the con-

1.9. F0 value tents of the sterilization validation procedure. Assume a value of 109CfortheZ value defined as the 2.3. Persons Responsible for the Validation number of degrees of temperature required for a 10-fold The manufacturer must assign persons to be responsible change in the D value. The F0 value indicates the time for the sterilization validation. The responsible parties must (minutes) required to give the equivalent lethality at Tb of the perform each of the duties listed below according to the sterilization heat obtained by integrating the lethality rate (L) sterilization validation procedure. over an entire heating cycle. 2.3.1. For products that are to be produced according to the sterilization validation procedure, a written sterilization T0 － Tb T0－Tb L ＝ log－1 ＝ 10 Z validation implementation plan must be prepared. The im- Z plementation plan will specify the following points based on

T0 ＝ Temperature inside the chamber or inside the a consideration of the implementation details of the steriliza- product to be sterilized tion validation.

Tb ＝ Reference temperature (1219C) a) Subject pharmaceutical name (product name)

t1 b) Purpose of the applicable sterilization validation F0 ＝fLdt c) Expected results t0 d) Verification methods (including inspection results and

t1 － t0 ＝ Processing time (minutes) evaluation methods) e) Period of verification implementation 1.10. Control device f) Names of persons performing the sterilization valida- A general term for the devices and measurement equip- tion (persons-in-charge) ment, including the equipment for controlling, measuring g) Names of the persons who created the plan, creation and recording the physical parameters that can be measured date, and in the event of revisions, the names of the revisers, (temperature, humidity, pressure, time, radiation dose, etc.). date of the revisions, revised sections, and reasons for revi- 1.11. Parametric release sion. A release procedure based on an evaluation of the produc- h) Technical requirements for the applicable sterilization tion records and critical parameters of the sterilization validation process (temperature, humidity, pressure, time, radiation i) Other required matters for the implementation of the dose, etc.) based on the results of validation, in lieu of applicable sterilization validation release based on testing results of the final product. 2.3.2. The following sterilization validation is implemented according to the plan defining the items above. 2. Sterilization Validation a) When the manufacturing license and additional 2.1. Subject of the Implementation (modification) licenses for product production are obtained, A manufacturer of sterile pharmaceuticals (hereafter, implementation items for the sterilization validation to be ``manufacturer'') must establish a quality system, implement executed product sterilization validation for the categories below as a 1 Product qualification general rule, and continuously control the sterilization 2 Facility/equipment qualification process based on the results of the sterilization validation. 1) Installation qualification a) Sterilization process 2) Operation qualification b) Sterilization process support system 3 Performance qualification 2.2. Documenting Sterilization Validation Procedure 1) Physical performance qualification 2.2.1. The manufacturer must prepare a ``Sterilization 2) Microbiological performance qualification Validation Procedure'' defining the items listed below b) Sterilization validation to be executed until it is time regarding the procedures for managing the sterilization to renew the manufacturing license JP XVI General Information / Microorganisms 2223

1 Re-validation when there are changes c) Bioburden data for the raw materials and product be- 2 Periodic re-validation (The items implemented, etc. fore sterilization must be determined based on a consideration of d) Datarelatedtothesterilization indicators relevant factors such as the sterilization method.) e) Data on the maintenance management of the steriliza- 2.3.3. Evaluate the results of the sterilization validation tion process and sterilization process support systems and verify that sterility is assured. f) Data on the management of sterilization parameters 2.3.4. Make a written report of the results of the steriliza- g) Data on the calibrations of measurement equipment tion validation to the manufacturer's authorized person. h) Re-validation data 2.3.5. Perform the day-to-day management of the steriliza- i) Other tion process. 7. Critical Control Points 3. Microorganism Control Program The important control points for each sterilization method When parametric release is adopted, it is important to are presented. control the bioburden in the raw materials of the product, 7.1. Moist heat sterilization the containers and stoppers, and in the product before Moist heat sterilization is a method for killing microorgan- sterilization. The bioburden is measured with a previously isms in which saturated water vapor is generated or intro- specified method and frequency, and when required, surveys duced into a sterilization chamber at the appropriate temper- of the characteristics of the isolated microorganisms are ature and pressure, and the chamber is then heated for a cer- made to investigate their resistance to the applicable steriliza- tain period of time. It is roughly classified into saturated tion method. Refer to the ``Microbiological Evaluation of vapor sterilization, in which the target microorganisms are Process Areas for Sterile Pharmaceutical Products'' regard- directly exposed to the saturated vapor, and unsaturated ing the method for evaluating the environmental microor- vapor sterilization, in which the fluid inside a container, ganisms in the processing areas of pharmaceutical products. such as an ampule, is subjected to moist heat energy or highfrequency energy from the outside. 4. Sterilization Indicators 7.1.1. Important control points Biological indicators (BI), chemical indicators (CI), and A process control procedure must be created, specifying dosimeters are among the means used to monitor a steriliza- the process parameters that affect the sterile quality of the tion process and as indices of sterility (refer to Terminal pharmaceutical product, and the permissible range of varia- Sterilization and Sterilization Indicators). When using sterili- tion for each parameter. The important control points for zation indicators it is important to consider environmental the moist heat sterilization are indicated below. and human safety, and to take all necessary precautions. The a) Heating history (usually indicated by F value) BI used for sterilization validation and daily process control 0 b) Temperature must be defined in the specification, and recorded in writing. c) Pressure When BI are used for daily process control it must be veri- d) Time fied that the loading pattern on the form, product, or simu- e) Product loading format/loading density lated product has a resistance equal to or greater than that f) Other necessary matters used for the microbiological performance qualification. 7.1.2. Utilities 5. Establishment of a Change Control System The utilities and control devices required for moist heat Changes which have a large effect on the sterile quality, sterilization determine the quality and precision. such as changes in sterilization equipment, loading pattern, a) Quality of the vapor used and sterilization conditions, correspond to changes of the b) Quality of the air introduced into the sterilization parametric release conditions for the relevant pharmaceuti- chamber to restore pressure, etc. cal product. A change control system must be defined in the c) Quality of the water used for cooling sterilization validation procedure; and when there are d) Precision of the temperature control devices changes in the causes of variation that have been previously e) Precision of the pressure control devices specified, there must be an investigation of the causes of f) Precision of the time control devices variation and of acceptable conditions to verify that the g) Other pharmaceutical product is guaranteed always to conform to 7.2. Ethylene oxide gas sterilization the quality standards. Furthermore, before modifications are Ethylene oxide gas allows sterilization at low tempera- made to a sterilization process that has been validated, it is tures, so there is typically little injury to the substance being mandatory to obtain approval for the implementation of the sterilized; however, since the gas is toxic it must be handled modifications in question from the appropriate authorized with extreme caution. The sterilization process consists of person. preconditioning, a sterilization cycle, and aeration. The preconditioning is performed before the sterilization cycle to 6. Release Procedure process the product so that temperature and relative hu- A release procedure must be created to clarify the condi- midity in the room or container are within the range in the tions required for shipment based on parametric release of specifications. The sterilization cycle indicates the stage at terminally sterilized products. The following points must be which the actual sterilization is performed, and consists of evaluated and recorded when a product is released. removal of the air, conditioning (when used), injection of Depending on the sterilization method, some of these the sterilization gas, maintenance of the sterilization condi- items may be omitted or modified. tions, removal of the sterilization gas, and replacement of a) Batch record the air. The aeration is the process of eliminating the residual b) Microorganism evaluation data of production en- ethylene oxide gas from the product, either inside the sterili- vironment 2224 Microorganisms / General Information JP XVI zation chamber or in a separate location. by secondarily generated electrons, while in the case of the 7.2.1. Important control points electron beam, the cells are killed by the electrons generated The important control points for the ethylene oxide gas directly from the electron accelerator. For this reason, the sterilization are indicated below. processing time for electron beam sterilization is generally 7.2.1.1. Preconditioning (when performed) shorter than that for g-ray sterilization; but, since the a) Time, temperature, humidity penetration of the g-rays is better than that of the electron b) Product loading pattern/loading density beam, there must be appropriate consideration of the density c) Sterilization loading temperature and/or humidity and thickness of the substance being sterilized when choos- d) Time from the end of preconditioning until the start ing between these methods. For an irradiation sterilization of the sterilization process, the control procedures primarily make use of e) Other necessary matters dosimeters and measure the absorbed dose in the substance 7.2.1.2. Conditioning being sterilized. This is called dosimetric release. a) If pressure reduction is performed, the pressure 7.3.1. Important control points achieved and required time The important control points for the irradiation steriliza- b) Reduced pressure maintenance period tion are indicated below. c) Time, temperature, pressure, humidity 7.3.1.1. g-ray radiation d) Sterilization loading temperature and humidity a) Irradiation time (timer setting or conveyor speed) e) Other necessary matters b) Absorbed dose 7.2.1.3. Sterilization cycle c) Product loading pattern a) Pressure increase, injection time, and final pressure d) Other necessary matters for the injection of the sterilization gas 7.3.1.2. Electron beam and X-ray radiation b) Concentration of the ethylene oxide gas (it is desirable a) Electron beam characteristics (average electron beam to analyze directly the gas concentration inside the steriliza- current, electron energy, scan width) tion chamber, but the following alternatives are acceptable if b) Conveyor speed direct analysis is difficult) c) Absorbed dose i) Mass of gas used d) Product loading pattern ii) Volume of gas used e) Other necessary matters iii) Conversion calculation using the initial low pressure 7.3.2. Utilities level and the gas injection pressure A traceable calibration, performed according to national c) Temperature within the sterilization chamber standards, must be performed for the radiation devices and d) Temperature of the loaded products to be sterilized dose measurement systems. This calibration must be per- e) Effect time (exposure time) formed as specified in a written plan in order to verify that f) Product loading pattern/loading density the equipment is kept within the required range of accuracy. g) BI placement points and cultivation results 7.3.2.1. Required calibration items for gamma-radiation h) Other necessary matters equipment 7.2.1.4. Aeration a) Cycle time or conveyor speed a) Time, temperature b) Weighing device b) Loaded sterilized substance temperature c) Dose measurement system c) Pressure variation in the sterilization chamber and/or d) Other the aeration room 7.3.2.2. Required calibration items for electron-beam and d) Rate of change of the air or other gases in the aeration X-ray radiation equipment room a) Electron beam characteristics e) Other necessary matters b) Conveyor speed 7.2.2. Utilities c) Weighing device The utilities and control devices required for ethylene d) Dose measurement system oxide sterilization determine the quality and precision. e) Other a) Quality of the ethylene oxide gas b) Quality of the injected vapor or water References c) Quality of the replacement air after the completion of 1) Validation Standards, PAB Notification No.158, sterilization Ministry of Health and Welfare 1995 d) Quality of the BI 2) Sterilization Validation Standards, PMSB/IGD Notifi- e) Precision of the temperature control devices cation No.1, Ministry of Health and Welfare 1997 f) Precision of the pressure control devices 3) Quality Assurance Standards for Medical Devices, g) Precision of the humidity control devices PAB Notification No.1128, Ministry of Health and h) Precision of the time control devices Welfare 1994 i) Other 4) ISO 9000 series, International Standards for Quality 7.3. Irradiation Sterilization Assurance Irradiation sterilization refers to methods of killing 5) ISO 11134 Industrial moist heat sterilization microorganisms through exposure to ionizing radiation. The 6) ISO 11135 Ethylene oxide sterilization types of ionizing radiation used are gamma-rays (g-rays) 7) ISO 11137 Radiation sterilization emitted from a radioisotope such as 60Co or 137Cs, or elec- 8) ISO 11138 Biological indicators tron beams and bremsstrahling (X-ray) generated from an 9) ISO 11140 Chemical indicators electron accelerator. In the case of g-rays, the cells are killed 10) ISO 11737-1 Microbiological Methods Part 1: Estima- JP XVI General Information / Microorganisms 2225

tion of population of microorganisms on products (decimal reduction time) or absorbed dose (decimal reduc- 11) USP 〈1222〉 Terminally Sterilized Pharmaceutical tion dose) required to cause a 1-logarithm or 90z reduction Products - Parametric Release in the population of test microorganisms under stated exposure conditions. (vii) Sterilization indicator: Indicators used to monitor the sterilization process, or as an index of sterility, including Terminal Sterilization and biological indicators (BI), chemical indicators (CI), Sterilization Indicators dosimeters and the like. 2. Sterilization Sterilization is a process whereby the killing or removal of 2.1. Heat Method all forms of viable microorganisms in substances is accom- In the heat method, microorganisms are killed by heating. plished. It is achieved by terminal sterilization or a filtration 2.1.1. Moist heat method method. For substances to which terminal sterilization can Microorganisms are killed in saturated steam under pres- be applied, an appropriate sterilization method should be sure. In this method, factors which may affect the steriliza- selected in accordance with the properties of the product, in- tion include temperature, steam pressure and exposure time. cluding the packaging, after full consideration of the advan- Therefore, in routine sterilization process control, it is re- tages and disadvantages of each sterilization method, from quired to monitor continuously the temperature, steam pres- among the heat method, irradiation method and gas method. sure and exposure time, and they should be included in the After installation of the sterilizer (including design and de- specifications of the sterilizer. velopment of the sterilization process), validation is required 2.1.2. Dry-heat method to confirm that the sterilization process is properly per- Microorganisms are killed in dry heated air. This method forming its designed function, under conditions of loading is usually conducted in a batch-type dry heat sterilizer or a and unloading of the product, on the basis of sufficient tunnel-type dry heat sterilizer. In this method, factors which scientific evidence. After the process has been validated and may affect the sterilization include temperature and ex- the sterilization of the product commenced, the process must posure time. Therefore, in routine sterilization process con- be controlled correctly, and qualification tests of the equip- trol, it is required to monitor continuously the temperature ment and procedures must be performed regularly. and exposure time, and they should be included in the speci- The bioburden per product, prior to terminal sterilization, fications of the sterilizer. must be evaluated periodically or on the basis of batches. 2.2. Irradiation method Refer to the ISO standard (ISO 11737-1) relevant to biobur- Microorganisms are directly killed by ionizing radiation, den estimation. For a substance to which terminal steriliza- or by the heat generated by microwave radiation. tion can be applied, generally use sterilization conditions 2.2.1. Radiation method such that a sterility assurance level of less than 10－6 can be Ionizing radiations which may be used are gamma (g)rays obtained. The propriety of the sterilization should be judged emitted from a radioisotope such as cobalt 60, an electron by employing an appropriate sterilization process control, beam and bremsstrahlung (X rays) generated from an elec- with the use of a suitable sterilization indicator, and if neces- tron accelerator. Although any procedure can be applied to sary, based on the result of the sterility test. The filtration thermally unstable products with no radioactivity residue, it procedure is used for the sterilization of a liquid product, to is necessary to consider the possibility of material degrada- which terminal sterilization can not be applied. Concerning tion. Although a 25 kGy dose is traditionally used as a the disinfection and/or sterilization necessary for processing sterilization dose, there are some ways to calculate the dose equipment and areas of pharmaceutical products, and per- as follows: the bioburden of the substance to be sterilized is forming microbiological tests specified in the monographs, measured and the sterilization dose is calculated based on the see Disinfection and Sterilization Methods. mean bioburden and the standard resistance distribution 1. Definitions (Method 1 in ISO 11137), the dose is calculated from the The definitions of the terms used in this text are as fraction positive information from a sterility test in which follows. representative product samples are exposed to a substeriliz- (i) Terminal sterilization: A process whereby a product is ing dose (Method 2 in ISO 11137), or the dose is calculated sterilized in its final container or packaging, and which based on the bioburden and D value of the most resistant permits the measurement and evaluation of quantifiable microorganisms (Log method) (see 5.3.). In the case of the microbial lethality. radiation sterilization procedure, factors which may affect (ii) Product: A generic term used to describe raw mate- the sterilization include dose (absorbed dose) and exposure rials, intermediate products, and finished products, to be time. Therefore, in g ray sterilization process control, it is re- sterilized. quired to determine the dose (the absorbed dose) at ap- (iii) Bioburden: Numbers and types of viable microor- propriate intervals and to monitor continuously the exposure ganisms in a product to be sterilized. time in terms of the operating parameters (the conveyer (iv) Sterility assurance level (SAL): Probability of a via- speed, the cycle time). The dose control mechanism should ble microorganism being present in a product unit after ex- be included in the specifications of the sterilizer. In the case posure to the proper sterilization process, expressed as 10－n. of electron beam or bremsstrahlung irradiation, it is required (v) Integrity test: A non-destructive test which is used to to monitor the acceleration voltage, the beam current and predict the functional performance of a filter instead of the beam scanning width besides the above-mentioned items. microorganism challenge test. 2.2.2. Microwave method (vi) D value: The value which shows the exposure time Microorganisms are killed by the heat generated by micro- 2226 Microorganisms / General Information JP XVI wave radiation, usually at the frequency of 2450 ± 50 MHz. lent effect in the sterilization of biological indicator. This method is applied to liquids or water-rich products in However, when the spores are suspended in liquid, it is nec- sealed containers. Since a glass or plastic container may be essary to ensure that the resistance characteristics of the destroyed or deformed due to the rise of the inner pressure, spores are not affected due to germination. the containers must be certified to be able to withstand the heat and the inner pressure generated during microwave Typical examples of biological indicator sterilization. Leakage of electromagnetic radiation must be at a sufficiently low level to cause no harm to humans and Representative Sterilization Strain name no interference with radio communications and the like. In microorganisms* this method, factors which may affect the sterilization in- Moist heat Geobacillus ATCC 7953, NBRC clude temperature, processing time and microwave output method stearo- 13737, JCM 9488 power. Therefore, in routine sterilization process control, it thermophilus ATCC 12980, NBRC is required to monitor continuously the temperature, time 12550, JCM 2501 and the microwave output power, and they should be in- Dry heat Bacillus cluded in the specifications of the sterilizer. method atrophaeus ATCC 9372, NBRC 2.3. Gas method 13721 Ethylene oxide (EO) is widely used as a sterilization gas. Gas method Bacillus Since EO gas has an explosive nature, a 10 – 30z mixture atrophaeus ATCC 9372, NBRC with carbon dioxide is commonly used. Also, as EO gas is a 13721 strong alkylating agent, it can not be applied to the products which are likely to react with or absorb it. Furthermore, be- * In addition to these microorganisms, other microorgan- cause EO gas is toxic, the residual concentration of EO gas isms with the greatest resistance to the sterilization proce- and other secondarily generated toxic gases in products steri- dure concerned, found in the bioburden, can be used as lized with EO gas must be reduced to less than the safe levels the biological indicator. thereof by means of aeration and the like before the product is shipped. In this method, factors which may affect the sterilization include temperature, gas concentration (pres- 4.1.1. D value of BI sure), humidity and exposure time. Therefore, in routine Methods for determination of the D value include the sur- sterilization process control, it is required to monitor con- vival curve method and the fraction negative method tinuously the temperature, gas concentration (pressure), hu- (Stumbo, Murphy & Cochran procedure, Limited Spearman- midity and exposure time, and they should be included in the Karber procedure and the like). In using marketed BIs, it is specifications of the sterilizer. usually unnecessary to determine the D value before use if the D value indicated on the label has been determined by a 3. Filtration method standardized biological indicator evaluation resistometer Microorganisms are removed by using a sterilizing filter (BIER) under strictly prescribed conditions in accordance made of an appropriate material. However, this method is with ISO 11138-1. It is acceptable that the D value indicated not intended for microorganisms smaller than bacteria. on the label shows a scattering of not more than ±30 Generally, a sterilizing filter challenged with more than 107 seconds. microorganisms of a strain of Brevundimonas diminuta 4.1.2. Setting up procedure of BI (ATCC 19146, NBRC 14213, JCM 2428), cultured under the 4.1.2.1. In the case of dry materials appropriate conditions, per square centimeter of effective A Dry type BI is placed at predetermined cold spots in the filter area should provide a sterile effluent. In this method, product to be sterilized or a suitable product showing an factors which may affect the sterilization include pressure, equivalent effect in the sterilization. The BIs are usually pri- flow rate, filter unit characteristics and the like. In routine mary packaged in the same way as the product, including a filtration process control, it is required to perform integrity secondary packaging, if applicable. tests of the sterilizing filter after each filtration process (also 4.1.2.2. In the case of wet materials prior to the filtration process, if necessary). Spores are suspended as the BI in the same solution as the 4. Sterilization Indicators product or in an appropriate similar solution, and should be 4.1. Biological indicator (BI) placed at cold spots in the sterilizer. A BI is prepared from specific microorganisms resistant to 4.1.3. Culture conditions of BI the specified sterilization process and is used to develop and/ Soybean casein digest medium is generally used. General or validate a sterilization process. The dry type BI is classi- culture conditions are at 55 – 609C for 7 days in the case of fied into two kinds. In one, bacterial spores are added to a G. stearothermophilus andat30–359C for 7 days in the carrier such as filter paper, glass or plastic and then the car- case of B. atrophaeus. riers are dried and packaged. In the other, bacterial spores 4.2. Chemical indicator (CI) are added to representative units of the product to be steri- CI is an indicator which shows a color change of a sub- lized or to simulated products. Packaging materials of the BI stance applied to a paper slip, etc. as a result of physical should show good heat penetration in dry heat sterilization and/or chemical change due to exposure to heat, gas or ra- and good gas or steam penetration in ethylene oxide and diation. The CI can be classified into three types. The first is moist heat sterilizations. It should be confirmed that any employed to identify whether or not sterilization has already carrier does not affect the D value of the spores. In the case been implemented, the second is employed to control the of a liquid product, the spores may be suspended in the same sterilization process (for example, its color changes after solution as the product or in a solution showing an equiva- sterilization for a sufficient time), and the third is the Bowie JP XVI General Information / Crude Drugs 2227

&Dicktypeusedtoevaluatetheeffectivenessofairremoval D: D value of the BI during the pre-vacuum phase of the pre-vacuum sterilization N: Sterility assurance level cycle. N0: Maximum bioburden count in the product 4.3. Dosimeter 5.4. Absolute bioburden method In the radiation (g-ray) method, the sterilization effect de- The sterilization conditions are determined by employing pends on the absorbed radiation dose, so the sterilization the D value of the most resistant microorganism found in the process control is mainly performed by measuring the dose. product or environment by the resistant estimation and being A dosimeter is installed at a position corresponding to the based on the bioburden count in the product. Generally, a minimum dose region of an exposed container or a position count of mean bioburden added three times of its standard where the dose is in a known relation to that in the above deviation obtained by an extensive bioburden estimation is region. Measurement should be done for each radiation employed as the bioburden count. When this procedure is batch. If there are many containers in the same batch, used, it is required to make frequent counting and resistance dosimeters should be employed so that more than one determination of microorganisms in daily bioburden estima- dosimeter is always installed at the effective radiation section tion. of the irradiation chamber. It should be noted that dosimeters may be affected by environmental conditions 6. References (temperature, humidity, ultraviolet light, time until reading, ( i ) ISO 11134 Industrial moist heat sterilization etc.) before and during irradiation. Practical dosimeters for ( ii ) ISO 11135 Ethylene oxide sterilization g-ray and bremsstrahlung sterilization include the dyed poly- (iii) ISO 11137 Radiation sterilization methylmethacrylate dosimeter, clear polymethylmethacry- (iv) ISO 11138 Biological indicators late dosimeter, ceric-cerous sulfate dosimeter, alanine-EPR ( v ) ISO 11140 Chemical indicators dosimeter and the like. A dosimeter for gamma radiation (vi) ISO 11737 Microbiological methods can not generally be used for sterilization process control Part 1: Estimation of population of mi- with an electron beam of less than 3 MeV energy. Dosimeters croorganisms on products for electron beam sterilization include the cellulose acetate dosimeter, radiochromic film dosimeter and the like. A practical dosimeter must be calibrated against an appropriate national or international standard dosimetry system. G5 Crude Drugs 5. Determination of sterilization conditions using microorganism as an indicator Taking account of the characteristics upon the sterilization Aristolochic Acid concerned, bioburden, etc. of a product to be sterilized, Aristolochic acid, which occurs in plants of Aristolochia- chose a suitable method from the followings and determine ceae, is suspected to cause renal damage. It is also reported the conditions. to be oncogenic (see References). 5.1. Half-cycle method Aristolochic acid toxicity will not be a problem if crude In this method, a sterilization time of twice as long as that drugs of the origin and parts designated in the JP are used, required to inactive all of 106 counts of BI placed in the but there may be differences in crude drug nomenclature be- product is used, regardless of bioburden count in the tween different countries, and it is known that crude drug product being sterilized or the resistance of the objective preparations not meeting the specifications of the JP are cir- microorganisms to the sterilization. culating in some countries. Consequently, when crude drugs 5.2. Overkill method or their preparations are used, it is important that the mate- In this method, a sterilization condition giving a sterility rials should not include any plant containing aristolochic assurance level of not more than 10－6 counts is used, regard- acid. less of bioburden count in the product being sterilized or the Since Supplement I to JP14, the test for aristolochic acid I resistance of the objective microorganisms to the steriliza- was added to the Purity under Asiasarum Root, which con- tion. Generally, a sterilization condition providing 12 sists of the rhizome and root. Because the aerial part of the logarithmic reduction (12D) of a known count of BI of more plant may contain aristolochic acid and may have been im- than 1.0 D value is used. properly contaminated in Asiasarum Root. 5.3. Combination of BI and bioburden It is considered that Akebia Stem, Sinomenium Stem and Generally, a count of mean bioburden added three times Saussurea Root do not contain aristolochic acid, unless of its standard deviation obtained by an extensive bioburden plants of origin other than that designated in the JP are estimation is considered as the maximum bioburden count, used. However, contamination of aristolochic acid might and the sterilization time (or radiation dose) is calculated occur, as mentioned above. In this case, the test described in with the bioburden count based on an objective sterility as- the Purity under Asiasarum Root is useful for checking the surance level. When this procedure is used, it is required to presence of aristolochic acid. determine the resistance of the bioburden to the sterilization References: as well as the bioburden count in the product being steri- Drug & Medical Device Safety Information (No.161) lized. If a more resistant microorganism than the BI spore is (July, 2000). found in the bioburden estimation, it should be used as the New England Journal of Medicine (June 8, 2000). BI. Mutation Research 515, 63 – 72 (2002). N Sterilization time (or radiation dose) ＝ D × log 0 N 2228 Crude Drugs / General Information JP XVI

tion, which is an identification test. In the above scientific Purity Tests on Crude Drugs paper, it was shown that these 4 plant species can be clearly classified by comparing the nucleotide sequences of the ITS Using Genetic Information mentioned above, and that the species can be easily classified without performing sequence analysis by performing PCR The first step in the quality assurance of natural products using a species specific primer set or by using a restriction is the use of raw materials from the right part of the right enzyme which recognizes species specific sequence. origin (the right source). Therefore, it is clearly stated in Ar- In validation studies, the simplicity of the test is given ticle 4 of the General Rules For Crude Drugs that the source maximum consideration. We examined methods that observe of a crude drug is the approval or rejection criteria. There PCR amplification bands using species specific primer sets are various methods for differentiating the sources of crude (Mutant Allele Specific Amplification: Method 1) and that drugs, such as morphological methods, organoleptic tests, observe DNA fragments produced by restriction enzyme and chemical methods, and appropriate methods for each treatment of the PCR products, which are prepared using a are described in the individual monographs. Morphological primer set common to each plant source (PCR-Restriction methods, organoleptic tests, and chemical methods are Fragment Length Polymorphism: Method 2), and do not discrimination methods for species that are based on the involve nucleotide sequence analyses. In these methods phenotypic characteristics of the crude drugs. On the other based on PCR, an extremely small amount of template DNA hand, together with recent progress in molecular biology is amplified to billions to hundred-billions times. Therefore, techniques and the accumulation of genetic information on when using them as identification tests for powdered crude plants, differentiating methods of crude drugs based on drugs, the target DNA fragment can be observed even if the genotypes have been established. Unlike morphological and vast majority of the crude drug for analysis is not appro- other methods that are based on phenotypic characteristics, priate plant species and there is only a minute amount of the genotypic methods are not affected by environmental powder from a crude drug derived from a suitable plant. factors. Also, the methods have several advantages, such as (Consequently, in identification tests, either a cut or a whole specialized expertise and skill for classification are not need- crude drug must be used, as long as one is careful to avoid ed, and objective results are easily obtained. contamination by powder originating from other crude The evolution of living organisms is accomplished by drugs.) On the other hand, when used as a purity test, the genetic mutation, and differences among the nucleotide form of the crude drug is irrelevant as long as the gene sequences of genes of closely related species reflect the strain amplification is performed properly and the target gene is relationships between the species. Based on this theory, in not polymorphic, so if DNA fragments of an inappropriate recent years methods that classify species phylogenetically plant are confirmed in the purity test, regardless of the form using the nucleotide sequence of rDNA that codes for of the crude drug, it becomes clear there is contamination by ribosomal RNA (rRNA) on the nuclear genome have been an inappropriate crude drug. adopted. In the same way, the sequence of this rDNA is also The methods shown here are reference information and at most often used in the classification of higher plants based the present stage results obtained using the methods do not on the genotype. In particular, it is very easy to classify affect the approval or rejection of the crude drug in each closely related species using the intergenic transcriber space monograph. Furthermore, by performing the sequence ana- (ITS) region of the rDNA region, since by comparison with lysis outlined in the previous paper, it goes without saying the coded gene region nucleotide substitution is more often that more accurate decision concerning the source species undertaken. Furthermore, since the genes on the nuclear can be made. genome originate from the parents' genom, there is an advantage that interspecies hybrids can be detected. Higher 1. DNA Amplification Equipment plants also have mitochondrial genes and chloroplastic DNA amplification equipment is used to amplify the DNA genes. Although the genes on these genomes are also often which is extracted from a crude drug and then purified. used for classification, interspecies hybrids cannot be con- Since there are slight differences in the methods of tempera- firmed because the genes are normally uniparental in- ture control, and so on depending on the equipment used, heritance. there may be differences in the intensity, etc. of the PCR The two methods presented here have been developed amplification bands even if PCR is carried out under the based on the reported identification methods of Atractylodes stipulated conditions. Therefore, when judging results based Lancea Rhizome and Atractylodes Rhizome1,2) utilizing the solely on the presence or absence of PCR amplification gene sequence of the ITS of rDNA. Inter-laboratory valida- bands as in 3. Methods 1, confirm that only proper amplifi- tion study for purity test of Atractylodes Rhizome targeted cation bands are obtained when performing PCR using DNA for Atractylodes Lancea Rhizome have been completed. The obtained from samples confirmed beforehand to be the plant sources for Atractylodes Lancea Rhizome stipulated in source species. If proper amplification bands are not ob- the individual monographs are Atractylodes lancea De tained, the PCR temperature conditions should be slightly Candolle or A. chinensis Koidzumi (Compositae), while adjusted. This equipment can be used for the restriction en- those for Atractylodes Rhizome are A. japonica Koidzumi zyme treatment in 4. Method 2. ex Kitamura or A. ovata De Candolle (Compositae). The ap- 2. General precautions proval or rejection of the source of Atractylodes Lancea Crude drugs are different from fresh plants in that they Rhizome is, in principle, determined by the description of are dried products and a certain amount of time has passed the crude drug, including microscopy, while that of Atrac- since they were harvested. Therefore, in many cases the tylodes Rhizome is determined by the description of the DNA has undergone fragmentation. Furthermore, various crude drug, including microscopy, together with color reac- substances that can block or interfere with the PCR reaction JP XVI General Information / Crude Drugs 2229 may be present in the plant. For these reasons, the extraction and dNTP are provided as adjuncts to the enzyme. When and purification of template DNA is the process that should conducting purity tests on Atractylodes Lancea Rhizome in receive the greatest amount of attention. In the case of Atractylodes Rhizome, the primer sets used are C and D (C Atractylodes crude drugs, the periderm should be removed is positive with A. lancea, D is positive with A. chinensis)as using a clean scalpel or other clean instrument before pul- described in the paper mentioned above (J. Nat. Med.60, verizing the sample because very often there are inhibitory 149 – 156, 2006), however, when a combination of primer A substances present in the periderm. and B is used, it is possible to confirm the source species of each of the respective specimens. In order to confirm that 3. Method 1 (Mutant Allele Specific Amplification the DNA has been extracted correctly, the reaction solution Method) containing the positive control primer (Pf and Pr) as shown Generally, this method is referred to as Mutant Allele below should be prepared. In addition, the negative control Specific Amplification (MASA) or Amplification Refractory solutions which are not containing DNA sample or either of Mutation System (ARMS), and it provides nucleotide the primer sets should be prepared and simultaneously con- sequence information of sample-derived template DNA, duct PCR. based upon the presence or absence of DNA amplification in PCR using a species specific primer set. Pf: 5?-CAT TGT CGA AGC CTG CAC AGC A-3? 3.1. Procedure Pr: 5?-CGA TGC GTG AGC CGA GAT ATC C-3? The following is an example procedure. The PCR reaction is performed under the following condi- 3.1.1. Preparation of template DNA tions: starting the reaction at 959C for 10 minutes, 30 cycles There are various methods with which to extract and puri- of 0.5 minutes at 959C and 0.75 minutes at 689C(699C only fy DNA from the samples. It is recommended that commer- when using the primer set C), terminate reaction at 729Cfor cially available DNA extraction kits be used when consider- 7 minutes, and store at 49C. The resulting reaction mixture is ing their advantages of not using any noxious reagents and used for the following process as PCR amplification reaction not requiring any complicated purification procedures. In solution. this case, attention should be paid to the final amount (con- 3.1.4. Agarose gel electrophoresis and detection of PCR centration) of DNA obtained, and the initial amount of ini- products tial sample and the volume of liquid to elute the DNA need After completion of the PCR reaction, mix 5 mLofthe to be controlled. When extraction and purification are per- PCR amplification reaction solution with an appropriate formed using silica gel membrane type kits stipulated in volume of gel loading buffer solution, add the mixture to the notifications3) related to inspection methods of the foods wells of 2 w/vz agarose gel, and then perform electrophore- produced by recombinant DNA techniques, it is appropriate sis using 1-fold TAE buffer solution (refer to General Infor- to use 200 mg of sample, 1 mL of AP1 buffer solution, 2 mL mation, Rapid Identification of Microorganisms Based on of RNase A, and 325 mL of AP2 buffer solution. Also, the Molecular Biological Method). Run in parallel an appropri- most important things are that the supernatant loaded on the ate DNA molecular mass standard. Electrophoresis is termi- first column is clear and that there is no need to load 1 mL nated when the bromophenol blue dye in the gel loading unreasonably. Furthermore, 50 mL is an appropriate volume buffer has advanced to a point corresponding to 1/2 to 2/3 used in the final elution of the DNA, and normally the initial the length of the gel. eluate is used as the DNA sample stock solution. Stain the gel after electrophoresis when not using gel 3.1.2. Confirmation of purity of DNA in DNA sample stained in advance with ethidium bromide. Place the gel that stock solution and assay of DNA has undergone electrophoresis and staining in a gel image The purity of the DNA in the stock solution can be con- analyzer, irradiate with ultraviolet light (312 nm), and de- firmed by the OD /OD ratio using a spectropho- 260 nm 280 nm tected its electrophoresis pattern. Compare this to the DNA tometer. A ratio of 1.5 indicates that the DNA has been ade- molecular mass standard and determine the absence or quately purified. The amount of DNA is calculated using presence of the target amplification band. 1OD ＝ 50 mg/mL. The measurement mentioned above 260 nm 3.2. Judgement is performed using appropriately diluted DNA sample stock Confirm at first that a 305 bp band is found with the reac- solution. Based on the results obtained, dilute with water to tion solution to which the positive control primer set has the concentration needed for the subsequent PCR reactions, been added, and confirm there are no bands in a solution dispense the solution into micro tubes as the sample DNA with no primer sets and a solution with no sample DNA so- solution, and if necessary store frozen at not over －209C. lution. Next, if a 226 bp band is confirmed when the primer The dispensed DNA sample is used immediately after thaw- set C is added or if a 200 bp band is confirmed when the ing and any remaining solution should be discarded and not primer set D is added, the sample is judged to be Atrac- refrozen. If the concentration of the DNA sample stock so- tylodes Lancea Rhizome (in the case of cut crude drug, con- lution does not reach the concentration stipulated in PCR, it tamination of Atractylodes Lancea Rhizome is observed) is used as a DNA sample solution. and it is rejected. The sample is judged not to be Atrac- 3.1.3. PCR tylodes Lancea Rhizome (in the case of cut crude drug, there When a commercially available PCR enzyme mentioned in is no contamination of Atractylodes Lancea Rhizome) and the above notification4) is used, it is appropriate that 25 mL the purity test is acceptable if a 305 bp band is confirmed of reaction mixture consisting of 2.5 mL of the PCR buffer with the positive control primer set, bands are not observed solution containing magnesium, dNTP (0.2 mmol/L), 5?and in reaction solution without primer and reaction solution 3? primer (0.4 mmol/L), Taq DNA polymerase (1.25 units), without DNA sample solution, and a 226 bp band is not ob- and 5 mLof10ng/mL sample DNA solution (50 ng of DNA) served with the primer set C and a 200 bp band is not ob- is prepared on ice. Among them, the PCR buffer solution served with the primer set D. If a band is not observed with 2230 Crude Drugs / General Information JP XVI the positive control primer, it is to be concluded that the template DNA solution. DNA extraction failed and the procedure should be started The PCR reaction is performed under the following condi- over again from the DNA extraction step. If bands are con- tions: 959C for 10 minutes, 40 cycles of 959C for 0.5 minute, firmed in reaction solutions without primer sets or without 659C for 0.25 minute, and 729C for 0.25 minute and 729C DNA sample solution, it should be assumed that there was for 7 minutes. Store the solution at 49C, and use this solu- an error in the PCR procedure and therefore the procedure tion as the PCR amplified solution. A negative control (con- should be repeated again from the step 3.1.3. PCR. taining water instead of the template DNA solution) must be included in the procedure. 4. Method 2 (PCR-Restriction Fragment Length Polymor- The sequence of each primer is as follows: phism) Generally, this method is referred to as PCR-Restriction 5?-primer: 5?-GGC ACA ACA CGT GCC AAG GAA Fragment Length Polymorphism (RFLP), and it provides AA-3? nucleotide sequence information of sample-derived template 3?-primer: 5?-CGA TGC GTG AGC CGA GAT ATC C-3? DNA, based upon the DNA fragment pattern produced by 4.1.3. Restriction enzyme treatment restriction enzyme treatment of the PCR products, which are The treatment is performed on individual reaction solu- amplified by using a primer set common to the DNA se- tions using two enzymes, Fau IandMsp I. In the case of quence of the objective plant. Fau I, to an appropriate amount of the reaction solution, The test is performed with 25 samples randomly taken composed of a reaction buffer containing 1.0 unit of en- from a lot, and each sample is designated with a number zyme, add 3.0 mL of PCR products while cooling in an ice from 1 to 25. Differentiation of the sources is performed by bath to make 15.0 mL. In the case of Msp I, to an appropri- individual PCR-RFLP measurement of the samples, and de- ate amount of the reaction solution, composed of a reaction cision of the acceptability of the purity is dependent on how buffer containing 20.0 units of enzyme, add 3.0 mLofPCR many nonconforming samples are present in the first 20 sam- products while cooling in an ice bath to make 15.0 mL. Incu- ples, taken in numerical order, for which judgement is possi- bate these solutions at the temperature recommended by the ble as described below. manufacturer for 2 hours, and then inactivate the enzyme by 4.1. Procedure heating at 729C for 10 minutes. The negative control of the The following is an example procedure. PCR reaction is also treated in the same manner. 4.1.1. Preparation of template DNA 4.1.4. Agarose gel electrophoresis and detection of DNA There are various methods with which to extract and puri- fragments fy DNA from the samples. It is recommended that commer- After the restriction enzyme treatment, mix the total cially available DNA extraction kits be used, when consider- amount of the reaction solution and an appropriate amount ing their advantages of not using noxious reagents and not of the gel loading buffer solution, place it in a 4 w/vz requiring complicated purification procedures. Recently, agarose gel well, and carry out electrophoresis with 1-fold PCR reagents that inhibit the effect of PCR enzyme- concentrated TAE buffer solution (see ``Rapid Identification inhibiting substances present in samples have become com- of Microorganisms Based on Molecular Biological mercially available, and by using these reagents, it is possible Methods'' under General Information). Carry out the elec- to prepare the template DNA from the sample simply by in- trophoresis together with appropriate DNA molecular mass cubating the sample with the DNA extraction reagent. Here, standard. Stop the electrophoresis when the bromophenol a recommended DNA preparing procedure using such PCR blue included in the loading buffer solution has moved about reagents is described for the convenience of experimenters. 2 cm from the well. The 4 w/vz agarose gel is sticky, Cut 20 mg of the sample into small pieces with a clean difficult to prepare and hard to handle, so that it is better to knife, add 400 mL of the DNA extraction reagent, and incu- use a commercially available precast gel. bate at 559C overnight (16 – 18 hours). Then heat at 959C After the electrophoresis, stain the gel, if it is not already for 5 minutes to inactivate the enzyme in the reagent. Centri- stained, with ethidium bromide, and observe the gel on an fuge to precipitate the sample, and use 50 mL of the superna- illuminating device under ultraviolet light (312 nm) to con- tant liquid as the template DNA solution. The DNA solution firm the electrophoretic pattern. prepared in this method can not be used for concentration 4.2. Judgement measurement based on OD , because it contains many 260 nm 4.2.1. Judgment of each sample foreign substances affecting OD value from the sample. 260 Confirm that no band is obtained with the negative con- The composition of the DNA extraction reagent is as trol of the PCR, other than the primer dimer (about 40 bp) follows: band. A sample treated with Fau I, showing bands of about 2-Amino-2-hydroxymethyl-1.3- 20 mmol/L 80 bp and 60 bp, or that treated with Msp I, showing bands propanediol-hydrochloric acid (pH 8.0) of about 90 bp and 50 bp, is judged as Atractylodes Lancea Ethylenediamine tetraacetate 5 mmol/L Rhizome. A sample not showing any band other than a band Sodium chloride 400 mmol/L at about 140 bp and the primer dimer band is judged as Sodium dodecyl sulfate 0.3z Atractylodes Rhizome. If a sample does not show any band Proteinase K 200 mg/mL other than the primer dimer band, it is considered that PCR 4.1.2. PCR products were not obtained, and judgement is impossible for In the method using the PCR enzyme and PCR reagent as the sample. described2), the reaction mixture is prepared on an ice bath in 4.2.2. Judgment of the purity a total volume of 20 mL of a solution containing 10.0 mLof Judgment of the purity is based on the result of the judg- 2-fold concentrated PCR reagent, 5?-and3?-primers (0.5 ment of each sample. If there is no sample that is judged as mmol/L), Taq DNA polymerase (0.5 units) and 0.5 mLof Atractylodes Lancea Rhizome among 20 samples taken in JP XVI General Information / Crude Drugs 2231 order of the numbering, excluding any sample for which judgement is impossible, the lot is acceptable for purity. When On the Scientific Names of Crude there is one sample that is judged as Atractylodes Lancea Rhizome among the 20 samples, perform the same test with DrugsListedintheJP 25 newly taken samples from the lot, and if there is no sam- The notation system of the scientific names for the origi- ple that is judged as Atractylodes Lancea Rhizome, the lot is nal plants and animals of crude drugs listed in JP is not acceptable for purity. When there is a sample that is judged necessary the same as the taxonomic system used in the as Atractylodes Lancea Rhizome in the second test, or there literature. The reason for this is that the JP is not an aca- is more than one sample that is judged as Atractylodes Lan- demic text, but an ordinance. The relationship between the cea Rhizome in the first test, the lot is not acceptable for scientific names used in the JP and those generally used purity. taxonomically is indicated in the following table, to avoid misunderstanding by JP users owing to differences in the 5. Reference notation system. 1) Y. Guo, et al., J. Nat. Med. 60, 149 – 156 (2006). 2) K. Kondo, et al., J. Jpn. Bot. 84, 356 – 359 (2009). 3) Notification No. 110, Director of Food Health Depart- ment, March 2001; Partial Amendment: Notification No. 0629002, 2.2.1.2, Director of Food Safety Depart- ment, June 2006. 4) Notification No. 0629002, 2.1.3.1.1, Director of Food Safety Department, June 2006.

Scientific names used in the JP and the corresponding taxonomic names

Scientific names used in the JP ＝Scientific names being used taxonomically (Combined notation, Stan- dard form for author or authors)

Crude Drug Scientific names that are different from those written in JP but identical Family to them taxonomically or being regarded as identical, and typical sub- classified groups belonging to their species. The names marked with ``*'' are those being written together in JP.

Acacia Acacia senegal Willdenow アラビアゴム＝Acacia senegal (L.) Willd. Leguminosae Other species of the same genus

Achyranthes Root Achyranthes fauriei Leveille et Vaniot ゴシツ＝Achyranthes fauriei H. Lev. & Vaniot Amaranthaceae Achyranthes bidentata Blume

Agar Gelidium elegans Kuetzing カンテン Other species of the same genus Gelidiaceae

Other red algae

Akebia Stem Akebia quinata Decaisne モクツウ＝Akebia quinata (Thunb. ex Houtt.) Decne. Lardizabalaceae Akebia trifoliata Koidzumi ＝Akebia trifoliata (Thunb.) Koidz.

Alisma Rhizome Alisma orientale Juzepczuk タクシャ＝Alisma orientale (Sam.) Juz. Alismataceae Alisma plantago-aquatica L. var. orientale Sam. 2232 Crude Drugs / General Information JP XVI

Aloe Aloe ferox Miller アロエ＝Aloe ferox Mill.

Hybrid between Aloe ferox Miller and Aloe africana Miller Liliaceae Aloe africana Miller ＝Aloe africana Mill.

Hybrid between Aloe ferox Miller and Aloe spicata Baker

Alpinia Officinarum Rhizome Alpinia officinarum Hance Zingiberaceae リョウキョウ

Amomum Seed Amomum xanthioides Wallich シュクシャ＝Amomum xanthioides Wall. Zingiberaceae Amomum villosum Lour. var. xanthioides (Wall.)T.L.Wu&Senjen

Anemarrhena Rhizome Anemarrhena asphodeloides Bunge Liliaceae チモ

Angelica Dahurica Root Angelica dahurica Bentham et Hooker filius ex Franchet et Savatier Umbelliferae ビャクシ＝Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav.

Apricot Kernel Prunus armeniaca Linneá キョウニン＝Prunus armeniaca L.

Prunus armeniaca Linnevar.á ansu Maximowicz Rosaceae ＝Prunus armeniaca L. var. ansu Maxim.

Prunus sibirica Linneá ＝Prunus sibirica L.

Aralia Rhizome Aralia cordata Thunberg Araliaceae ドクカツ＝Aralia cordata Thunb.

Areca Areca catechu Linneá Palmae ビンロウジ＝Areca catechu L.

Artemisia Capillaris Flower Artemisia capillaris Thunberg Compositae インチンコウ＝Artemisia capillaris Thunb.

Asiasarum Root Asiasarum sieboldii F. Maekawa サイシン＝Asiasarum sieboldii (Miq.) F. Maek.

Asarum sieboldii Miq.

Asarum sieboldii Miq. var. seoulense Nakai Aristolochiaceae Asiasarum heterotropoides F. Maekawa var. mandshuricum F. Maekawa ＝Asiasarum heterotropoides (F.Schmidt)F.Maek.var.mandshuricum (Maxim.) F. Maek.

Asarum heterotropoides F. Schmidt var. mandshuricum (Maxim.) Kitag.

Asparagus Tuber Asparagus cochinchinensis Merrill Liliaceae テンモンドウ＝Asparagus cochinchinensis (Lour.) Merr.

Astragalus Root Astragalus membranaceus Bunge オウギ＝Astragalus membranaceus (Fisch.) Bunge

Astragalus mongholicus Bunge Leguminosae

Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao JP XVI General Information / Crude Drugs 2233

Atractylodes Lancea Rhizome Atractylodes lancea De Candolle ソウジュツ＝Atractylodes lancea (Thunb.) DC.

Atractylodes chinensis Koidzumi Compositae ＝Atractylodes chinensis (DC.) Koidz.

Hybrid between above species

Atractylodes Rhizome Atractylodes japonica Koidzumi ex Kitamura ビャクジュツ＝Atractylodes japonica Koidz. ex Kitam.

Atractylodes macrocephala Koidzumi Compositae ＝Atractylodes macrocephala Koidz.

* Atractylodes ovata De Candolle ＝Atractylodes ovata (Thunb.) DC.

Bear Bile Ursus arctos Linneá ユウタン＝Ursus arctos L. Ursidae Other animals of the related genus

Bearberry Leaf Arctostaphylos uva-ursi Sprengel Ericaceae ウワウルシ＝Arctostaphylos uva-ursi (L.) Spreng.

Belladonna Root Atropa belladonna Linneá Solanaceae ベラドンナコン＝Atropa belladonna L.

Benincasa Seed Benincasa cerifera Savi トウガシ Benincasa hispida (Thunb.) Cogn. Cucurbitaceae Benincasa cerifera Savi forma emarginata K. Kimura et Sugiyama ＝Benincasa cerifera Savi f. emarginata K. Kimura & Sugiyama

Benzoin Styrax benzoin Dryander アンソッコウ＝Styrax benzoin Dryand. Styracaceae Other species of the same genus

Bitter Cardamon Alpinia oxyphylla Miquel Zingiberaceae ヤクチ＝Alpinia oxyphylla Miq.

Bitter Orange Peel Citrus aurantium Linneá トウヒ＝Citrus aurantium L.

Citrus aurantium Linneá var. daidai Makino Rutaceae ＝Citrus aurantium L. var. daidai Makino

Citrus aurantium L. `Daidai'

Brown Rice Oryza sativa Linneá Gramineae コウベイ＝Oryza sativa L.

Bupleurum Root Bupleurum falcatum Linneá サイコ＝Bupleurum falcatum L. Umbelliferae Bupleurum chinense DC. Bupleurum scorzonerifolium Willd.

Burdock Fruit Arctium lappa Linneá Compositae ゴボウシ＝Arctium lappa L.

Calumba Jateorhiza columba Miers Menispermaceae コロンボ

Capsicum Capsicum annuum Linneá Solanaceae トウガラシ＝Capsicum annuum L. 2234 Crude Drugs / General Information JP XVI

Cardamon Elettaria cardamomum Maton Zingiberaceae ショウズク

Cassia Seed Cassia obtusifolia Linneá ケツメイシ＝Cassia obtusifolia L. Leguminosae Cassia tora Linneá ＝Cassia tora L.

Catalpa Fruit Catalpa ovata G. Don キササゲ Catalpa bungei C. A. Meyer Bignoniaceae ＝Catalpa bungei C. A. Mey.

Chrysanthemum Flower Chrysanthemum morifolium Ramatuelle キクカ＝Chrysanthemum morifolium Ramat. Compositae Chrysanthemum indicum Linneá ＝Chrysanthemum indicum L.

Cimicifuga Rhizome Cimicifuga simplex Turczaninow ショウマ＝Cimicifuga simplex (DC.) Turcz.

Cimicifuga dahurica Maximowicz ＝Cimicifuga dahurica (Turcz.) Maxim. Ranunculaceae Cimisifuga heracleifolia Komarov ＝Cimisifuga heracleifolia Kom.

Cimicifuga foetida Linneá ＝Cimicifuga foetida L.

Cinnamon Bark Cinnamomum cassia Blume Lauraceae ケイヒ

Cinnamon Oil Cinnamomum cassia Blume ケイヒ油 Lauraceae Cinnamomum zeylanicum Nees

Citrus Unshiu Peel Citrus unshiu Marcowicz チンピ＝Citrus unshiu (Swingle) Marcow. Rutaceae Citrus reticulata Blanco `Unshiu'

Citrus reticulata Blanco

Clematis Root Clematis chinensis Osbeck イレイセン Clematis mandshurica Ruprecht ＝Clematis mandshurica Rupr. Ranunculaceae

Clematis hexapetala Pallas ＝Clematis hexapetala Pall.

Clove Syzygium aromaticum Merrill et Perry チョウジ＝Syzygium aromaticum (L.) Merr. & M. L. Perry Clove oil Myrtaceae チョウジ油 * Eugenia caryophyllata Thunberg ＝Eugenia caryophyllata Thunb. Eugenia caryophyllus (Spreng.)Bullock&S.G.Harrison

Cnidium Monnieri Fruit Cnidium monnieri Cusson Umbelliferae ジャショウシ＝Cnidium monnieri (L.) Cusson

Cnidium Rhizome Cnidium officinale Makino Umbelliferae センキュウ

Coix Seed Coix lacryma-jobi Linneá var. mayuen Stapf Gramineae ヨクイニン＝Coix lacryma-jobi L. var. mayuen (Rom. Caill.) Stapf JP XVI General Information / Crude Drugs 2235

Condurango Marsdenia cundurango Reichenbach filius Asclepiadaceae コンズランゴ＝Marsdenia cundurango Rchb. f.

Coptis Rhizome Coptis japonica Makino オウレン＝Coptis japonica (Thunb.) Makino

Coptis japonica (Thunb.) Makino var. dissecta (Yatabe) Nakai Coptis japonica (Thunb.) Makino var. japonica Coptis japonica (Thunb.) Makino var. major (Miq.) Satake Ranunculaceae Coptis chinensis Franchet ＝Coptis chinensis Franch.

Coptis deltoidea C. Y. Cheng et Hsiao

Coptis teeta Wallich ＝Coptis teeta Wall.

Cornus Fruit Cornus officinalis Siebold et Zuccarini Cornaceae サンシュユ＝Cornus officinalis Siebold & Zucc.

Corydalis Tuber Corydalis turtschaninovii Besser forma yanhusuo Y. H. Chou et C. C. Hsu エンゴサク＝Corydalis turtschaninovii Besser f. yanhusuo (W. T. Wang) Y. H. Chou & C. C. Hsu Papaveraceae

Corydalis yanhusuo W. T. Wang

Crataegus Fruit Crataegus cuneata Siebold et Zuccarini サンザシ＝Crataegus cuneata Siebold & Zucc. Rosaceae Crataegus pinnatifida Bunge var. major N. E. Brown ＝Crataegus pinnatifida Bunge var. major N. E. Br.

Cyperus Rhizome Cyperus rotundus Linneá Cyperaceae コウブシ＝Cyperus rotundus L.

Digenea Digenea simplex C. Agardh Rhodomelaceae マクリ＝Digenea simplex (Wulfen) C. Agardh

Dioscorea Rhizome Dioscorea japonica Thunberg サンヤク＝Dioscorea japonica Thunb.

Dioscorea batatas Decaisne Dioscoreaceae ＝Dioscorea batatas Decne.

Dioscorea opposita Thunb.

Dolichos Seed Dolichos lablab Linneá Leguminosae ヘンズ＝Dolichos lablab L.

Eleutherococcus Senticosus Eleutherococcus senticosus Maximowicz Rhizome ＝Eleutherococcus senticosus (Rupr.&Maxim.)Maxim. シゴカ Araliaceae * Acanthopanax senticosus Harms ＝Acanthopanax senticosus (Rupr. & Maxim.) Harms

Ephedra Herb Ephedra sinica Stapf マオウ Ephedra intermedia Schrenk et C. A. Meyer Ephedraceae ＝Ephedra intermedia Schrenk & C. A. Mey.

Ephedra equisetina Bunge 2236 Crude Drugs / General Information JP XVI

Epimedium Herb Epimedium pubescens Maximowicz インヨウカク＝Epimedium pubescens Maxim.

Epimedium brevicornu Maximowicz ＝Epimedium brevicornu Maxim.

Epimedium wushanense T. S. Ying

Epimedium sagittatum Maximowicz Berberidaceae ＝Epimedium sagittatum (Siebold & Zucc.) Maxim.

Epimedium koreanum Nakai

Epimedium grandiflorum Morren var. thunbergianum Nakai ＝Epimedium grandiflorum Morr. var. thunbergianum (Miq.) Nakai

Epimedium sempervirens Nakai

Eucommia Bark Eucommia ulmoides Oliver Eucommiaceae トチュウ＝Eucommia ulmoides Oliv.

Euodia Fruit Euodia ruticarpa Hooker filius et Thomson ゴシュユ＝Euodia ruticarpa (A. Juss.) Hook. f. & Thomson

* Evodia rutaecarpa Bentham ＝Evodia rutaecarpa (A. Juss.) Benth. Tetradium ruticarpum (A.Juss.)Hartley

Euodia officinalis Dode Rutaceae * Evodia officinalis Dode Evodia rutaecarpa (A.Juss.)Benth.var.officinalis (Dode) Huang

Euodia bodinieri Dode

* Evodia bodinieri Dode Evodia rutaecarpa (A.Juss.)Benth.var.bodinieri (Dode) Huang

Fennel Foeniculum vulgare Miller Umbelliferae ウイキョウ＝Foeniculum vulgare Mill.

Fennel Oil Foeniculum vulgare Miller Umbelliferae ウイキョウ油＝Foeniculum vulgare Mill.

Illicium verum Hooker filius Illiciaceae ＝Illicium verum Hook. f.

Forsythia Fruit Forsythia suspensa Vahl レンギョウ＝Forsythia suspensa (Thunb.) Vahl Oleaceae Forsythia viridissima Lindley ＝Forsythia viridissima Lindl.

Fritillaria Bulb Fritillaria verticillata Willdenow var. thunbergii Baker バイモ＝Fritillaria verticillata Willd. var. thunbergii (Miq.) Baker Liliaceae Fritillaria thunbergii Miq.

Gambir Uncaria gambir Roxburgh Rubiaceae アセンヤク＝Uncaria gambir (Hunter) Roxb.

Gardenia Fruit Gardenia jasminoides Ellis サンシシ Rubiaceae Gardenia jasminoides Ellis f. longicarpa Z. W. Xie & Okada

Gastrodia Tuber Gastrodia elata Blume Orchidaceae テンマ

Gentian Gentiana lutea Linneá Gentianaceae ゲンチアナ＝Gentiana lutea L. JP XVI General Information / Crude Drugs 2237

Geranium Herb Geranium thunbergii Siebold et Zuccarini Geraniaceae ゲンノショウコ＝Geranium thunbergii Siebold & Zucc.

Ginger Zingiber officinale Roscoe Zingiberaceae ショウキョウ

Ginseng Panax ginseng C. A. Meyer ニンジン＝Panax ginseng C. A. Mey. Araliaceae * Panax schinseng Nees

Glehnia Root and Rhizome Glehnia littoralis Fr. Schmidt ex Miquel Umbelliferae ハマボウフウ＝Glehnia littoralis F. Schmidt ex Miq.

Glycyrrhiza Glycyrrhiza uralensis Fischer カンゾウ＝Glycyrrhiza uralensis Fisch. Leguminosae Glycyrrhiza glabra Linneá ＝Glycyrrhiza glabra L.

Hemp Fruit Cannabis sativa Linneá Moracea マシニン＝Cannabis sativa L.

Honey Apis mellifera Linneá ハチミツ＝Apis mellifera L. Apidae Apis cerana Fabricius

Houttuynia Herb Houttuynia cordata Thunberg Saururaceae ジュウヤク＝Houttuynia cordata Thunb.

Immature Orange Citrus aurantium Linneá var. daidai Makino キジツ＝Citrus aurantium L. var. daidai Makino

Citrus aurantium L. `Daidai'

Citrus natsudaidai Hayata Rutaceae Citrus aurantium Linneá ＝Citrus aurantium L.

Citrus aurantium L. subsp. hassaku (Tanaka) Hiroe ＝Citrus hassaku hort. ex Tanaka

Imperata Rhizome Imperata cylindrica Beauvois ボウコン＝Imperata cylindrica (L.) P. Beauv. Gramineae Imperata cylindrica (L.) P. Beauv. var. major (Nees) C. E. Hubb.

Ipecac Cephaelis ipecacuanha A. Richard トコン＝Cephaelis ipecacuanha (Brot.) A. Rich. Rubiaceae Cephaelis acuminata Karsten ＝Cephaelis acuminata H. Karst.

Japanese Angelica Root Angelica acutiloba Kitagawa トウキ＝Angelica acutiloba (Siebold & Zucc.) Kitag. Umbelliferae Angelica acutiloba Kitagawa var. sugiyamae Hikino ＝Angelica acutiloba (Siebold & Zucc.) Kitag. var. sugiyamae Hikino 2238 Crude Drugs / General Information JP XVI

Japanese Gentian Gentiana scabra Bunge リュウタン Gentiana scabra Bunge var. buergeri (Miq.) Maxim.

Gentiana manshurica Kitagawa ＝Gentiana manshurica Kitag. Gentianaceae

Gentiana triflora Pallas ＝Gentiana triflora Pall.

Gentiana triflora Pall. var. japonica Hara

Japanese Valerian Valeriana fauriei Briquet カノコソウ＝Valeriana fauriei Briq. Valerianaceae Valeriana fauriei Briq. f. yezoensis Hara

Jujube Seed Zizyphus jujuba Miller var. spinosa Hu ex H. F. Chou Rhamnaceae サンソウニン＝Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou

Jujube Zizyphus jujuba Miller var. inermis Rehder Rhamnaceae タイソウ＝Zizyphus jujuba Mill. var. inermis (Bunge) Rehder

Koi Zea mays Linneá Gramineae コウイ＝Zea mays L.

Manihot esculenta Crantz Euphorbiacaea

Solanum tuberosum Linneá Solanaceae ＝Solanum tuberosum L.

Ipomoea batatas Poiret ＝Ipomoea batatas (L.) Poir Convolvulaceae Ipomoea batatas (L.) Lam.

Oryza sativa Linneá Gramineae ＝Oryza sativa L.

Leonurus Herb Leonurus japonicus Houttuyn ヤクモソウ＝Leonurus japonicus Houtt. Labiatae Leonurus sibiricus Linneá ＝Leonurus sibiricus L.

Lilium Bulb Lilium lancifolium Thunberg ビャクゴウ＝Lilium lancifolium Thunb.

Lilium brownii F. E. Brown var. colchesteri Wilosn ＝Lilium brownii F. E. Br. var. colchesteri (VanHoutte)E.H.Wilsonex Elwes Liliaceae Lilium brownii F. E. Brown var. viridulum Baker

Lilium brownii F. E. Brown ＝Lilium brownii F. E. Br.

Lilium pumilum De Candolle ＝Lilium pumilum DC.

Lindera Root Lindera strychnifolia Fernandez-Villar ウヤク＝Lindera strychnifolia (Siebold & Zucc.) Fern.-Vill. Lauraceae Lindera aggregata (Sims) Kosterm.

Lithospermum Root Lithospermum erythrorhizon Siebold et Zuccarini Boraginaceae シコン＝Lithospermum erythrorhizon Siebold & Zucc. JP XVI General Information / Crude Drugs 2239

Longan Aril Euphoria longana Lamarck リュウガンニク＝Euphoria longana Lam. Sapindaceae Dimocarpus longan Lour.

Lonicera Leaf and Stem Lonicera japonica Thunberg Caprifoliaceae ニンドウ＝Lonicera japonica Thunb.

Loquat Leaf Eriobotrya japonica Lindley Rosaceae ビワヨウ＝Eriobotrya japonica (Thunb.) Lindl.

Lycium Bark Lycium chinense Miller ジコッピ＝Lycium chinense Mill. Solanaceae Lycium barbarum Linneá ＝Lycium barbarum L.

Lycium Fruit Lycium chinense Miller クコシ＝Lycium chinense Mill. Solanaceae Lycium barbarum Linneá ＝Lycium barbarum L.

Magnolia Bark Magnolia obovata Thunberg コウボク＝Magnolia obovata Thunb.

* Magnolia hypoleuca Siebold et Zuccarini ＝Magnolia hypoleuca Siebold & Zucc.

Magnolia officinalis Rehder et Wilson Magnoliaceae ＝Magnolia officinalis Rehder & E. H. Wilson

Magnolia officinalis Rehder et Wilson var. biloba Rehder et Wilson ＝Magnolia officinalis Rehder & E. H. Wilson var. biloba Rehder & E. H. Wilson

Magnolia Flower Magnolia salicifolia Maximowicz シンイ＝Magnolia salicifolia (Siebold & Zucc.) Maxim.

Magnolia kobus De Candolle ＝Magnolia kobus DC.

Magnolia biondii Pampanini ＝Magnolia biondii Pamp.

Magnolia sprengeri Pampanini Magnoliaceae ＝Magnolia sprengeri Pamp.

Magnolia heptapeta Dandy ＝Magnolia heptapeta (Buchoz) Dandy

* Magnolia denudata Desrousseaux ＝Magnolia denudata Desr.

Mallotus Bark Mallotus japonicus Mueller Argoviensis Euphorbiaceae アカメガシワ＝Mallotus japonicus (Thunb.) Mull.ä Arg.

Mentha Herb Mentha arvensis Linneá var. piperascens Malinvaud ハッカ＝Mentha arvensis L. var. piperascens Malinv. Mentha Oil ハッカ油 Mentha haplocalyx Briq. Labiatae Hybrid originated from Mentha arvensis L. var. piperascens Malinv. as the mother species

Moutan Bark Paeonia suffruticosa Andrews ボタンピ Paeoniaceae * Paeonia moutan Sims 2240 Crude Drugs / General Information JP XVI

Mulberry Bark Morus alba Linneá Moraceae ソウハクヒ＝Morus alba L.

Nelumbo Seed Nelumbo nucifera Gaertner Nymphaeaceae レンニク＝Nelumbo nucifera Gaertn.

Notopterygium Notopterygium incisum Ting ex H. T. Chang キョウカツ Umbelliferae Notopterygium forbesii Boissieu

Nuphar Rhizome Nuphar japonicum De Candolle Nymphaeaceae センコツ＝Nuphar japonicum DC.

Nutmeg Myristica fragrans Houttuyn Myristicaceae ニクズク＝Myristica fragrans Houtt.

Nux Vomica Strychnos nux-vomica Linneá Loganiaceae ホミカ＝Strychnos nux-vomica L.

Ophiopogon Tuber Ophiopogon japonicus Ker-Gawler Liliaceae バクモンドウ＝Ophiopogon japonicus (L. f.) Ker Gawl.

Oriental Bezoar Bos taurus Linneá var. domesticus Gmelin Bovidae ゴオウ＝Bos taurus L. var. domesticus Gmelin

Oyster Shell Ostrea gigas Thunberg Ostreidae ボレイ＝Ostrea gigas Thunb.

Panax Japonicus Rhizome Panax japonicus C. A. Meyer Araliaceae チクセツニンジン＝Panax japonicus C. A. Mey.

Peach Kernel Prunus persica Batsch トウニン＝Prunus persica (L.) Batsch

Prunus persica Batsch var. davidiana Maximowicz Rosaceae ＝Prunus persica (L.) Batsch var. davidiana (Carriere)à Maxim.

Prunus davidiana (Carriere)à Franch.

Peony Root Paeonia lactiflora Pallas Paeoniaceae シャクヤク＝Paeonia lactiflora Pall.

Perilla Herb Perilla frutescens Britton var. acuta Kudo ソヨウ＝Perilla frutescens (L.) Britton var. acuta (Thunb.) Kudo Labiatae Perilla frutescens Britton var. crispa Decaisne ＝Perilla frutescens (L.) Britton var. crispa (Thunb.) Decne.

Peucedanum Root Peucedanum praeruptorum Dunn ゼンコ Angelica decursiva Franchet et Savatier ＝Angelica decursiva (Miq.) Franch. & Sav. Umbelliferae

* Peucedanum decursivum Maximowicz ＝Peucedanum decursivum (Miq.) Maxim.

Pharbitis Seed Pharbitis nil Choisy Convolvulaceae ケンゴシ＝Pharbitis nil (L.) Choisy

Phellodendron Bark Phellodendron amurense Ruprecht オウバク＝Phellodendron amurense Rupr.

Phellodendron amurense Rupr. var. sachalinense F. Schmidt Phellodendron amurense Rupr. var. japonicum (Maxim.) Ohwi Rutaceae Phellodendron amurense Rupr. var. lavallei (Dode) Sprangue

Phellodendron chinense Schneider ＝Phellodendron chinense C. K. Schneid. JP XVI General Information / Crude Drugs 2241

Picrasma Wood Picrasma quassioides Bennet Simaroubaceae ニガキ＝Picrasma quassioides (D. Don) Benn.

Pinellia Tuber Pinellia ternata Breitenbach Araceae ハンゲ＝Pinellia ternata (Thunb.) Breitenb.

Plantago Herb Plantago asiatica Linneá Plantaginaceae シャゼンソウ＝Plantago asiatica L.

Plantago Seed Plantago asiatica Linneá Plantaginaceae シャゼンシ＝Plantago asiatica L.

Platycodon Root Platycodon grandiflorum A. De Candolle Campanulaceae キキョウ＝Platycodon grandiflorum (Jacq.)A.DC.

Pogostemon Herb Pogostemon cablin Bentham Labiatae カッコウ＝Pogostemon cablin (Blanco) Benth.

Polygala Root Polygala tenuifolia Willdenow Polygalaceae オンジ＝Polygala tenuifolia Willd.

Polygonatum Rhizome Polygonatum falcatum A. Gray オウセイ Polygonatum sibiricum Redouteá

Polygonatum kingianum Collett et Hemsley Liliaceae ＝Polygonatum kingianum Collett & Hemsl.

Polygonatum cyrtonema Hua

Polygonum Root Polygonum multiflorum Thunberg Polygonaceae カシュウ＝Polygonum multiflorum Thunb.

Polyporus Sclerotium Polyporus umbellatus Fries Polyporaceae チョレイ＝Polyporus umbellatus (Pers.) Fries

Poria Sclerotium Wolfiporia cocos Ryvarden et Gilbertson ブクリョウ＝Wolfiporia cocos (Schw.) Ryv. & Gilbn. Polyporaceae * Poria cocos Wolf ＝Poria cocos (Schw.) Wolf

Processed Aconite Root Aconitum carmichaeli Debeaux ブシ Aconitum japonicum Thunberg Ranunculaceae ＝Aconitum japonicum Thunb.

Processed Ginger Zingiber officinale Roscoe Zingiberaceae カンキョウ

Prunella Spike Prunella vulgaris Linnevar.á lilacina Nakai Labiatae カゴソウ＝Prunella vulgaris L. var. lilacina Nakai

Pueraria Root Pueraria lobata Ohwi Leguminosae カッコン＝Pueraria lobata (Willd.) Ohwi

Quercus Bark Quercus acutissima Carruthers ボクソク＝Quercus acutissima Carruth.

Quercus serrata Murray Fagaceae Quercus mongolica Fischer ex Ledebour var. crispula Ohashi ＝Quercus mongolica Fisch. ex Ledeb. var. crispula (Blume) Ohashi

Quercus variabilis Blume

Red Ginseng Panax ginseng C. A. Meyer コウジン＝Panax ginseng C. A. Mey. Araliaceae * Panax schinseng Nees 2242 Crude Drugs / General Information JP XVI

Rehmannia Root Rehmannia glutinosa Liboschitz var. purpurea Makino ジオウ＝Rehmannia glutinosa Libosch. var. purpurea Makino Scrophulariaceae Rehmannia glutinosa Liboschitz ＝Rehmannia glutinosa Libosch.

Rhubarb Rheum palmatum Linneá ダイオウ＝Rheum palmatum L.

Rheum tanguticum Maximowicz ＝Rheum tanguticum Maxim. Polygonaceae Rheum officinale Baillon ＝Rheum officinale Baill.

Rheum coreanum Nakai

Hybrid between above species

Rose Fruit Rosa multiflora Thunberg Rosaceae エイジツ＝Rosa multiflora Thunb.

Rosin Several plants of Pinus genus Pinaceae ロジン

Royal Jelly Apis mellifera Linneá ローヤルゼリー＝Apis mellifera L. Apidae Apis cerana Fabricius

Safflower Carthamus tinctorius Linneá Compositae コウカ＝Carthamus tinctorius L.

Saffron Crocus sativus Linneá Iridaceae サフラン＝Crocus sativus L.

Saposhnikovia Root and Rhi- Saposhnikovia divaricata Schischkin zome Umbelliferae ＝Saposhnikovia divaricata (Turcz.) Schischk. ボウフウ

Sappan Wood Caesalpinia sappan Linneá Leguminosae ソボク＝Caesalpinia sappan L.

Saussurea Root Saussurea lappa Clarke モッコウ＝Saussurea lappa (Decne.)C.B.Clarke Compositae Aucklandia lappa Decne.

Schisandra Fruit Schisandra chinensis Baillon Schisandraceae ゴミシ＝Schisandra chinensis (Turcz.) Baill.

Schizonepeta Spike Schizonepeta tenuifolia Briquet Labiatae ケイガイ＝Schizonepeta tenuifolia Briq.

Scopolia Rhizome Scopolia japonica Maximowicz ロートコン＝Scopolia japonica Maxim.

Scopolia carniolica Jacquin Solanaceae ＝Scopolia carniolica Jacq.

Scopolia parviflora Nakai ＝Scopolia parviflora (Dunn) Nakai

Scutellaria Root Scutellaria baicalensis Georgi Labiatae オウゴン JP XVI General Information / Crude Drugs 2243

Senega Polygala senega Linneá セネガ＝Polygala senega L. Polygalaceae Polygala senega Linneá var. latifolia Torrey et Gray ＝Polygala senega L. var. latifolia Torr. & A. Gray

Senna Leaf Cassia angustifolia Vahl センナ Leguminosae Cassia acutifolia Delile

Sesame Sesamum indicum Linneá Pedaliaceae ゴマ＝Sesamum indicum L.

Sinomenium Stem and Rhi- Sinomenium acutum Rehder et Wilson zome Menispermaceae ＝Sinomenium acutum (Thunb.) Rehder & E. H. Wilson ボウイ

Smilax Rhizome Smilax glabra Roxburgh Liliaceae サンキライ＝Smilax glabra Roxb.

Sophora Root Sophora flavescens Aiton Leguminosae クジン

Sweet Hydrangea Leaf Hydrangea macrophylla Seringe var. thunbergii Makino Saxifragaceae アマチャ＝Hydrangea macrophylla (Thunb.) Ser. var. thunbergii (Siebold) Makino

Swertia Herb Swertia japonica Makino Gentianaceae センブリ＝Swertia japonica (Shult.) Makino

Toad Venom Bufo bufo gargarizans Cantor センソ Bufonidae Bufo melanostictus Schneider

Tragacanth Astragalus gummifer Labillardiereá Leguminosae トラガント＝Astragalus gummifer Labill.

Tribulus Fruit Tribulus terrestris Linneá Zygophyllaceae シツリシ＝Tribulus terrestris L.

Trichosanthes Root Trichosanthes kirilowii Maximowicz カロコン＝Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maximowicz var. japonica Kitamura Cucurbitaceae ＝Trichosanthes kirilowii Maxim. var. japonica (Miq.) Kitam.

Trichosanthes bracteata Voigt ＝Trichosanthes bracteata (Lam.) Voigt

Turmeric Curcuma longa Linneá Zingiberaceae ウコン＝Curcuma longa L.

Uncaria Hook Uncaria rhynchophylla Miquel チョウトウコウ＝Uncaria rhynchophylla (Miq.) Miq.

Uncaria sinensis Haviland Rubiaceae ＝Uncaria sinensis (Oliv.) Havil.

Uncaria macrophylla Wallich ＝Uncaria macrophylla Wall.

Zanthoxylum Fruit Zanthoxylum piperitum De Candolle サンショウ＝Zanthoxylum piperitum (L.) DC. Rutaceae Zanthoxylum piperitum (L.) DC. f. inerme Makino

Zedoary Curcuma zedoaria Roscoe Zingiberaceae ガジュツ

When ``Other species of the same genus'' is included as its original plants the scientific name is not written in Monograph, however, it is written in this table. Reference Terabayashi S. et al.: Pharmaceutical and Medical Device Regulatory Science, 41(5), 407 – 418 (2010). 2244 Drug Formulation / General Information JP XVI

drum base so that the base forms an angle of about 109with the horizontal and the tablets no longer bind together when G6 Drug Formulation lying next to each other, which prevents them from falling freely. Effervescent tablets and chewable tablets may have differ- Tablet Friability Test ent specifications as far as friability is concerned. In the case of hygroscopic tablets, an appropriate humidity-controlled This test is harmonized with the European Pharmacopoeia environment is required for testing. and the U.S. Pharmacopeia. Drums with dual scooping projections, or apparatus with more than one drum, for the running of multiple samples at The Tablet Friability Test is a method to determine the one time, are also permitted. friability of compressed uncoated tablets. The test procedure presented in this chapter is generally applicable to most compressed tablets. Measurement of tablets friability supplements other physical strength measurement, such as tablet G7 Containers and Package crushing strength. Use a drum, with an internal diameter between 283 and 291 mm and a depth between 36 and 40 mm, of transparent synthetic polymer with polished internal surfaces, and sub- Plastic Containers for ject to minimum static build-up (see figure for a typical ap- Pharmaceutical Products paratus). One side of the drum is removable. The tablets are tumbled at each turn of the drum by a curved projection Various kinds of plastics are used as the materials for with an inside radius between 75.5 and 85.5 mm that extends manufacturing containers for pharmaceutical products. from the middle of the drum to the outer wall. The outer di- Such plastics should not have the properties to deteriorate ameter of the central ring is between 24.5 and 25.5 mm. The the efficacy, safety or stability of the pharmaceutical prod- drum is attached to the horizontal axis of a device that ucts to be packed in the container. In selecting a suitable rotates at 25 ± 1 rpm. Thus, at each turn the tablets roll or plastic container, it is desirable to have sufficient informa- slide and fall onto the drum wall or onto each other. tion on the manufacturing processes of the plastic container Fortabletswithaunitmassequaltoorlessthan650mg, including that on the substances added. Since each plastic take a sample of whole tablets n corresponding as near as has specific properties and a wide variety of pharmaceutical possible to 6.5 g. For tablets with a unit mass of more than products may be packed in plastic containers, the com- 650 mg, take a sample of 10 whole tablets. The tablets patibility of plastic containers with pharmaceutical products should be carefully dedusted prior to testing. Accurately should be judged for each combination of container and the weigh the tablet sample, and place the tablets in the drum. specific pharmaceutical product to be contained therein. Rotate the drum 100 times, and remove the tablets. Remove Such judgement should be performed through a verification any loose dust from the tablets as before, and accurately that the container for the pharmaceutical preparation can weigh. comply with the essential requirements for the container, Generally, the test is run once. If obviously cracked, i.e., the design specifications, based on the data from the ex- cleaved, or broken tablets are present in the tablet sample periments on the prototype products of the container and/or after tumbling, the sample fails the test. If the results are information from scientific documentation, etc. In addition, difficult to interpret or if the mass loss is greater than the such compatibility must be ensured based upon an appropri- targeted value, the test should be repeated twice and the ate quality assurance system. mean of the three tests determined. A maximum mean mass Furthermore, in introducing a plastic container, it is loss from the three samples of not more than 1.0z is consi- desirable that proper disposal method after use is taken into dered acceptable for most products. consideration. If tablet size or shape causes irregular tumbling, adjust the 1. Essential Requirements in Designing Plastic Containers for Pharmaceutical Products The plastic material used for the container should be of high quality. Therefore, recycled plastic materials, which are of unknown constitution, must not be used. The leachables or migrants from the container should not deteriorate the efficacy or stability of the pharmaceutical products contained therein. In addition, the possible toxic hazards due to the leachables or migrants should not exceed a given level. Furthermore, the amounts of leachable or migratable chemical substances, such as monomers and additives, from the containers to the pharmaceutical products contained therein must be sufficiently small from the viewpoint of safety. The container should have a certain level of physical properties such as hardness, flexibility, shock resistance, tensile strength, tear strength, bending strength, heat resistance JP XVI General Information / Containers and Package 2245 and the like, in accordance with its intended usage. test and hemolysis test The quality of the pharmaceutical products packed in the ( ii ) preparations in contact with skin or mucous mem- container must not deteriorate during storage. For example, branes: in the case of pharmaceutical products which are unstable to Cytotoxicity test and sensitization test light, the container should provide a sufficient level of light (iii) liquid orally administered preparations: shielding. In the case of pharmaceutical products which are Cytotoxicity test easily oxidized, the container material should not allow the It is recommended to conduct the tests in accordance with permeation of oxygen. In the case of aqueous pharmaceuti- the latest versions of the standard test methods on medical cal products and pharmaceutical products that must be kept devices and materials published in Japan and other coun- dry, the container material should not allow the permeation tries. of water vapor. In addition, it is necessary to pay attention Those standard test methods are listed for information: to the permeability of solvents other than water through the 2.1. Examplification of Standard Tests container. The concentration of the pharmaceuticals must 2.1.1. Selection of Tests not be decreased by more than a certain level due to the ad- (i) Guidelines for Basic Biological Tests of Medical sorption of the pharmaceuticals on the surface of the con- Devices and Materials (PAB Notification, YAKU-KI NO.99, tainer, the migration of the pharmaceuticals into the inside June 27, 1995), Principles and selection of tests of the material of the container, or the loss of pharmaceuti- (ii) ISO 10993-1: Biological evaluation of medical cals through the container. Also, the pharmaceutical prod- devices—Evaluation and testing ucts contained therein must not be degraded by an interac- 2.1.2. Acute Toxicity Test tion with the material of the container. (i) ASTM F750-82: Standard practice for evaluating ma- The container should not be deformed, should not de- terial extracts by systemic injection in the mice teriorate and should not be degraded by the pharmaceutical (ii) BS5736: Part 3 Method of test for systemic toxicity; products contained therein. Unacceptable loss of function of assessment of acute toxicity of extracts from medical devices the container should not result from a possible high tempera- (iii) USP 24 <88> Biological reactivity tests, in vivo ture or low temperature or their repetitions encountered dur- 2.1.3. Cytotoxicity Test ing storage or transportation. (i) Guidelines for Basic Biological Tests of Medical The container should be of a required level of transparen- Devices and Materials, I. Cytotoxicity Test 10. Cytotoxicity cy,whenitisnecessarytoexamine foreign insoluble matter test using extract of medical device or material and/or turbidity of the pharmaceutical products by visual (ii) ISO 10993-5: Biological evaluation of medical observation. devices—Tests for cytotoxicity : in vitro methods In the case of pharmaceutical products which must be (iii) USP 24 <87> Biological reactivity tests, in vitro sterilized, it is required to satisfy the above-mentioned essen- 2.1.4. Hemolysis Test tial requirements of the container after the sterilization, if (i) Guidelines for Basic Biological Tests of Medical there is a possibility that the quality of the container may Devices and Materials, VII. Hemolysis Test change after the sterilization. There should not be any (ii) ISO 10993-4: Biological evaluation of medical residue or generation of new toxic substances of more than devices—Selection of tests for interaction with blood. Annex certain risk level after the sterilization. In addition, the con- D tainer should not have any inappropriate structure and/or (iii) ASTM F756-82: Standard practice for assessment of material that might result in any bacterial contamination of hemolytic properties of materials the pharmaceutical products contained therein during 2.1.5. Sensitization Test storage and transportation after sterilization. (i) Guidelines for Basic Biological Tests of Medical Devices and Materials, II. Sensitization Test 2. Toxicity Evaluation of Container at Design Phase (ii) ISO 10993-10: Biological evaluation of medical For design verification, the toxicity of the container devices—Tests for irritation and sensitization should be evaluated. For the toxicity evaluation, it is desirable to select appropriate test methods and acceptance criteria 3. Test Results to be Recorded per Production Unit for the evaluation, and to explain the rationale for the selec- At the commercial production phase, it is required to tion clearly. The tests should be conducted using samples of establish acceptance criteria on at least the test items listed the whole or a part of the prototype products of the con- below and to record the test results of each production unit tainer. If the container consists of plural parts of different of plastic containers for pharmaceutical products. In addi- materials, each part should be tested separately. Such mate- tion, it is desirable to explain the rationale for setting the rials as laminates, composites, and the like are regarded as a acceptance criteria clearly. However, these requirements single material. To test containers made of such materials, it should not be applied to orally administered preparations is recommended to expose the inner surface of the container, except for liquid preparations. which is in contact with the pharmaceutical products con- (i) Combustion Tests: Residue of ignition, heavy metals. tained therein, to the extraction media used in the tests as far If necessary, the amounts of specified metals (lead, cad- as possible. mium, etc.) The tests required for the toxicity evaluation of the con- (ii) Extraction Tests: pH, ultraviolet absorption spectra, tainer are different depending upon the tissue to which the potassium permanganate-reducing substances, foaming, pharmaceutical products contained therein are to be applied. non-volatile residue The following tests are required for containers for (iii) Cytotoxicity Test ( i ) preparations in contact with blood: (iv) Any other tests necessary for the specific container Acute toxicity test, cytotoxicity test, sensitization for aqueous infusions. 2246 Water / General Information JP XVI

prescribed in the JP monograph. Sterile Purified Water in Container is prepared from Puri- G8 Water fied Water by 1) introducing it into a hermetic container, sealing up the container, then sterilizing the product, or 2) making it sterile by using a suitable method, introducing the Quality Control of Water for sterilized water into a sterile hermetic container by applying aseptic manipulation, then sealing up the container. Pharmaceutical Use Plastic containers for aqueous injections may be used in place of hermetic containers. Water used for manufacturing pharmaceutical products 1.4. Water for Injection and for cleaning their containers and equipments used in the The specifications for ``Water for Injection'' and ``Sterile manufacture of the products is referred to as ``pharmaceuti- Water for Injection in Containers'' are prescribed in the JP cal water''. For assuring the quality of pharmaceutical water monographs. Water for Injection is prepared by distillation consistently, it is important to verify through appropriate or reverse osmosis and/or ultrafiltration (RO/UF), either process validation of water processing system that water with from Water after applying some adequate pretreatments the quality suitable for its intended use is produced and sup- such as ion exchange, RO, etc., or from Purified Water. plied, and to keep the quality of produced water through In the case of water processing systems based on distilla- routine works for controlling the water processing system. tion, it is necessary to take care for avoiding contamination 1. Types of Pharmaceutical Water of produced water by the impurities accompanied with the 1.1. Water entrain. The specification for ``Water'' is prescribed in the In the case of water processing system based on RO/UF, it Japanese Pharmacopoeia (JP) monograph. It is required for is required to provide water with equivalent quality to that Water to meet the Quality Standards for Drinking Water prepared by distillation consistently, based on substantial provided under the Article 4 of the Japanese Water Supply process validation through long-term operation and Law.InthecasethatWater is produced at individual facili- elaborate routine control of the system. It is essential to ties using well water or industrial water as source water, it is ensure consistent production of water suitable for Water for necessary for produced water to meet the Quality Standards Injection by the entire water processing system including for Drinking Water and an additional requirement for am- pretreatment facilities, in any systems based on RO/UF. For monium of ``not more than 0.05 mg/L''. Furthermore, the water supplied to the system, it is also required to keep when Water is to be used after storing for a period of time, it the quality suitable as source water through adequate valida- is necessary to prevent microbial proliferation. tion and routine control on the water. Water is used as source water for Purified Water and For the water processing system based on RO/UF, routine Water for Injection. It is also used for manufacturing inter- control should be performed by analyzing water specimens, mediates of active pharmaceutical ingredients (APIs), and monitoring some quality attributes using in-line apparatus for pre-washing of the equipments used in the manufacture and checking the volume of water passed through the sys- of pharmaceutical products. tem. In addition, it is recommended to carry out periodical 1.2. Purified Water appearance observation and air-leaktestonthemembranes The specifications for ``Purified Water'' and ``Purified being currently used. It is also recommended to establish Water in Containers'' are prescribed in the JP monographs. protocols for keeping the performance of membrane mod- Purified Water is prepared by distillation, ion-exchange, ules within appropriate control ranges and for estimating the reverse osmosis (RO), ultrafiltration (UF) capable of remov- timing to exchange the modules, through diagnosis on the ing substances with molecular masses of not less than ap- degree of deterioration based on the results of tensile proximately 6000, or a combination of these processes from strength test on the used membrane modules, and visual ob- Water, after applying some adequate pretreatments if neces- servation on those modules whether any leakages of mem- sary. For the production of Purified Water, appropriate con- branes have occurred or not, and to what extent they have trol of microorganisms is required. Particularly, in the case occurred. Furthermore, it is desirable to establish the fre- that Purified Water is prepared by ion-exchange, RO or UF, quency of membrane exchange considering with its actual it is necessary to apply the treatments adequate for prevent- condition of use. ing microbial proliferation, or to sanitize the system periodi- In the case that Water for Injection is stored in the water cally. processing system temporarily, a stringent control for micro- When Purified Water is treated with chemical agents for organisms and endotoxins should be taken. An acceptable sterilizing, preventing microbial proliferation, or maintain- criterion of lower than 0.25 EU/mL for endotoxins is speci- ing the endotoxin level within an appropriate control range, fied in the JP monograph of Water for Injection. a specification suitable for the intended use of treated water ``Sterile Water for Injection in Container'' is prepared should be established individually, and a process control for from Water for Injection by 1) introducing it into a hermetic keeping the quality of treated water in compliance with the container, sealing up the container, then sterilizing the specification thus established should be performed. product, or 2) making it sterile by using a suitable method, ``Purified Water in Containers'' is prepared from Purified introducing the sterilized water into a sterile hermetic con- Water by introducing it in a tight container. tainer by applying aseptic manipulation, then sealing up the 1.3. Sterile Purified Water container. The specification for ``Sterile Purified Water in Contain- Plastic containers for aqueous injections may be used in ers'' (its alternative name is Sterile Purified Water)is place of hermetic containers. JP XVI General Information / Water 2247

2. Reverse Osmosis and/or Ultrafiltration (RO/UF) contamination with microorganisms or endotoxins is not RO/UF are the methods for refining water by using mem- permissible, Water for Injection (or Sterile Water for Injec- brane modules based on either reverse osmosis or ultrafiltration in Containers) should be used. For the manufacture of tion, or the modules combining them, and used as the alter- ophthalmics and eye ointments, Purified Water (or Purified native methods for distillation in the production of Purified Water in Containers) can also be used. Water or Water for Injection. For the manufacture of non-sterile drug products, water When Water for Injection is produced by RO/UF, a water with a quality not lower than that of Purified Water (or processing system equipped with pretreatment facilities, Purified Water in Containers) should be used. However, out facilities for producing Water for Injection and facilities for of the non-sterile drug products such as liquids, ointments, supplying Water for Injection is usually used. The pretreat- suspensions, emulsions, suppositories and aerosols, for those ment facilities are used to remove solid particles, dissolved which require care against microbiological contamination, salts and colloids in source water, and placed before the Purified Water (or Purified Water in Containers) adequately facilities for producing Water for Injection so as to reduce controlled from microbiological viewpoints should be used the load on the facilities for producing Water for Injection. in consideration of the possible impacts of preservatives They consist of apparatus properly selected from aggrega- formulated in the drug products. For the manufacture of tion apparatus, precipitation-separation apparatus, filtration products containing crude drugs, it is recommended to select apparatus, chorine sterilization apparatus, oxidation-reduc- adequate type of water considering with viable counts of the tion apparatus, residual chlorine-removing apparatus, crude drugs used for manufacturing the product and precise filtration apparatus, reverse osmosis apparatus, microbial limit required for the product. ultrafiltration apparatus, ion exchange apparatus, etc., Water used for pre-washing of containers or equipment depending on the quality of source water. The facilities for surfaces that comes in direct contact with the drug products producing Water for Injection consist of apparatus for sup- should have the quality not lower than that of Water.Water plying pretreated water, sterilization apparatus with ultravio- used for final rinsing should have an equivalent quality to let rays, heat exchange apparatus, membrane modules, ap- that of water used for manufacturing drug products. paratus for cleaning and sterilizing the facilities, etc. The 3.2. Active Pharmaceutical Ingredient (API) facilities for supplying Water for Injection consist of a reser- Water used for manufacturing active pharmaceutical in- voir tank for storing Water for Injection in the facilities tem- gredient (API) should be selected in consideration of the porarily, pipe lines, heat exchange apparatus, a pump for characteristics of drug product for which the API is to be circulating Water for Injection in the facilities, pressure con- used, and its manufacturing process, so that the quality of trol apparatus, etc. the final drug product is assured. InthecasethatWater for Injection is stored in the water Water used for manufacturing API or for cleaning con- processing system temporarily, it should usually be circu- tainers or equipment surfaces that comes in direct contact lated in a loop consisting of a reservoir tank and pipe lines at with the raw materials or API intermediates, should have the a temperature not lower than 809C for preventing microbial quality not lower than that of Water adequately controlled proliferation. from the chemical and microbiological viewpoints, even if When Purified Water is produced by RO/UF, basic com- the water is used at an earlier stage of synthetic or extraction position of water processing system is almost the same as process in the manufacture of API. Water used in the final that for Water for Injection described above. purification process should have the quality equal to or When RO/UF is utilized for preparing pharmaceutical higher than that of Purified Water (or Purified Water in water, it is necessary to select the most appropriate combina- Containers). Water used for final rinsing of containers or tion of membrane modules in consideration of the quality of equipment surfaces that comes in direct contact with the source water and the quality of produced water required for APIs should have an equivalent quality to that of water used its intended use. When the ultrafiltration membrane is used for manufacturing the APIs. to prepare Purified Water or Water for Injection,membrane For manufacturing sterile API, Sterile Purified Water in modules capable of removing microorganisms and sub- Containers or Water for Injection (or Sterile Water for stances with molecular masses not less than approximately Injection in Containers) should be used. Similarly, for 6000 should be used. manufacturing APIs used for drug products where endotoxin control is required and there are no subsequent 3. Selection of Pharmaceutical Water processes capable of removing endotoxins, Water for Injec- Depending on the intended use of pharmaceutical water, tion (or Sterile Water for Injection in Containers), or Puri- the water suitable for assuring the quality of final products fied Water (or Purified Water in Containers)forwhich without causing any trouble during their manufacturing endotoxins are controlled at a low level, should be used. processes, should be selected from the above 4 types (1.1. ¿ 1.4.) of pharmaceutical water specified in the JP. Table 1 ex- 4. Quality Control of Pharmaceutical Water emplifies a protocol for such selection (in the case of phar- 4.1. Outline maceutical water used for the manufacture of drug prod- Verification that water with the quality required for its in- ucts). tended use has been produced by the pharmaceutical water Sterile Purified Water in Containers or Water for Injec- processing system through substantial validation studies at tion (or Sterile Water for Injection in Containers)maybe an earlier stage of its operation, is the prerequisite for con- used in place of Purified Water or Purified Water in Con- ducting quality control on pharmaceutical water in a routine tainers. and periodical manner. If this prerequisite is fulfilled, the 3.1. Drug Products following methods are applicable for quality control of phar- For the manufacture of sterile drug products, for which maceutical water. 2248 Water / General Information JP XVI

Table 1 An Exemplified Protocol for Selecting Pharmaceutical Water (Water Used in the Manufacture of Drug Products or APIs)

Classification Class of Pharmaceutical Water Application Remarks

Drug Product Water for Injection or Sterile Injections, Ophthalmics, Eye Water for Injection in Contain- Ointments ers

Purified Water or Purified Ophthalmics, Eye Ointment For ophthalmics and eye oint- Water in Containers ments for which precautions should be taken against microbial contamination, Purified Water or Purified Water in Containers kept its viable counts at low levels through sterilization, UF filtration, etc. should be used.

Aerosols, Liquids and Solutions, For liquids, solutions, ointments, Extracts, Elixirs, Capsules, Gran- suspensions, emulsions, supposi- ules, Pills, Suspensions and tories, aerosols and so on for Emulsions, Suppositories, Pow- which precautions should be ders, Spirits, Tablets, Syrups, In- taken against microbial contami- fusions and Decoctions, Plasters, nation, Purified Water or Puri- Tinctures, Troches, Ointments, fied Water in Containers ade- Cataplasms, Aromatic Waters, quately controlled from Liniments, Lemonades, Fluidex- microbiological viewpoints tracts, Lotions, and Pharmaceuti- should be used. cal preparations of percutaneous absorption type

Active Water for Injection or Sterile Sterile APIs, and APIs rendered Pharmaceutical Water for Injection in Contain- sterile in the formulation process Ingredient (API) ers

Purified Water or Purified APIs, APIs rendered sterile in the In the manufacture of APIs to be Water in Containers formulation process, and API in- rendered sterile in the formula- termediates tion process and have no subsequent processes capable of removing endotoxins, Purified Water or Purified Water in Con- tainers controlled endotoxins at a low level should be used.

Water API Intermediates

For routine control, it is very useful to control quality of tinuously produced and supplied. Specimens should be col- produced water based on the monitoring of electrical con- lected at the representative locations in the facilities for ductivity (conductivity) and total organic carbon (TOC). In producing and supplying water, with particular care so that addition, items to be monitored periodically, such as some collected specimens reflect the operating condition of the specified impurities, viable counts, endotoxins, insoluble pharmaceutical water processing system. An adequate pro- particulate matters, etc., should be determined according to tocol for the control of microorganisms at the sampling site the intended use of pharmaceutical water. The frequency of should be established considering with the situation around measurement should be determined considering with the the site. variation in the quality of water to be monitored. Sampling frequency should be established based on the The following are points to consider in controlling the data from validation studies on the system. For microbiolog- quality of produced water from microbiological and physical monitoring, it is adequate to use the water specimens for icochemical (conductivity and TOC) viewpoints. It is neces- the test within 2 hours after sampling. In the case that it is sary to monitor other items if necessary, and to confirm that not possible to test within 2 hours, the specimens should be they meet the specifications established individually. kept at 2 – 89C and be used for the test within 12 hours. 4.2. Sampling 4.3. Alert and Action Levels Monitoring should be conducted at an adequate frequency In producing pharmaceutical water using a water process- to ensure that the pharmaceutical water processing system is ing system, microbiological and physicochemical monitoring well-controlled and that water with acceptable quality is con- is usually carried out to assure that water with required qual- JP XVI General Information / Water 2249

Table 2 Methods for Assessment of Viable Counts in Pharmaceutical Water

Pharmaceutical Water Method Water Purified Water Water for Injection

Measurement Method Pour Plate Method or Mem- Pour Plate Method or Mem- Membrane Filtration brane Filtration brane Filtration

Minimum Sample Size 1.0 mL 1.0 mL 100 mL

Media Standard Agar Medium R2A Agar Medium, Standard R2A Agar Medium, Standard Agar Medium Agar Medium

Incubation Period Standard Agar Medium: 48 – R2A Agar Medium: 4 – 7 days R2AAgarMedium:4–7days 72 hours (or longer) (or longer) (or longer) Standard Agar Medium: 48 – Standard Agar Medium: 48 – 72 hours (or longer) 72 hours (or longer)

Incubation Temperature Standard Agar Medium: 30 – R2A Agar Medium: 20 – 259C R2AAgarMedium:20–259C 359C or30–359C or30–359C Standard Agar Medium: 30 – Standard Agar Medium: 30 – 359C 359C

ity is being continuously produced when the system is oper- The followings indicate incubation-based microbiological ating as it designed. The operating condition of the system monitoring techniques for pharmaceutical water processing can be estimated by the comparison of monitoring data thus systems. To adopt a rapid microorganism detection tech- obtained against the alert level, action level, other levels for nique, it is necessary to confirm in advance that the microbi- controlling the system, and acceptance criteria specified for al counts obtained by such techniques are equivalent to those the water required for its intended use, and also by the trend obtained by the incubation-based monitoring techniques. analysis of monitoring data through plotting them in a con- 4.4.1. Media and Incubation Conditions trol chart. In this manner, the alert level and action level are There are many mesophilic bacteria of heterotrophic type used for controlling the process of water production, and that are adapted to poor nutrient water environments. not used for judging pass/fail of produced water. Heterotrophic bacteria may form bio-films in many phar- 4.3.1. Definition of Alert Level maceutical water processing systems, and to cause quality ``Alert level'' indicates that, when exceeded it, the system deterioration of the produced water. Therefore, it is useful is threatening to deviate from its normal operating range. to monitor microbiological quality of water by using the Alert levels are used for giving a warning, and exceeding R2A Agar Medium, which is excellent for growing bacteria them does not necessarily require a corrective action. Alert of oligotrophic type. On the other hand, in routine level is generally established either at a mean＋2s on the microbiological monitoring, an approach by using the basis of past trend analysis, or at a level of 70z (50z for Standard Agar Medium, prescribed in the Quality Standards viable counts) of action level, whichever is smaller. for Drinking Water provided under the Article 4 of the 4.3.2. Definition of Action Level Japanese Water Supply Law, is widely employed. In this ``Action level'' indicates that, when exceeded it, the sys- approach, the trend in microbiological change of water tem has deviated from its normal operating range. Exceeding processing system is estimated from the number of viable it indicates that corrective action must be taken to bring the microorganisms capable of proliferating at 30 – 359Cinthe system back within its normal operating range. Standard Agar Medium in a comparatively short period of Alert and action levels should be established within the time. specified acceptance criteria of the water required for its in- Table 2 shows examples of measurement methods, tended use in consideration of available technologies and the minimum sample sizes, media, and incubation periods for quality required for the water. Consequently, exceeding an estimating viable counts. alert or action level does not necessarily indicate that the The media shown in Table 2 are as follows. quality of produced water has become inadequate for its in- (i) Standard Agar Medium tended use. Casein peptone 5.0 g 4.4. Microbiological Monitoring Yeast extract 2.5 g The main purpose of microbiological monitoring program Glucose 1.0 g for pharmaceutical water processing system is to foresee any Agar 15.0 g microbiological quality deterioration of the produced water, Water 1000 mL and to prevent any adverse effects on the quality of phar- Mix all the ingredients, and sterilize by heating in an maceutical products. Consequently, detecting all of the autoclave at 1219C for 15 – 20 minutes. pH after sterili- microorganisms present in the water to be monitored may zation: 6.9 – 7.1. not be necessary. However it is required to adopt a monitor- (ii) R2A Agar Medium ing technique able to detect a wide range of microorganisms, Peptone (casein and animal tissue) 0.5 g including slow growing microorganisms. 2250 Water / General Information JP XVI

Casamino acid 0.5 g considered equivalent to these strains. Prepare the fluid con- Yeast extract 0.5 g taining the strain according to the procedure prescribed in Sodium pyruvate 0.3 g the Microbial Limit Test <4.05>. When pipetting 1 mL of the Glucose 0.5 g fluid onto the Standard Agar Medium and incubating at 30 – Magnesium sulfate heptahydrate 0.05 g 359C for 48 hours, sufficient proliferation of the inoculated Soluble starch 0.5 g strain must be observed. Dipotassium hydrogen phosphate 0.3 g Staphylococcus aureus: ATCC 6538, NCIMB 9518, CIP Agar 15.0 g 4.83 or NBRC 13276 Water 1000 mL Pseudomonas aeruginosa: ATCC 9027, NCIMB 8626, Mix all the ingredients, and sterilize by heating in an CIP 82.118 or NBRC 13275 autoclave at 1219C for 15 – 20 minutes. pH after sterili- Colon bacillus (Escherichia coli): ATCC 8739, NCIMB zation: 7.1 – 7.3. 8545, CIP 53.126 or NBRC 3972 4.4.3. Action Levels for Microorganisms for Pharmaceuti- The following reagents should be used for preparing the cal Water Processing System R2A Agar Medium. The following action levels are considered appropriate and (i) Casamino acid Prepared for microbial test, by the generally applicable to pharmaceutical water processing sys- acid hydrolysis of casein. tems. Loss on drying <2.41>:Notmorethan8z (0.5 g, 1059C, constant mass) Action Levels for viable counts in various types of phar- Residue on ignition <2.44>: Not more than 55z (0.5 g) maceutical water Nitrogen content <1.08>:Notlessthan7z (1059C, con- Water:100CFU/mL* (Acceptance criterion prescribed in stant mass, after drying) the Quality Standards for Drinking Water provided under

(ii) Sodium pyruvate CH3COCOONa White to pale the Article 4 of the Water Supply Law) yellow crystalline powder. Freely soluble in water, and Purified Water:100CFU/mL** slightly soluble in ethanol (99.5) and in acetone. Water for Injection: 10 CFU/100 mL** Identification (1) Determine the infrared absorption (*Viable counts obtained using the Standard Agar Medi- spectrum as directed in the potassium bromide disk method um, **Viable counts obtained using the R2A Agar Medium) under Infrared Spectrophotometry <2.25>: it exhibits absorp- Although the action level for Purified Water shown above tion maxima at the wave numbers around 1710 cm－1,1630 issetatthesamelevelasthatforWater, in near future, an cm－1, 1410 cm－1,1360cm－1,1190cm－1,1020cm－1,980 adequate action level would be set for Purified Water in con- cm－1,830cm－1,750cm－1,630cm－1 and 430 cm－1. sideration of the current level of technologies. Therefore, for (2) A solution (1 in 20) responds to the Qualitative Tests the time being, it is recommended for each facility to per- <1.09> for sodium salt (1). form a higher level of microbiological control of water Content: Not less than 97.0z. Assay—Weigh accurately processing system based on the action level established indi- about 0.4 g of sodium pyruvate and dissolve it in 200 mL of vidually. water. Transfer 20 mL of this solution into an iodine bottle, When actual counts in validation studies or routine con- and cool to 109C or lower. Add 40 mL of 0.05 mol/L iodine trol exceed the above action levels, it is necessary to isolate VS and 20 mL of sodium hydroxide solution (17 in 100), and identify the microorganisms present in the water, and to then allow to stand in a dark place for 2 hours, and add 15 sanitize or disinfect the affected system. mL of diluted sulfuric acid (1 in 6). Titrate <2.50> with 0.1 4.5. Physicochemical Monitoring mol/L sodium thiosulfate VS (indicator: starch TS). Per- Physicochemical monitoring of a pharmaceutical water form a blank determination in the same manner, and make processing system is usually performed using conductivity any necessary correction. and TOC as the indicators for water quality. By monitoring Each mL of 0.05 mol/L iodine VS conductivity, total amounts of inorganic salts present in the

＝ 1.834 mg of C3H3NaO3 water can be estimated, and by monitoring TOC, total amount of organic compounds present in the water can be 4.4.2. Media Growth Promotion Test estimated. Normally, the Conductivity Measurements <2.51> In the media growth promotion test with the R2A Agar and the Test for Total Organic Carbon <2.59> specified in Medium, use the strains listed below or other strains consi- the General Tests, Processes and Apparatus of the JP should dered equivalent to these strains. Prior to the test, inoculate be applied to these physicochemical monitoring. However, these strains into Sterile Purified Water and incubate at 20 – since tests for monitoring are performed in the situations 259Cfor3days. different from those for judging pass/fail to the acceptance Methylobacterium extorquens: NBRC 15911 criteria prescribed in the monographs, supplements neces- Pseudomonas fluorescens: NBRC 15842, ATCC 17386, sary to cover the situations to which the JP general tests can- etc. not be applied, are described below. Dilute the fluid containing the strain starved in Purified To adopt the monitoring using conductivity and TOC as Water with Sterile Purified Water to prepare a fluid contain- the indicators for inorganic and organic impurities at in- ing about 50 – 200 CFU/mL of viable counts. When pipet- dividual facility, appropriate alert and action levels, and ting 1 mL of the diluted fluid onto the R2A Agar Medium countermeasures against unexpected apparatus failures and incubating at 20 – 259C for 4 – 7 days, sufficient should be established for each indicator. proliferation of the inoculated strain must be observed. 4.5.1. Monitoring of Conductivity as the Indicator for In the media growth promotion test with the Standard Inorganic Impurities Agar Medium, use the strains listed below or other strains Measurement of conductivity for monitoring is usually JP XVI General Information / Water 2251 conducted continuously using an in-line apparatus with a Table 3 Stage 1 Allowable Conductivity for Different flow-through type or pipe-insertion type cell. Alternatively, Temperatures* offline batch testing may be performed using a dip type cell Allowable Allowable with water specimens taken at point-of-use sites or other ap- Temperature Temperature propriate locations of the pharmaceutical water processing Conductivity Conductivity (9C) －1 (9C) －1 system. For the operation control of a pharmaceutical water (mS･cm ) (mS･cm ) processing system, guides for judging whether it is adequate 00.6 to continue the operation of the system or not, based on the 5 0.8 55 2.1 results from monitoring of conductivity, are shown below, 10 0.9 60 2.2 both for the cases of monitoring at the standard temperature 15 1.0 65 2.4 (209C) by applying Conductivity Measurements <2.51> of the 20 1.1 70 2.5 JP and monitoring at temperatures other than 209Cby 25 1.3 75 2.7 applying <645> WATER CONDUCTIVITY of the United 30 1.4 80 2.7 States Pharmacopeia (USP) with some modifications. 35 1.5 85 2.7 4.5.1.1. Monitoring of Conductivity by applying the Con- 40 1.7 90 2.7 ductivity Measurements <2.51> of the JP 45 1.8 95 2.9 The Conductivity Measurements <2.51> of the JP princi- 50 1.9 100 3.1 pally requires to measure the conductivity at the standard temperature (209C). However, measurement at a tempera- * Applicable only to non-temperature-compensated con- ture within a range of 15 – 309C may also be acceptable, ductivity measurements. when the results are corrected using the equation prescribed in the Conductivity Measurements <2.51>.Inthiscase,the recommended allowable conductivity (action level) for Puri- observing the conductivity periodically. When the fied Water and Water for Injection is as follows. change in conductivity, due to the uptake of at- ・Action Level 1.0 mS･cm－1 (209C) mospheric carbon dioxide, becomes not greater than Since the above allowable conductivity is established for 0.1 mS･cm－1 per 5 minutes, adopt the observed in-line monitoring, an alternative action level may be used value as the conductivity (259C) of the water speci- for the monitoring based on offline batch testing. men. 4.5.1.2. Monitoring of Conductivity by applying the <645> (iii) If the conductivity of the water specimen at 259C WATER CONDUCTIVITY of the USP with some modifi- obtained above is not greater than 2.1 mS･cm－1,the cations water tested meets the requirement for monitoring Usually, it is somewhat difficult to control the tempera- conductivity. If the observed value exceeds 2.1 mS･ ture exactly in in-line conductivity monitoring. Therefore, cm－1, it should be judged that the water tested does the following approach can be applied for the monitoring at not meet the requirement for monitoring conduc- temperatures other than the standard temperature (209C) of tivity. the JP. This approach is based on the Stages 1 and 2 of the 4.5.2. Monitoring of TOC as the Indicator for Organic Im- three-stage approach described in ``<645> WATER CON- purities DUCTIVITY'' of the USP. The acceptance criterion of TOC is specified as ``not Stage 1 (In-line Measurement) greater than 0.50 mg/L (500 ppb)'' in the monographs of ( i ) Determine the temperature and the conductivity of Purified Water and Water for Injection. However it is the water specimens using a non-temperature-com- recommended for each facility preparing pharmaceutical pensated conductivity reading. water to conduct operation control of pharmaceutical water ( ii ) From the Table 3, find the temperature value equal processing system through TOC monitoring on produced to or just lower than the measured temperature. water based on its own alert and action levels for TOC deter- Adopt the corresponding conductivity value on this mined individually. The followings are the recommended table as the allowable conductivity at the measured action levels for TOC. temperature. ・Action Level: ≦300 ppb (in-line) (iii) If the observed conductivity is not greater than the ≦400 ppb (off-line) allowable conductivity adopted above, the water The Quality Standards for Drinking Water provided under tested meets the requirement for monitoring conduc- the Article 4 of the Japanese Water Supply Law require that tivity. If the observed conductivity exceeds the TOC should be ``not greater than 3 mg/L (3 ppm)''. Taking allowable conductivity, proceed with Stage 2. the above recommended action levels into consideration, it is also recommended for each facility to conduct quality con- Stage 2 (Off-line Measurement) trol of source water through TOC monitoring based on its ( i ) Measure the conductivity of the water specimen, by own alert and action levels for TOC determined individually. transferring it into a container and agitating it vigor- The JP specifies the Test for Total Organic Carbon <2.59>, ously in order to attain equilibrium between the and normally, TOC measurement should be conducted using water specimen and the atmosphere on absorbing/ an apparatus which meets the requirements described in the desorbing carbon dioxide. JP method. However, if a TOC apparatus conforms to the ( ii ) Transfer a sufficient amount of water to be tested apparatus suitability test requirements described in ``<643> into a suitable container, and stir the water speci- TOTAL ORGANIC CARBON'' of the USP, or those men. Adjust the temperature to 25 ± 19C, and described in the ``Methods of Analysis 2.2.44. TOTAL begin agitating the water specimen vigorously, while 2252 Water / General Information JP XVI

ORGANIC CARBON IN WATER FOR PHARMACEU- ration and that passed through plastic layer of the containers TICAL USE'' of the European Pharmacopoeia (EP), the during storage, and also due to ionic substances released apparatus can be used for the monitoring of pharmaceutical from the containers, even if the conductivity of Purified water processing system, if sufficiently pure water not con- Water or Water for Injection used for its production is taminated with ionic organic substances, or organic sub- maintained at the level not more than 1.0 mS･cm－1.Particu- stances having nitrogen, sulfur, phosphorus or halogen larly in the cases of pharmaceutical water products packed in atoms in their chemical structures, is used as the source small scale glass containers, it is necessary to pay attention to water supplied to the system. the change of conductivity during storage. A TOC apparatus, characterized by calculating the 5.2.2. Potassium Permanganate-reducing Substances or amount of organic carbon from the difference in conduc- Total Organic Carbon (TOC) (as the indicator for organic tivity before and after the decomposition of organic sub- impurities) stances without separating carbon dioxide from the sample JP specifies the classical test of potassium permanganate- solution, may be influenced negatively or positively, when reducing substances in the monographs of Purified Water in applied to the water specimens containing ionic organic sub- Containers, Sterile Purified Water in Containers and Sterile stances, or organic substances having nitrogen, sulfur, phos- Water for Injection in Containers for controlling organic phorus or halogen atoms in their chemical structures. There- impurities in pharmaceutical water in containers. It forms a fore, the apparatus used for TOC monitoring should be remarkable contrast to the specifications of Purified Water selected appropriately in consideration of the purity of the and Water for Injection, in which JP requires to control watertobemonitoredandthecontaminationriskinthecase organic impurities in pharmaceutical water in bulk based on of apparatus failure. the test of TOC (acceptance criterion: not more than 0.5 4.6. Storage of Water for Injection mg/L (500 ppb)). This is because that it is considered In storing Water for Injection temporarily, adequate difficult to establish the specification of pharmaceutical measures able to prevent microbial proliferation stringently, water in containers for organic impurities based on the test such as circulating it in a loop at a high temperature must be of TOC from the facts that there were many cases of taken, and an appropriate storage time should also be estab- remarkable increases in TOC values after storage of water in lished based on the validation studies, in consideration of the containers. Particularly in the cases of pharmaceutical water risks of contamination and quality deterioration. products packed in small scale plastic containers, it is necessary to pay attention to the increase of materials released 5. Points to Consider for Assuring the Quality of Phar- from containers during storage. maceutical Water in Containers The test of potassium permanganate-reducing substances There are some specific points to consider for assuring the is retained in the specifications of pharmaceutical water in quality of pharmaceutical water in containers (Purified containers, not as the most suitable method for the test of Water in Containers, Sterile Purified Water in Containers organic impurities present in the water in containers, but as a and Sterile Water for Injection in Containers), which are counter measure for performing the test of the water in con- available as commercially products. tainers with the same test method despite of the material 5.1. Methods for Preparing Sterile Pharmaceutical Water (glass, polyethylene or polypropylene, etc) and the size (0.5 – in Containers and Their Sterilization Validation 2000 mL) of the containers, and the duration of storage. The following 2 different preparation methods are Therefore, it is recommended to adopt the test of TOC as described in the monographs of Sterile Purified Water in the alternative for the test of potassium permanganate- Containers and Sterile Water for Injection in Containers. reducing substances, and to perform quality control of phar- ( i ) Introduce Purified Water (or Water for Injection) maceutical water in containers based on TOC measurements into a hermetic container, seal up the container, then under the responsibility of each manufacturer, if possible. sterilize the product. In such cases, it is recommended to adopt the following (ii) Make Purified Water (or Water for Injection)sterile values as the levels preferable to attain. by using a suitable method, introduce the sterilized For products containing not more than 10 mL of water: water into a sterile hermetic container by applying TOC not greater than 1500 ppb aseptic manipulation, then seal up the container. For products containing more than 10 mL of water: For assuring the sterility of pharmaceutical water prod- TOC not greater than 1000 ppb ucts, only the validation of final sterilization process is required in the case of preparation method (i), whereas valida- As for the pharmaceutical water packed in the plastic con- tions of all the processes are indispensable in the case of tainers made of polyethylene, polypropylene, etc., in addi- preparation method (ii), since the latter is based on the idea tion to the concern for the release of materials such as to assure the sterility of pharmaceutical water products by monomer, oligomers, plasticizers, etc. from plastics, it is ``aseptically'' introducing Purified Water (or Water for In- necessary to pay attention to the storage environment of the jection) treated in advance with filtration sterilization, etc. products to avoid the contaminations with low molecular into a sterile hermetic container, and sealing it up. volatile organics such as ethanol, or low molecular air pol- 5.2. Deterioration of Water Quality during the Storage in lutants such as nitrogen oxides, since these plastics have the Containers properties of permeating various gases. 5.2.1. Conductivity (as the indicator for inorganic impuri- 5.2.3. Microbial Limit (Total Aerobic Viable Counts) ties) For Purified Water in Containers, it is not required to as- The conductivity of pharmaceutical water in containers sure the sterility, but it is necessary to produce it by using may increase to some higher levels due to the absorption of sanitary or aseptic processes in order to meet the acceptance carbon dioxide from the atmosphere at the time of its prepa- criterion of ``102 CFU/mL'' for total aerobic viable counts JP XVI General Information / Others 2253 throughout the period of their storage. It is also necessary to ultrafiltration, may be used for these purposes. Water pro- take special care against microbial contamination during its duced for these purposes at other individual facilities may circulation. In addition, it is recommended to use them as also be used. soon as possible after opening their seals. The water for tests specified in General Tests in the JP is The acceptance criterion of ``102 CFU/mL'' for total aer- as follows: obic viable counts of Purified Water in Containers is at the ・Water for ammonium limit test: <1.02> Ammonium same level as the action level for viable counts in the produc- Limit Test (Standard Ammonium Solution) tion of Purified Water (in bulk). However, different from ・Water for bacterial endotoxins test: <4.01> Bacterial En- the case of microbiological monitoring of Purified Water, dotoxins Test Soybean-Casein Digest Agar Medium is used for the test of ・Water for particulate matter test (for injections): <6.07> total aerobic viable counts of Purified Water in Containers Insoluble Particulate Matter Test for Injections to detect microorganisms contaminated from the surround- ・Water for particulate matter test (for ophthalmic solu- ings during its storage and circulation. tions): <6.08> Insoluble Particulate Matter Test for Oph- 5.3. Points to consider in the case that commercially avail- thalmic Solutions able products of pharmaceutical water in containers are used ・Water for particulate matter test (for plastic containers): for the manufacture of pharmaceutical products <7.02> Test Methods for Plastic Containers It is allowable to use commercially available products of The water for tests specified in General Information in the pharmaceutical water in containers (Purified Water in Con- JP is as follows: tainers, Sterile Purified Water in Containers or Sterile Water ・Water for aluminum test: Test for Trace Amounts of for Injection in Containers) for the manufacture of phar- Aluminum in Trans Parenteral Nutrition (TPN) Solu- maceutical products and products for clinical trial, and for tions the tests of pharmaceutical products. In such cases, it is nec- ・Water for ICP analysis: Inductively Coupled Plasma essary to consider the following points. Emission Spectral Analysis ( i ) When such products are used for manufacturing The term ``water'' described in the text concerning tests of pharmaceutical products, it is recommended to use drugs means ``the water to be used in the tests of drugs'' as them soon after confirming their compliances to the defined in the paragraph 20 under General Notices. requirements of JP monograph from the test results at the time of its receipt or those offered from the supplier of the products. ( ii ) In the case that such products are used for manufac- G9 Others turing pharmaceutical products, it is necessary to validate the process in which the water was used as a part of process validation of pharmaceutical prod- International Harmonization ucts. In the case that they are used for manufacturing products for clinical trial, it is necessary to con- Implemented in the Japanese firm that the water doesn't give any adverse effects Pharmacopoeia Sixteenth Edition on the quality of the products. (iii) The products of sterile pharmaceutical water in con- Items for which harmonization has been agreed among the tainers should be used only once after opening their European Pharmacopoeia, the United States Pharmacopeia seals, and it must be avoided to use them again after and the Japanese Pharmacopoeia are implemented in the storage. Japanese Pharmacopoeia Sixteenth Edition (JP 16). They (iv) It is recommended to prepare a standard operation are shown in the tables below. practice (SOP) adequate for its intended use, con- The column headed Harmonized items shows the har- sidering that the contamination and quality deterio- monized items written in the Pharmacopoeial Harmoniza- ration of the water due to human and laboratory en- tion Agreement Document, and the column headed JP 16 vironmental origins might goonrapidlyimmediately shows the items as they appear in JP 16. In the Remarks after opening the product seal. column, notes on any differences between JP 16 and the agreement are shown as occasion demands. The date on which the agreement has been signed is shown on the top pf each table. In the case where the harmonized Watertobeusedinthe items have been revised and/or corrected, this is indicated in Tests of Drugs parenthesis.

The water to be used in the tests of drugs is defined as ``the water suitable for performing the relevant test'' in the paragraph 20 under General Notices of the JP. Therefore, it is necessary to confirm that the water to be used in a test of a drug is suitable for the purpose of the relevant test before its use. Unless otherwise specified in the individual test method, Purified Water, Purified Water in Containers or the water produced by an appropriate process, such as ion exchange or 2254 Others / General Information JP XVI

Aug. 2005 (Rev. 2)