Quick viewing(Text Mode)

General Information

General Information

GENERAL INFORMATION

use, keeping solvent reservoirs covered, and not placing ami- 1. AminoAcidAnalysis no analysis instrumentation in direct sunlight. Laboratory practices can determine the quality of the analysis. Place the instrumentation in a low tra‹c This test is harmonized with the European area of the laboratory. Keep the laboratory clean. Clean and and the U.S. Pharmacopeia. calibrate pipets according to a maintenance schedule. Keep Amino acid analysis refers to the methodology used to de- pipet tips in a covered box; the analysts may not handle pipet termine the amino acid composition or content of , tips with their hands. The analysts may wear powder-free la- , and other pharmaceutical preparations. Proteins tex or equivalent gloves. Limit the number of times a test and peptides are macromolecules consisting of covalently sample vial is opened and closed because dust can contribute bonded amino acid residues organized as a linear polymer. to elevated levels of , , and . The sequence of the amino in a or A well-maintained instrument is necessary for acceptable determines the properties of the molecule. Proteins are amino acid analysis results. If the instrument is used on a considered large molecules that commonly exist as folded routine basis, it is to be checked daily for leaks, detector and structures with a speciˆc conformation, while peptides are lamp stability, and the ability of the column to maintain reso- smaller and may consist of only a few amino acids. Amino lution of the individual amino acids. Clean or replace all acid analysis can be used to quantify protein and peptides, to instrument ˆlters and other maintenance items on a routine determine the identity of proteins or peptides based on their schedule. amino acid composition, to support protein and peptide structure analysis, to evaluate fragmentation strategies for Reference Standard Material peptide mapping, and to detect atypical amino acids that Acceptable amino acid standards are commercially might be present in a protein or peptide. It is necessary to available for amino acid analysis and typically consist of an hydrolyze a protein/peptide to its individual amino acid aqueous mixture of amino acids. When determining amino constituents before amino acid analysis. Following pro- acid composition, protein or peptide standards are analyzed tein/peptide hydrolysis, the amino acid analysis procedure with the test material as a control to demonstrate the integrity can be the same as that practiced for free amino acids in other of the entire procedure. Highly puriˆed bovine serum pharmaceutical preparations. The amino acid constituents of albumin has been used as a protein standard for this purpose. the test sample are typically derivatized for analysis. Calibration of Instrumentation Apparatus Calibration of amino acid analysis instrumentation Methods used for amino acid analysis are usually based on typically involves analyzing the amino acid standard, which a chromatographic separation of the amino acids present in consists of a mixture of amino acids at a number of concen- the test sample. Current techniques take advantage of the au- trations, to determine the response factor and range of tomated chromatographic instrumentation designed for ana- analysis for each amino acid. The concentration of each ami- lytical methodologies. An amino acid analysis instrument will no acid in the standard is known. In the calibration proce- typically be a low-pressure or high-pressure liquid chromato- dure, the analyst dilutes the amino acid standard to several graph capable of generating mobile phase gradients that diŠerent analyte levels within the expected linear range of the separate the amino acid analytes on a chromatographic amino acid analysis technique. Then, replicates at each of the column. The instrument must have postcolumn derivatiza- diŠerent analyte levels can be analyzed. Peak areas obtained tion capability, unless the sample is analyzed using for each amino acid are plotted versus the known concentra- precolumn derivatization. The detector is usually an ultrav- tion for each of the amino acids in the standard dilution. iolet-visible or ‰uorescence detector depending on the These results will allow the analyst to determine the range of derivatization method used. A recording device (e.g., amino acid concentrations where the peak area of a given integrator) is used for transforming the analog signal from amino acid is an approximately linear function of the amino the detector and for quantitation. It is preferred that acid concentration. It is important that the analyst prepare instrumentation be dedicated particularly for amino acid the samples for amino acid analysis so that they are within analysis. the analytical limits (e.g., linear working range) of the tech- nique employed in order to obtain accurate and repeatable General Precautions results. Background contamination is always a concern for the Four to six amino acid standard levels are analyzed to analyst in performing amino acid analysis. High purity determine a response factor for each amino acid. The reagents are necessary (e.g., low purity can response factor is calculated as the average peak area or peak contribute to glycine contamination). Analytical reagents are height per nmol of amino acid present in the standard. A changed routinely every few weeks using only high-pressure calibration ˆle consisting of the response factor for each ami- liquid (HPLC) grade solvents. Potential no acid is prepared and used to calculate the concentration of microbial contamination and foreign material that might be each amino acid present in the test sample. This calculation present in the solvents are reduced by ˆltering solvents before involves dividing the peak area corresponding to a given ami- 1655 1656 AminoAcidAnalysis/ General Information JP XV no acid by the response factor for that amino acid to give the standard can be added to a protein prior to hydroly- nmol of the amino acid. For routine analysis, a single-point sis. The recovery of the internal standard gives the general calibration may be su‹cient; however, the calibration ˆle is recovery of the amino acids of the protein solution. Free ami- updated frequently and tested by the analysis of analytical no acids, however, do not behave in the same way as protein- controls to ensure its integrity. bound amino acids during hydrolysis because their rates of release or destruction are variable. Therefore, the use of an Repeatability internal standard to correct for losses during hydrolysis may Consistent high quality amino acid analysis results from an give unreliable results. It will be necessary to take this point analytical laboratory require attention to the repeatability of under consideration when interpreting the results. Internal the assay. During analysis of the chromatographic separation standards can also be added to the mixture of amino acids af- of the amino acids or their derivatives, numerous peaks can ter hydrolysis to correct for diŠerences in sample application be observed on the chromatogram that correspond to the and changes in reagent stability and ‰ow rates. Ideally, an in- amino acids. The large number of peaks makes it necessary to ternal standard is an unnaturally occurring primary amino have an amino acid analysis system that can repeatedly iden- acid that is commercially available and inexpensive. It should tify the peaks based on retention time and integrate the peak also be stable during hydrolysis, its response factor should be areas for quantitation. A typical repeatability evaluation in- linear with concentration, and it needs to elute with a unique volves preparing a standard amino acid solution and analyz- retention time without overlapping other amino acids. Com- ing many replicates (i.e., six analyses or more) of the same monly used amino acid standards include norleucine, standard solution. The relative standard deviation (RSD) is nitrotyrosine, and a-aminobutyric acid. determined for the retention time and integrated peak area of each amino acid. An evaluation of the repeatability is ex- Protein Hydrolysis panded to include multiple assays conducted over several Hydrolysis of protein and peptide samples is necessary for days by diŠerent analysts. Multiple assays include the prepa- amino acid analysis of these molecules. The glassware used ration of standard dilutions from starting materials to deter- for hydrolysis must be very clean to avoid erroneous results. mine the variation due to sample handling. Often the amino Glove powders and ˆngerprints on hydrolysis tubes may acid composition of a standard protein (e.g., bovine serum cause contamination. To clean glass hydrolysis tubes, boil albumin) is analyzed as part of the repeatability evaluation. tubes for 1 hour in 1 mol/L hydrochloric acid or soak tubes By evaluating the replicate variation (i.e., RSD), the labora- in concentrated nitric acid or in a mixture of concentrated tory can establish analytical limits to ensure that the analyses hydrochloric acid and concentrated nitric acid (1:1). Clean from the laboratory are under control. It is desirable to estab- hydrolysis tubes are rinsed with high-purity water followed lish the lowest practical variation limits to ensure the best by a rinse with HPLC grade , dried overnight in an results. Areas to focus on to lower the variability of the ami- oven, and stored covered until use. Alternatively, pyrolysis of no acid analysis include sample preparation, high back- clean glassware at 5009C for 4 hours may also be used to ground spectral interference due to quality of reagents eliminate contamination from hydrolysis tubes. Adequate and/or laboratory practices, instrument performance and disposable laboratory material can also be used. maintenance, data analysis and interpretation, and analyst Acid hydrolysis is the most common method for hydrolyz- performance and habits. All parameters involved are fully in- ing a protein sample before amino acid analysis. The acid vestigated in the scope of the validation work. hydrolysis technique can contribute to the variation of the analysis due to complete or partial destruction of several ami- Sample Preparation no acids. is destroyed; serine and are Accurate results from amino acid analysis require puriˆed partially destroyed; might undergo oxidation; protein and peptide samples. BuŠer components (e.g., salts, and is typically recovered as cystine (but cystine , detergents) can interfere with the amino acid analysis recovery is usually poor because of partial destruction or and are removed from the sample before analysis. Methods reduction to cysteine). Application of adequate vacuum that utilize postcolumn derivatization of the amino acids are (≦less than 200 mmofmercuryor26.7Pa)orintroductionof generally not aŠected by buŠer components to the extent seen an inert (argon) in the headspace of the reaction vessel with precolumn derivatization methods. It is desirable to can reduce the level of oxidative destruction. In peptide limit the number of sample manipulations to reduce potential bonds involving and the amido bonds of Ile- background contamination, to improve analyte recovery, Ile, Val-Val, Ile-Val, and Val-Ile are partially cleaved; and and to reduce labor. Common techniques used to remove and are deamidated, resulting in aspar- buŠer components from protein samples include the follow- tic acid and , respectively. The loss of trypto- ing methods: (1) injecting the protein sample onto a reversed- phan, asparagine, and glutamine during an acid hydrolysis phase HPLC system, eluting the protein with a volatile sol- limits quantitation to 17 amino acids. Some of the hydrolysis vent containing a su‹cient organic component, and drying techniques described are used to address these concerns. the sample in a vacuum centrifuge; (2) dialysis against a vola- Some of the hydrolysis techniques described (i.e., Methods tile buŠer or water; (3) centrifugal ultraˆltration for buŠer 4-11) may cause modiˆcations to other amino acids. There- replacement with a volatile buŠer or water; (4) precipitating fore, the beneˆts of using a given hydrolysis technique are the protein from the buŠer using an organic solvent (e.g., weighed against the concerns with the technique and are test- ); and (5) gel ˆltration. ed adequately before employing a method other than acid Internal Standards hydrolysis. Itisrecommendedthataninternalstandardbeusedtomo- A time-course study (i.e., amino acid analysis at acid nitor physical and chemical losses and variations during ami- hydrolysis times of 24, 48, and 72 hours) is often employed to no acid analysis. An accurately known amount of internal analyze the starting concentration of amino acids that are JP XV General Information / AminoAcidAnalysis 1657 partially destroyed or slow to cleave. By plotting the observed or 26.7 Pa) to the headspace of the vessel, and heat concentration of labile amino acids (i.e., serine and threo- to about 1109C for a 24-hour hydrolysis time. Acid vapor nine) versus hydrolysis time, the line can be extrapolated to hydrolyzes the dried sample. Any condensation of the acid in the origin to determine the starting concentration of these the sample vials is minimized. After hydrolysis, dry the test amino acids. Time-course hydrolysis studies are also used sample in vacuum to remove any residual acid. with amino acids that are slow to cleave (e.g., isoleucine and Method 2 valine). During the hydrolysis time course, the analyst will Tryptophan oxidation during hydrolysis is decreased by us- observe a plateau in these residues. The level of this plateau is ing mercaptoethanesulfonic acid (MESA) as the reducing taken as the residue concentration. If the hydrolysis time is acid. too long, the residue concentration of the sample will begin Hydrolysis Solution 2.5 mol/L MESA solution. to decrease, indicating destruction by the hydrolysis condi- Vapor Phase Hydrolysis About 1 to 100 mgofthe tions. protein/peptide under test is dried in a hydrolysis tube. The An acceptable alternative to the time-course study is to hydrolysis tube is placed in a larger tube with about 200 mLof subject an amino acid calibration standard to the same the Hydrolysis Solution. The larger tube is sealed in vacuum hydrolysis conditions as the test sample. The amino acid in (about 50 mm of mercury or 6.7 Pa) to vaporize the Hydroly- free form may not completely represent the rate of destruc- sis Solution. The hydrolysis tube is heated to 1709Cto1859C tion of labile amino acids within a peptide or protein during for about 12.5 minutes. After hydrolysis, the hydrolysis tube the hydrolysis. This is especially true for peptide bonds that is dried in vacuum for 15 minutes to remove the residual acid. are slow to cleave (e.g., Ile-Val bonds). However, this technique will allow the analyst to account for some residue Method 3 destruction. Microwave acid hydrolysis has been used and is Tryptophan oxidation during hydrolysis is prevented by rapid but requires special equipment as well as special precau- using thioglycolic acid (TGA) as the reducing acid. tions. The optimal conditions for microwave hydrolysis must Hydrolysis Solution A solution containing 7 mol/L be investigated for each individual protein/peptide sample. hydrochloric acid, 10z of tri‰uoroacetic acid, 20z of The microwave hydrolysis technique typically requires only a thioglycolic acid, and 1z of . few minutes, but even a deviation of one minute may give in- Vapor Phase Hydrolysis About 10 to 50 mgofthe adequate results (e.g., incomplete hydrolysis or destruction protein/peptide under test is dried in a sample tube. The sam- of labile amino acids). Complete , using a mixture ple tube is placed in a larger tube with about 200 mLofthe of , has been used but can be complicated, requires Hydrolysis Solution. The larger tube is sealed in vacuum the proper controls, and is typically more applicable to pep- (about 50 mm of mercury or 6.7 Pa) to vaporize the TGA. tides than proteins. The sample tube is heated to 1669C for about 15 to 30 Note: During initial analyses of an unknown protein, minutes. After hydrolysis, the sample tube is dried in vacuum experiments with various hydrolysis time and temperature for 5 minutes to remove the residual acid. Recovery of tryp- conditions are conducted to determine the optimal condi- tophan by this method may be dependent on the amount of tions. sample present. Method 1 Method 4 Acid hydrolysis using hydrochloric acid containing phenol Cysteine-cystine and methionine oxidation is performed is the most common procedure used for protein/peptide with performic acid before the protein hydrolysis. hydrolysis preceding amino acid analysis. The addition of Oxidation Solution The performic acid is prepared fresh phenol to the reaction prevents the halogenation of . by mixing formic acid and 30 percent peroxide Hydrolysis Solution 6 mol/L hydrochloric acid contain- (9:1), and incubated at room temperature for 1 hour. ing 0.1z to 1.0z of phenol. Procedure The protein/peptide sample is dissolved in Procedure— 20 mLofformicacid,andheatedat509C for 5 minutes; then Liquid Phase Hydrolysis Place the protein or peptide 100 mLoftheOxidation Solution is added. The oxidation is sample in a hydrolysis tube, and dry. [Note: The sample is d- allowed to proceed for 10 to 30 minutes. In this reaction, cys- ried so that water in the sample will not dilute the acid used teine is converted to cysteic acid and methionine is converted for the hydrolysis.] Add 200 mLofHydrolysis Solution per to methionine sulfone. The excess reagent is removed from 500 mg of lyophilized protein. Freeze the sample tube in a dry the sample in a vacuum centrifuge. This technique may cause ice-acetone bath, and ‰ame seal in vacuum. Samples are typi- modiˆcations to tyrosine residues in the presence of halides. cally hydrolyzed at 1109C for 24 hours in vacuum or inert at- The oxidized protein can then be acid hydrolyzed using mosphere to prevent oxidation. Longer hydrolysis times Method 1 or Method 2. (e.g., 48 and 72 hours) are investigated if there is a concern Method 5 that the protein is not completely hydrolyzed. Cysteine-cystine oxidation is accomplished during the Vapor Phase Hydrolysis This is one of the most common liquid phase hydrolysis with azide. acid hydrolysis procedures, and it is preferred for microanal- Hydrolysis Solution 6 mol/L hydrochloric acid contain- ysis when only small amounts of the sample are available. ing 0.2z of phenol, to which is added sodium azide to obtain Contamination of the sample from the acid reagent is also a ˆnal concentration of 0.2z (w/v). The added phenol pre- minimized by using vapor phase hydrolysis. Place vials con- vents halogenation of tyrosine. taining the dried samples in a vessel that contains an ap- Liquid Phase Hydrolysis The protein/peptide hydrolysis propriate amount of Hydrolysis Solution.TheHydrolysis is conducted at about 1109C for 24 hours. During the hydrol- Solution does not come in contact with the test sample. App- ysis, the cysteine-cystine present in the sample is converted to ly an inert atmosphere or vacuum (≦ less than 200 mmof cysteic acid by the sodium azide present in the Hydrolysis So- 1658 AminoAcidAnalysis/ General Information JP XV lution. This technique allows better tyrosine recovery than room temperature in the dark. To achieve the pyridylethyla- Method 4, but it is not quantitative for methionine. Methio- tion reaction, add about 2 mL of 4-vinylpyridine to the nine is converted to a mixture of the parent methionine and protein solution, and incubate for an additional 2 hours at its two oxidative products, methionine sulfoxide and methio- room temperature in the dark. The protein/peptide is nine sulfone. desalted by collecting the protein/peptide fraction from a reversed-phase HPLC separation. The collected sample can Method 6 be dried in a 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 ˆnal concentration of 2z (v/v). Stock 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 hydrol- taining 100 mg of iodoacetamide per mL of (95). ysis, the cysteine-cystine present in the sample is converted to BuŠer Solution Use the Reducing Solution,preparedas cysteic acid by the DMSO present in the Hydrolysis Solution. directed for Method 8. As an approach to limit variability and compensate for par- Procedure Dissolve the test sample in 50 mLoftheBuŠer tial destruction, it is recommended to evaluate the cysteic Solution, and add about 2.5 mLofStock Solution C.Store acid recovery from oxidative hydrolyses of standard proteins under or argon for 2 hours at room temperature in containing 1 to 8 mol of cysteine. The response factors from the dark. Add the Carboxymethylation Solution in a ratio 1.5 protein/peptide hydrolysates are typically about 30z lower fold per total theoretical content of , and incubate for than those for nonhydrolyzed cysteic acid standards. Because an additional 30 minutes at room temperature in the dark. , methionine, tyrosine, and tryptophan are also mo- [Note: If the content of the protein is unknown, then diˆed, a complete compositional analysis is not obtained with add 5 mL of 100 mmol/L iodoacetamide for every 20 nmol of this technique. protein present.] The reaction is stopped by adding excess of 2-mercaptoethanol. The protein/peptide is desalted by col- Method 7 lecting the protein/peptide fraction from a reversed-phase Cysteine-cystine reduction and alkylation is accomplished HPLC separation. The collected sample can be dried in a by a vapor phase pyridylethylation reaction. vacuum centrifuge before acid hydrolysis. The Reducing Solution Transfer 83.3 mL of , 16.7 mL S-carboxyamidomethyl-cysteine formed will be converted to of 4-vinylpyridine, 16.7 mL of tributylphosphine, and 83.3 S-carboxymethylcysteine during acid hydrolysis. mL of water to a suitable container, and mix. Procedure Add the protein/peptide (between 1 and 100 Method 10 mg) to a hydrolysis tube, and place in a larger tube. Transfer Cysteine-cystine is reacted with dithiodiglycolic acid or the Reducing Solution to the large tube, seal in vacuum dithiodipropionic acid to produce a mixed disulˆde. [Note: (about 50 mm of mercury or 6.7 Pa), and incubate at about The choice of dithiodiglycolic acid or dithiodipropionic acid 1009C for 5 minutes. Then remove the inner hydrolysis tube, depends on the required resolution of the amino acid analysis and dry it in a vacuum desiccator for 15 minutes to remove method.] residual reagents. The pyridylethylated protein/peptide can Reducing Solution A solution containing 10 mg of then be acid hydrolyzed using previously described dithiodiglycolic acid (or dithiodipropionic acid) per mL of procedures. The pyridylethylation reaction is performed 0.2 mol/L . simultaneously with a protein standard sample containing 1 Procedure Transfer about 20 mg of the test sample to a to 8 mol of cysteine to improve accuracy in the pyridylethyl- hydrolysis tube, and add 5 mLoftheReducing Solution.Add cysteine recovery. Longer incubation times for the 10 mL of isopropyl , and then remove all of the sample pyridylethylation reaction can cause modiˆcations to the liquid by vacuum centrifugation. The sample is then hydro- a-amino terminal group and the e-amino group of in lyzed using Method 1. This method has the advantage that the protein. other amino acid residues are not derivatized by side reac- tions, and the sample does not need to be desalted prior to Method 8 hydrolysis. Cysteine-cystine reduction and alkylation is accomplished by a liquid phase pyridylethylation reaction. Method 11 Stock Solutions Prepare and ˆlter three solutions: Asparagine and glutamine are converted to 1 mol/L Tris hydrochloride (pH 8.5) containing 4 mmol/L and glutamic acid, respectively, during acid hydrolysis. disodium dihydrogen ethylendiamine tetraacetate (Stock So- Asparagine and aspartic acid residues are added and lution A), 8 mol/L guanidine hydrochloride (Stock Solution represented by Asx, while glutamine and glutamic acid B), and 10z of 2-mercaptoethanol in water (Stock Solution residues are added and represented by Glx. Proteins/peptides C). can be reacted with bis(1,1-tri‰uoroacetoxy)iodobenzene Reducing Solution PrepareamixtureofStock Solution B (BTI) to convert the asparagine and glutamine residues to and Stock Solution A (3:1)toobtainabuŠeredsolution diaminopropionic acid and diaminobutyric acid residues, of 6 mol/L guanidine hydrochloride in 0.25 mol/L Tris respectively, upon acid hydrolysis. These conversions allow hydrochloride. the analyst to determine the asparagine and glutamine Procedure Dissolve about 10 mg of the test sample in 50 content of a protein/peptide in the presence of aspartic acid mLoftheReducing Solution, and add about 2.5 mLofStock andglutamicacidresidues. Solution C. Store under nitrogen or argon for 2 hours at Reducing Solutions Prepare and ˆlter three solutions: a JP XV General Information / AminoAcidAnalysis 1659 solution of 10 mmol/L tri‰uoroacetic acid (Solution A), a tem, and the peak areas are integrated for quantiˆcation pur- solution of 5 mol/L guanidine hydrochloride and 10 mmol/L poses. tri‰uoroacetic acid (Solution B), and a freshly prepared Method 1—Postcolumn Detection General solution of N,N-dimethylformamide containing 36 mg of Principle BTI per mL (Solution C). -exchange chromatography with postcolumn ninhydrin Procedure In a clean hydrolysis tube, transfer about detection is one of the most common methods employed for 200 mg of the test sample, and add 2 mL of Solution A or quantitative amino acid analysis. As a rule, a Li-based Solution B and 2 mL of Solution C. Seal the hydrolysis tube cation-exchange system is employed for the analysis of the in vacuum. Heat the sample at 609C for 4 hours in the dark. more complex physiological samples, and the faster Na-based The sample is then dialyzed with water to remove the excess cation-exchange system is used for the more simplistic amino reagents. Extract the dialyzed sample three times with equal acid mixtures obtained with protein hydrolysates (typically volumes of n-butyl , and then lyophilize. The protein containing 17 amino acid components). Separation of the can then be acid hydrolyzed using previously described proce- amino acids on an ion-exchange column is accomplished dures. The a,b-diaminopropionic and a,g-diaminobutyric through a combination of changes in pH and cation strength. acid residues do not typically resolve from the lysine residues A temperature gradient is often employed to enhance separa- upon ion-exchange chromatography based on amino acid tion. analysis. Therefore, when using ion-exchange as the mode of When the amino acid reacts with ninhydrin, the reactant amino acid separation, the asparagine and glutamine con- has characteristic purple or yellow color. Amino acids, except tents are the quantitative diŠerence in the aspartic acid and , give a purple color, and show the maximum ab- glutamic acid content assayed with underivatized and BTI- sorption at 570 nm. The imino acids such as give a derivatized acid hydrolysis. [Note: The threonine, methio- yellow color, and show the maximum absorption at 440 nm. nine, cysteine, tyrosine, and histidine assayed content can be The postcolumn reaction between ninhydrin and amino acid altered by BTI derivatization; a hydrolysis without BTI will eluted from column is monitored at 440 and 570 nm, and the have to be performed if the analyst is interested in the compo- chromatogram obtained is used for the determination of ami- sition of these other amino acid residues of the protein/pep- no acid composition. tide.] Detection limit is considered to be 10 pmol for most of the amino acid derivatives, but 50 pmol for proline. Response Methodologies of Amino Acid Analysis General Principles linearity is obtained in the range of 20 to 500 pmol with Many amino acid analysis techniques exist, and the choice correlation coe‹cients exceeding 0.999. To obtain good com- of any one technique often depends on the sensitivity position data, samples larger than 1 mg before hydrolysis are required from the assay. In general, about one-half of the best suited for this amino acid analysis of protein/peptide. amino acid analysis techniques employed rely on the separation of the free amino acids by ion-exchange Method 2—Postcolumn OPA Fluorometric Detection chromatography followed by postcolumn derivatization General Principle (e.g., with ninhydrin or o-phthalaldehyde). Postcolumn o-Phthalaldehyde (OPA) reacts with primary in the detection techniques can be used with samples that contain presence of thiol compound, to form highly ‰uorescent isoin- small amounts of buŠer components, such as salts and dole products. This reaction is utilized for the postcolumn urea, and generally require between 5 and 10 mgofprotein derivatization in analysis of amino acids by ion-exchange sample per analysis. The remaining amino acid techniques chromatography. The rule of the separation is the same as typically involve precolumn derivatization of the free amino Method 1. Instruments and reagents for this form of amino acids (e.g., phenyl isothiocyanate; 6-aminoquinolyl-N- acid analysis are available commercially. Many modiˆcations hydroxysuccinimidyl or o-phthalaldehyde; of this methodology exist. (dimethylamino)azobenzenesulfonyl chloride; 9- Although OPA does not react with secondary amines ‰uorenylmethylchloroformate; and, 7-‰uoro-4-nitrobenzo-2- (imino acids such as proline) to form ‰uorescent substances, oxa-1,3-diazole) followed by reversed-phase HPLC. the oxidation with sodium hypochlorite allows secondary Precolumn derivatization techniques are very sensitive and amines to react with OPA. The procedure employs a strongly usually require between 0.5 and 1.0 mg of protein sample per acidic cation-exchange column for separation of free amino analysis but may be in‰uenced by buŠer salts in the samples. acids followed by postcolumn oxidation with sodium Precolumn derivatization techniques may also result in hypochlorite and postcolumn derivatization using OPA and multiple derivatives of a given amino acid, which complicates thiol compound such as N-acetyl-L-cysteine and 2-mercap- the result interpretation. Postcolumn derivatization tech- toethanol. The derivatization of primary amino acids are not niques are generally in‰uenced less by performance variation noticeably aŠected by the continuous supply of sodium of the assay than precolumn derivatization techniques. hypochlorite. The following Methods may be used for quantitative Separation of the amino acids on an ion-exchange column amino acid analysis. Instruments and reagents for these is accomplished through a combination of changes in pH and procedures are available commercially. Furthermore, many cation strength. After postcolumn derivatization of eluted modiˆcations of these methodologies exist with diŠerent amino acids with OPA, the reactant passes through the reagent preparations, reaction procedures, chromatographic ‰uorometric detector. Fluorescence intensity of OPA-deriva- systems, etc. Speciˆc parameters may vary according to the tizedaminoacidsaremonitoredwithanexcitation exact equipment and procedure used. Many laboratories will wavelength of 348 nm and an emission wavelength of utilize more than one amino acid analysis technique to exploit 450 nm. the advantages oŠered by each. In each of these Methods,the Detection limit is considered to be a few tens of picomole analog signal is visualized by means of a data acquisition sys- level for most of the amino acid derivatives. Response lineari- 1660 AminoAcidAnalysis/ General Information JP XV ty is obtained in the range of a few picomole level to a few be obtained from the analysis of derivatized protein hydroly- tens of nanomole level. To obtain good compositional data, sates containing as little as 30 ng of protein/peptide. the starting with greater than 500 ng of sample before hydrol- Method 5—Precolumn OPA Derivatization General ysis is best suited for the amino acid analysis of Principle protein/peptide. Precolumn derivatization of amino acids with o-phthalal- dehyde (OPA) followed by reversed-phase HPLC separation Method 3—Precolumn PITC Derivatization General with ‰uorometric detection is used. This technique does not Principle detect amino acids that exist as secondary amines (e.g., Phenylisothiocyanate (PITC) reacts with amino acids to proline). form phenylthiocarbamyl (PTC) derivatives which can be de- o-Phthalaldehyde (OPA) in conjunction with a thiol tected with high sensitivity at 245 nm. Therefore, precolumn reagent reacts with primary groups to form highly derivatization of amino acids with PITC followed by a rever- ‰uorescent isoindole products. 2-Mercaptoethanol or 3-mer- sed-phase HPLC separation with UV detection is used to captopropionic acid can be used as the thiol. OPA itself does analyze the amino acid composition. not ‰uoresce and consequently produces no interfering After the reagent is removed under vacuum, the deriva- peaks. In addition, its and stability in aqueous tized amino acids can be stored dry and frozen for several solution, along with the rapid kinetics for the reaction, make weeks with no signiˆcant degradation. If the solution for in- it amenable to automated derivatization and analysis using jection is kept cold, no noticeable loss in chromatographic an autosampler to mix the sample with the reagent. However, response occurs after three days. lack of reactivity with secondary amino acids has been Separation of the PTC-amino acids on a reversed-phase predominant drawback. This method does not detect amino HPLC with ODS column is accomplished through a combi- acids that exist as secondary amines (e.g., proline). To com- nation of changes in concentrations of acetonitrile and buŠer pensate for this drawback, this technique may be combined ionic strength. PTC-amino acids eluted from column are with another technique described in Method 7 or Method 8. monitored at 254 nm. Precolumn derivatization of amino acids with OPA is Detection limit is considered to be 1 pmol for most of the followed by a reversed-phase HPLC separation. Because of amino acid derivatives. Response linearity is obtained in the the instability of the OPA-amino acid derivative, HPLC range of 20 to 500 pmol with correlation coe‹cients exceed- separation and analysis are performed immediately following ing 0.999. To obtain good compositional data, samples larger derivatization. The liquid chromatograph is equipped with a than 500 ng of protein/peptide before hydrolysis is best suit- ‰uorometric detector for the detection of derivatized amino ed for this amino acid analysis of proteins/peptides. acids. Fluorescence intensity of OPA-derivatized amino acids Method 4—Precolumn AQC Derivatization General is monitored with an excitation wavelength of 348 nm and an Principle emission wavelength of 450 nm. Precolumn derivatization of amino acids with 6-amino- Detection limits as low as 50 fmol via ‰uorescence have quinolyl-N-hydroxysuccinimidyl carbamate (AQC) followed been reported, although the practical limit of analysis by reversed-phase HPLC separation with ‰uorometric remains at 1 pmol. detection is used. Method 6—Precolumn DABS-Cl Derivatization General 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate Principle (AQC) reacts with amino acids to form stable, ‰uorescent un- Precolumn derivatization of amino acids with symmetric urea derivatives (AQC-amino acids) which are rea- (dimethylamino)azobenzenesulfonyl chloride (DABS-Cl) fol- dily amenable to analysis by reversed-phase HPLC. There- lowed by reversed-phase HPLC separation with visible light fore, precolumn derivatization of amino acids with AQC fol- detection is used. lowed by reversed-phase HPLC separation is used to analyze (Dimethylamino)azobenzenesulfonyl chloride (DABS-Cl) the amino acid composition. is a chromophoric reagent employed for the labeling of Separation of the AQC-amino acids on ODS column is amino acids. Amino acids labeled with DABS-Cl (DABS- accomplished through a combination of changes in concen- amino acids) are highly stable and show the maximum trations of acetonitrile and . Selective ‰uorescence detec- absorption at 436 nm. tion of the derivatives with excitation wavelength at 250 nm DABS-amino acids, all 19 naturally occurring amino acids and emission wavelength at 395 nm allows for the direct derivatives, can be separated on an ODS column of a of the reaction mixture with no signiˆcant interfer- reversed-phase HPLC by employing gradient systems consist- ence from the only major ‰uorescent reagent by-product, ing of acetonitrile and aqueous buŠer mixture. Separated 6-aminoquinoline. Excess reagent is rapidly hydrolyzed (t 1/2 DABS-amino acids eluted from column are detected at º15 seconds) to yield 6-aminoquinoline, N-hydroxysuc- 436 nm in the visible region. cinimide and dioxide, and after 1 minute no further This Method can analyze the imino acids such as proline derivatization can take place. together with the amino acids at the same degree of sensitivi- Peak areas for AQC-amino acids are essentially unchanged ty, DABS-Cl derivatization method permits the simultaneous for at least 1 week at room temperature, and the derivatives quantiˆcation of tryptophan residues by previous hydrolysis have more than su‹cient stability to allow for overnight au- of the protein/peptide with sulfonic acids such as mercap- tomated chromatographic analysis. toethanesulfonic acid, p-toluenesulfonic acid or methanesul- Detection limit is considered to be ranging from ca. 40 to fonic acid described under Method 2 in ``Protein Hydroly- 320 fmol for each amino acid, except for Cys. Detection limit sis''. The other acid-labile residues, asparagine and gluta- for Cys is approximately 800 fmol. Response linearity is ob- mine, can also be analysed by previous conversion into di- tained in the range of 2.5 to 200 mmol/L with correlation aminopropionic acid and diaminobutyric acid, respectively, coe‹cients exceeding 0.999. Good compositional data could JP XV General Information / AminoAcidAnalysis 1661 by treatment of protein/peptide with BTI described under achieved for about 1.5 mg of protein hydrolysates in the ˆnal Method 11 in ``Protein Hydrolysis''. precolumn labeling reaction mixture for HPLC. The non-, norleucine cannot be Data Calculation and Analysis used as internal standard in this method, as this compound is When determining the amino acid content of a pro- eluted in a chromatographic region crowded with peaks of tein/peptide hydrolysate, it should be noted that the acid primary amino acids. Nitrotyrosine can be used as an internal hydrolysis step destroys tryptophan and cysteine. Serine and standard, because it is eluted in a clean region. threonine are partially destroyed by acid hydrolysis, while Detection limit of DABS-amino acid is about 1 pmol. As isoleucine and valine residues may be only partially cleaved. little as 2 to 5 pmol of an individual DABS-amino acid can be Methionine can undergo oxidation during acid hydrolysis, quantitatively analysed with reliability, and only 10 to 30 ng and some amino acids (e.g., glycine and serine) are common of the dabsylated protein hydrolysate is required for each contaminants. Application of adequate vacuum (≦0.0267 analysis. kPa) or introduction of inert gas (argon) in the headspace of the reaction vessel during vapor phase hydrolysis can reduce Method 7—Precolumn FMOC-Cl Derivatization General the level of oxidative destruction. Therefore, the quantitative Principle results obtained for cysteine, tryptophan, threonine, isoleu- Precolumn derivatization of amino acids with 9- cine, valine, methionine, glycine, and serine from a pro- ‰uorenylmethyl chloroformate (FMOC-Cl) followed by tein/peptide hydrolysate may be variable and may warrant reversed-phase HPLC separation with ‰uorometric detection further investigation and consideration. is used. 9-Fluorenylmethyl chloroformate (FMOC-Cl) reacts with Calculations both primary and secondary amino acids to form highly Amino Acid Mole Percent Thisisthenumberofspeciˆc ‰uorescent products. The reaction of FMOC-Cl with amino amino acid residues per 100 residues in a protein. This result acid proceeds under mild conditions in and may be useful for evaluating amino acid analysis data when is completed in 30 seconds. The derivatives are stable, only the molecular weight of the protein under investigation is the histidine derivative showing any breakdown. Although unknown. This information can be used to corroborate the FMOC-Cl is ‰uorescent itself, the reagent excess and identity of a protein/peptide and has other applications. ‰uorescent side-products can be eliminated without loss of Carefully identify and integrate the peaks obtained as direct- FMOC-amino acids. ed for each Procedure. Calculate the mole percent for each FMOC-amino acids are separated by a reversed-phase amino acid present in the test sample by the formula:

HPLC using ODS column. The separation is carried out by 100rU/r, gradient elution varied linearly from a mixture of acetonitrile in which rU is the peak response, in nmol, of the amino acid methanol and buŠer (10:40:50) to a mixture of under test; and r is the sum of peak responses, in nmol, for all acetonitrile and acetic acid buŠer (50:50), and 20 amino acid amino acids present in the test sample. Comparison of the derivatives are separated in 20 minutes. Each derivative elut- mole percent of the amino acids under test to data from ed from column is monitored by a ‰uorometric detector set at known proteins can help establish or corroborate the identity an excitation wavelength of 260 nm and an emission of the sample protein. wavelength of 313 nm. Unknown Protein Samples This data analysis technique The detection limit is in the low fmol range. A linearity can be used to estimate the protein concentration of an range of 0.1 to 50 mmol/L is obtained for most of the amino unknown protein sample using the amino acid analysis data. acids. Calculate the mass, in mg, of each recovered amino acid by the formula: Method 8—Precolumn NBD-F Derivatization General mM /1000, Principle W in which m is the recovered quantity, in nmol, of the amino Precolumn derivatization of amino acids with 7-‰uoro-4- acid under test; and M is the average molecular weight for nitrobenzo-2-oxa-1.3-diazole (NBD-F) followed by reversed- W that amino acid, corrected for the weight of the water phase HPLC separation with ‰uorometric detection is used. molecule that was eliminated during formation. 7-‰uoro-4-nitrobenzo-2-oxa-1.3-diazole (NBD-F) reacts Thesumofthemassesoftherecoveredaminoacidswillgive with both primary and secondary amino acids to form highly an estimate of the total mass of the protein analyzed after ap- ‰uorescent products. Amino acids are derivatized with NBD- propriate correction for partially and completely destroyed Fbyheatingto609C for 5 minutes. amino acids. If the molecular weight of the unknown protein NBD-amino acid derivatives are separated on an ODS is available (i.e., by SDS-PAGE analysis or mass spec- column of a reversed-phase HPLC by employing gradient troscopy), the amino acid composition of the unknown pro- elution system consisting of acetonitrile and aqueous buŠer tein can be predicted. Calculate the number of residues of mixture, and 17 amino acid derivatives are separated in 35 each amino acid by the formula: minutes. e- can be used as an internal m/(1000M/M ), standard, because it is eluted in a clean chromatographic WT in which m is the recovered quantity, in nmol, of the amino region. Each derivative eluted from column is monitored by a acid under test; M is the total mass, in mg, of the protein; and ‰uorometric detector set at an excitation wavelength of M is the molecular weight of the unknown protein. 480 nm and an emission wavelength of 530 nm. WT Known Protein Samples This data analysis technique can The sensitivity of this method is almost the same as be used to investigate the amino acid composition and pro- for precolumn OPA derivatization method (Method 5), tein concentration of a protein sample of known molecular excluding proline to which OPA is not reactive, and might be weight and amino acid composition using the amino acid advantageous for NBD-F against OPA. The detection limit analysis data. When the composition of the protein being for each amino acid is about 10 fmol. Proˆle analysis was 1662 Aristolochic Acid / General Information JP XV analyzed is known, one can exploit the fact that some amino It is considered that Akebia Stem, Sinomenium Stem and acids are recovered well, while other amino acid recoveries Saussurea Root do not contain aristolochic acid, unless may be compromised because of complete or partial destruc- of origin other than that designated in the JP are used. tion (e.g., tryptophan, cysteine, threonine, serine, methio- However, contamination of aristolochic acid might occur, as nine), incomplete bond cleavage (i.e., for isoleucine and mentioned above. In this case, the test described in the Purity valine) and free amino acid contamination (i.e., by glycine under Asiasarum Root is useful for checking the presence of and serine). aristolochic acid. Because those amino acids that are recovered best References: represent the protein, these amino acids are chosen to & Medical Device Safety Information (No.161) (July, quantify the amount of protein. Well-recovered amino acids 2000). are, typically, aspartate-asparagine, glutamate-glutamine, New England Journal of Medicine (June 8, 2000). alanine, , , lysine, and . This list Mutation Research 515, 63–72 (2002). can be modiˆed based on experience with one's own analysis system. Divide the quantity, in nmol, of each of the well- recovered amino acids by the expected number of residues for 3. Basic Requirements for Viral that amino acid to obtain the protein content based on each well-recovered amino acid. Average the protein content Safety of BiotechnologicalW results calculated. The protein content determined for each of the well-recovered amino acids should be evenly distribut- Biological Products listed in ed about the mean. Discard protein content values for those Japanese Pharmacopoeia amino acids that have an unacceptable deviation from the mean. Typically »greater than 5z variation from the mean Introduction is considered unacceptable. Recalculate the mean protein The primary role of speciˆcation of biotechnologicalWbio- content from the remaining values to obtain the protein con- logical products listed in Japanese Pharmacopoeia (JP) is not tent of the sample. Divide the content of each amino acid by only for securing quality control or consistency of the quality the calculated mean protein content to determine the amino but also for assuring their e‹cacy and safety. In the mean- acid composition of the sample by analysis. time, the requirements to assure quality and safety of Calculate the relative compositional error, in percentage, have come to be quite strict recently, and a rigid attitude by the formula: addressing safety assurance is expected for biotechnologicalW 100m/m , S biological products. The key points for quality and safety as- in which m is the experimentally determined quantity, in surance of biotechnologicalWbiological products are selection nmol per amino acid residue, of the amino acid under test; and appropriate evaluation of source material, appropriate and m is the known residue value for that amino acid. The S evaluation of manufacturing process and maintenance of average relative compositional error is the average of the manufacturing consistency, and control of speciˆc physical absolute values of the relative compositional errors of the properties of the products. Now, how to assure quality and individual amino acids, typically excluding tryptophan and safety of such drugs within a scope of JP has come to be cysteine from this calculation. The average relative composi- questioned. This General Information describes what sorts of tional error can provide important information on the stabil- approaches are available to overcome these issues. ity of analysis run over time. The agreement in the amino It is desired that quality and safety assurance of JP listed acid composition between the protein sample and the known products are achieved by state-of-the-art methods and con- composition can be used to corroborate the identity and puri- cepts which re‰ect progress of science and accumulation of ty of the protein in the sample. experiences. This General Information challenges to show the highest level of current scientiˆc speculation. It is expected that this information will contribute to promotion of scien- 2. Aristolochic Acid tiˆc understanding of quality and safety assurance of not only JP listed products but also the other biotechnologicalW Aristolochic acid, which occurs in plants of Aristolochia- biological products and to promotion of active discussion of ceae, is suspected to cause renal damage. It is also reported to each O‹cial Monograph in JP. be oncogenic (see References). 1. Fundamental measures to ensure viral safety of JP listed Aristolochic acid toxicity will not be a problem if crude biotechnologicalWbiological products drugs of the origin and parts designated in the JP are used, The biotechnologicalWbiological product JP includes the but there may be diŠerences in crude be- products derived from living tissue and body ‰uid (, tween diŠerent countries, and it is known that crude drug , etc.) of , etc. Protein drugs derived from cell preparations not meeting the speciˆcations of the JP are cir- lines of human or animal origin (e.g., recombinant DNA culating in some countries. Consequently, when crude drugs drug, drug) are also included. The fundamental or their preparations are used, it is important that the materi- measures required for comprehensive viral safety of JP listed als should not include any containing aristolochic acid. biotechnologicalWbiological products are as follows: 1) ac- Since Supplement I to JP14, the test for aristolochic acid I quaintance of possible contamination (source of con- was added to the Purity under Asiasarum Root, which con- tamination); 2) careful examination of eligibility of raw sists of the rhizome and root. Because the aerial part of the materials and their sources, e.g. humanWanimal, and plant may contain aristolochic acid and may have been thorough analysis and screening of the sample chosen as a improperly contaminated in Asiasarum Root. for drug production (e.g., pooled body ‰uid, cell JP XV General Information / Basic Requirements for Viral 1663 bank, etc.) to determine any virus contamination and deter- Table 1. Items described in General Information mination of type and nature of the virus, if contaminated; 3) for Viral Safety Assurance of JP listed evaluation to determine virus titer and virus-like particles BiotechnologicalWBiological Product hazardous to human, if exists; 4) selection of production related material (e.g., reagent, immune antibody column) I. Introduction free from infectious or pathogenic virus; 5) performance of 1. Fundamental measures to ensure viral safety of JP listed virus free test at an appropriate stage of manufacturing in- biotechnologicalWbiological products cluding the ˆnal product, if necessary; 6) adoption of eŠec- 2. Safety assurance measures described in the O‹cial tive viral clearance method in the manufacturing process to Monograph and this General Information removeWinactivate virus. Combined method sometimes 3. Items and contents described in this General Informa- achieves higher level of clearance; 7) development of a tion deliberate viral clearance scheme; 8) performance of the test II. General Matters to evaluate viral removal and inactivation. It is considered 1. Purpose that the stepwise and supplemental adoption of the said 2. Background measures will contribute to ensure viral safety and its im- 3. Unknown risk on the measures taken for ensuring viral provement. safety 4. Applicable range 2. Safety assurance measures described in the O‹cial 5. Possible viral contamination to a JP listed biotechno- Monograph and this General Information logical biological product (source of virus contamina- As mentioned in above 1, this General Information W tion) describes, in package, points to be concerned with and con- 6. Basis for ensuring viral safety crete information on the measures taken for viral safety of JP 7. Limit of virus test listed products. Except where any speciˆc caution is provided 8. Roles of viral clearance studies in O‹cial Monograph of a product in question, O‹cial III. Raw material substrate for drug production Monograph provides in general that ``Any raw material, sub- W 1. Issues relating to animal species and its region as a strate for drug production and production related material source of raw material substrate for drug production used for production of drug should be derived from healthy W and countermeasures to be taken thereto animals and should be shown to be free of latent virus which 2. Qualiˆcation evaluation test on human or animal as a is infectious or pathogenic to human'', ``Cell line and culture source of raw material substrate for drug production method well evaluated in aspects of appropriateness and ra- W IV. Points of concern with respect to manufacturing and vi- tionality on viral safety are used for production, and the rus testing presence of infectious or pathogenic latent virus to human in 1. Virus test conducted in advance of puriˆcation process process related materials derived from living organisms 2. Virus test as an acceptance test of an intermediate should be denied''. and ``biotechnologicalWbiological drug material, etc. should be produced through a manufacturing process which 3. Virus test on a ˆnal product is capable of removing infectious or pathogenic virus'', etc., V. Process evaluation on viral clearance to raise awareness on viral safety and on necessity to conduct 1. Rationale, objective and general items to be concerned test and process evaluation for viral safety. with respect to viral clearance process evaluation 3. Items and contents described in this General Information 2. Selection of virus As for viral safety of protein drug derived from cell line of 3. Design of viral clearance studies human or animal origin, there is a Notice in entitled 4. Interpretation of viral clearance studies ``Viral safety evaluation of biotechnology products derived 1) Evaluation of viral clearance factor from cell lines of human or animal origin'' (Iyakushin No. 2) Calculation of viral clearance index 329 issued on February 22, 2000 by Director, Evaluation and 3) Interpretation of results and items to be concerned at Licensing Division, Pharmaceutical and Medical Safety evaluation Bureau, Ministry of Health and Welfare) to re‰ect the inter- VI. Statistics nationally harmonized ICH Guideline, and as for blood plas- 1. Statistical considerations for assessing virus assays ma protein fraction preparations, there is a document enti- 2. Reproducibility and conˆdence limit of viral clearance tled ``Guideline for ensuring viral safety of blood plasma studies protein fraction preparations''. This General Information VII. Re-evaluation of viral clearance for ensuring viral safety of JP listed biotechnologicalWbiolog- VIII. Measurement for viral clearance studies ical products has been written, referencing the contents of 1. Measurement of virus infective titer those guidelines, to cover general points and their details to 2. Testing by nucleic-acid ampliˆcation test (NAT) be concerned for ensuring viral safety of not only JP listed IX. Reporting and preservation biotechnologicalWbiological products but also all products X. Others which would be listed in JP in future, i.e., biological products derived from living tissue and body ‰uid, such as urine, and protein drugs derived from cell line of human or from living tissue or body ‰uid of mammals, etc. and of pro- animal origin (Table 1). tein drugs derived from cell lines of human or animal origin. 3.1 Purpose That is to say, this document describes the measures and the The purpose of this document is to propose the compre- points of concern on the items, such as  consideration of hensive concepts of the measures to be taken for ensuring the source of virus contamination;  appropriate evaluation viral safety of biotechnologicalWbiological products derived on eligibility at selecting the raw material and on qualiˆcation 1664 Basic Requirements for Viral / General Information JP XV of its source, e.g. human or animal;  virus test, and its and test not having scientiˆc rationality. As the results, eŠec- analysis and evaluation at a stage of cell substrate for drug tive and prompt supply of an important drug, already having production;  appropriate evaluation to choose product enough accumulation of experiences, to the medical work related materials derived from living organisms (e.g. reagent, front will be hampered, and the drug, a social asset, may not immune antibody column, etc.);  conduct of necessary vi- to be utilized eŠectively. Medicine is a sword used in medical rus test on the product at an appropriate stage of manufac- ˆeld having double-edge named eŠectiveness and safety. turing;  development of viral clearance test scheme;  per- EŠectiveness and safety factors have to be derived as the formance and evaluation of viral clearance test. This docu- fruits of leading edge of science, and relatively evaluated on a ment is also purposed to comprehensively describe in details balance sheet of usefulness. Usefulness evaluation should not that supplemental and combining adoption of the said meas- be unbalanced in a way that too much emphasis is placed on ures will contribute to secure viral safety and its improve- safety concern without back-up of appropriate scientiˆc ra- ment. tionality. A drug can play an important role as a social asset 3.2 Background only when well balanced appropriate scientiˆc usefulness One of the most important issues to be cautioned for safety evaluation in addition to social concern of the age are given. of a biological product, which is directly derived from human In other words, drug is a common asset utilized by society for or animal, or of a protein drug, which is derived from cell as a fruit of science of the age, and the key point line of human or animal origin (recombinant DNA derived of its utilization lies on a balance of risk and beneˆt produced product, cell culture derived product, etc.), is risk of virus from scientiˆc and social evaluation. So, those factors have contamination. Virus contamination may cause serious situa- to be taken into account when target and pursuance levels for tion at clinical use once it occurs. Virus contamination may ensuring viral safety of a JP listed biotechnologicalWbiologi- be from a raw material or from a cell substrate for drug cal product are reviewed. production, or may be from an adventitious factor in- And, in general, the risk and beneˆt of drugs should be troduced to the manufacturing process. considered with the relative comparison to alternative drugs JP listed biological drugs or protein drugs derived from or medical treatment. The usefulness of certain drug should cell line have achieved drastic contribution to the medical be reviewed ˆnally after the competitive assessment on the society, and to date, there has not been any evidence of any risk and beneˆt on the alternative drugs, relevant drugs andW safety problem on them caused by virus. But, social require- or alternative medical treatment. ment of health hazard prevention is strong, and it is now very Under such background, the purpose of this article is to important to prevent accidental incidence, taking security describe the scientiˆc and rational measures to be taken for measures carefully supported by scientiˆc rationality. It is ensuring viral safety of JP listed biotechnologicalWbiological always great concern among the persons involved that under products. Giving scientiˆc and rational measures mean that; what sort of viewpoint and to what extent we have to pursue appropriate and eŠective measures, elaborated from the cur- for ensuring viral safety of a biotechnologicalWbiological rent scientiˆc level, are given to the issues assumable under product. Before discussing these issues, two fundamental the current scientiˆc knowledge. In other words, possible points have to be reconˆrmed. One is that; we have to con- contaminant virus is assumed to have the natures of genus, sider scientiˆc, medical, and social proˆles a drug has. In morph, particle size, physicalWchemical properties, etc. which other words, ``Medicine is a social asset which is utilized in are within the range of knowledge of existing virology, and is medical practice paying attention to the risk and beneˆt from those assumed to exist in human and animal, tissue and body the standpoints of science and society''. It is the destine and ‰uid, which are the source of biotechnologicalWbiological the mission of the medicalWpharmaceutical society to realize product, reagent, material, additives, etc. Accordingly, viral prompt and stable supply of such a social asset, drug, among clearance studies using a detection method which target those the medical work front to bring gospel to the patients. have to be designed. The other is that; issue of viral safety is independent from 3.3 Unknown risk on the measures taken for ensuring viral safety of the components of a drug per se (narrow sense of safety safety). It is important to consider that this is the matter of There are known and unknown risks. general safety of drug (broad sense of safety). In case of a It is easy to determine a test method and an evaluation drug which has been used for a long time in the medical standard on the known risk, which exists in the drug per se front, such as a JP listed product, its broad sense of safety is (pharmaceutical component) or inevitably exists due to a considered to have been established epidemiologically, and quality threshold, and quantiˆcation of such risk is possible. its usage past records have a great meaning. However, diŠer- In other words, it is easy to evaluate the known risk on a ent from safety of drug per se (its components), taking into balance sheet in relation to the beneˆt, and we can say that account any possibility of virus contamination, we have to valuation even in this respect has been established to some ex- say that only the results accumulated can not always assure tent. viral safety of a drug used in future. Accordingly, the basis On the other hand, as for the unknown risk which is inevit- for securing broad sense of viral safety of JP listed able for ensuring viral safety, the subject of the risk can not biotechnologicalWbiological products is to pay every attention be deˆned and quantitative concept is hard to introduce, and, to the measures to take for prevention, while evaluating the therefore, taking a counter measure and evaluating its eŠect accumulated results. are not so easy. Therefore, this is the subject to be challenged Adopting strict regulations and conducting tests at maxi- calling upon wisdom of the related parties among the society mum level to the extent theoretically considered may be the of drug. ways oŠ assuring safety, but applying such way generally, Talking about the unknown risk, there are view points that without su‹cient scientiˆc review of the ways and evaluation say ``It is risky because it is unknown.'' and ``What are the of usage results, causes excessive requirement of regulation unknowns, and how do we cope with them in ensuring safe- JP XV General Information / Basic Requirements for Viral 1665 ty?''. the subject of ``unknown viruses, which have a particle size The view of ``It 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 ``unknown viruses, which have special physicalWchemical to a ˆnal decision if it can be used as a drug. Such evaluationW properties that can not be matched to any of the currently decision has to be made based upon a rational, scientiˆc or knownviruses,mayexists''issetupasanarmchairtheory, social judgment. any experimental work can not be pursued under the current For example, in the case that ``In a manufacturing process scientiˆc 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 currently but its identiˆcation could not be conˆrmed, and, therefore, available methods and technologies will be meaningless ``for its risk can not be denied.'', the evaluation of ``It is risky be- ensuring safety'', since particle size or natures of such specu- cause it is unknown.'' is scientiˆcally rational and reasona- lated virus are unknown. Likewise, any counter measures can ble. On the other hand, however, if we a decision of ``It not be taken on the subject of ``unknown virus, which can is risky because it is unknown.'' due to the reason that ``In a not be detected by currently available screening method, may manufacturing process of drug, virus, virus-like particle or exist'', and conducting any virus detection test at any stage retrovirus was not detected, but there is a `concern' that will be useless ``for ensuring safety''. something unknown may exist.'', it can not be said that such The requirement of regulations or tests excessively over evaluation is based upon a rational, scientiˆc or social judg- scientiˆc rationality will raise human, economical and tem- ment. It goes without saying that the utmost care has to be poral burden to the pharmaceutical companies, and will ad- taken for viral safety, but the substance of `concern' has to versely aŠect prompt, eŠective and economical supply of a be at least clearly explainable. Otherwise, the `concern' may drug to the medical front. As drug is a sort of social asset, result in causing contradiction in the meaningful mission to which has to be scientiˆcally evaluated, how to assure max- utilize a social asset, drug, in medical practice. imization of its safety by means of scientiˆcally rational ap- From scientiˆc view point, we should not be narrow mind- proach at minimum human, economical and time resources is ed by saying ``it is risky'' because ``there is a `concern' that important. something unknown may exists'', but challenge to clarify the It is also important to reconˆrm 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 ``In a time is to deˆne ``what is unknown'' based upon current manufacturing process of drug, virus, virus-like particle or scientiˆc knowledge. Only through this way, is it possible for retrovirus was not detected, but there is a `concern' that us to elaborate the measures for ensuring safety. something unknown may exist.'', appropriateness of the test, Once we chase up the substance of unknown risk for viral which resulted in the judgment that ``virus, virus-like particle safety without premises of ``what is unknown'', ``unknown'' or retrovirus was not detected in a process of drug produc- will be an endless question because it theoretically remains tion'', should be a prerequisite premise when judged by unresolved forever. If this kind of approach is taken, the is- science standard ate the time. If there is any question on the sue and the measure can not be scientiˆcally connected to 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 eŠec- regulation and of test to be conducted. Yet, it is unlikely that tive. the measure which has no relation with science will be eŠec- 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 ``evaluation of a products, derived from living tissue or body ‰uid, and pro- puriˆcation process which can completely clear up every virus tein drugs, derived from human or animal cell line, that in that contaminated in a manufacturing process'' should be the Japan. In the case of protein drugs derived from human or subject of ``what sort of existing virus that contaminated is animal cell line, the products developed and approved before unknown'', not on the subject of ``what sort of virus that ex- enforcement of the Notice Iyakushin No. 329 entitled ``Viral ist in the world is unknown. In the former subject, the safety evaluation of biotechnology products derived from cell premise of the study is based on all the knowledge on viruses lines of human or animal origin'' should have been treated including DNAWRNA-virus, virus withWwithout envelope, under the Notice had there been one, and it is inevitable that particle size, physicalWchemical properties, etc. The premise some products approved after the Notice might not have been is that the virus contaminated should be within range of exist- su‹ciently treated. It is expected that such biodrug will be ing wisdom and knowledge of virus such as species, type, na- su‹ciently examined to meet such General Information be- ture, etc., even though the virus that contaminated is fore being listed in JP. On the other hand, blood prepara- unknown. Under such premise, when evaluation is made on a tions listed in the biological products standard and covered puriˆcation process to decide its capability of clearing a der- by ``Guideline for securing safety of blood plasma protein ived virus, which is within the range of existing wisdom and fraction preparations against virus'', are out of the scope of , speciˆc viral clearance studies designed to combine this General Information. Further, in case of a relatively low- a few model viruses with diŠerent natures, such as type of er molecular biogenous substance, such as amino acid, sac- nucleic acid, withWwithout envelope, particle size, physicalW charide and glycerin, and of , which is even classiˆed chemical properties, etc., would be enough to simulate every as infectious or pathogenic polymer, there are cases that viral sort of the virus already known, and will be a ``good measure contamination can not be considered due to its manufactur- for ensuring safety''. ing or puriˆcation process, and that potent viral inactivationW The issue of ``the sort of viruses that exist in the world is removal procedure that can not be applied to protein, can be unknown'' may be a future study item, but it is not an ap- used, and, therefore, it is considered reasonable to omit such propriate subject for the viral clearance test. Further, even if substances from the subject for application. However, some 1666 Basic Requirements for Viral / General Information JP XV part of this General Information may be used as reference. taminate to raw materials, etc. of drug and have to be cau- Further, a comprehensive assurance measure for viral safety tioned, are HIV, HAV, B Virus (HBV), HCV, Hu- is recommendable on a biotechnologicalWbiological product man T-Lymphotropic Virus (HTLV-IWII), Human Parvovi- not listed in JP using this document as a reference so long as rus B19, Cytomegalovirus (CMV), etc. BiotechnologicalWbio- it is similar to the biotechnologicalWbiological product JP. logical products produced from raw materialWcell substrate 3.5 Possible viral contamination to a JP listed biotechno- derived from tissue or body ‰uid of human or animal origin logicalWbiological product (source of virus contamination) always have a risk of contamination of pathogenic or other Promoting awareness of virus contamination to a JP listed latent virus. Therefore, safety measures should be thoroughly biotechnologicalWbiological product (source of virus con- taken. There is also the case that a material, other than the tamination) and citing countermeasure thereof are important biological component such as raw materialWsubstrate, causes for eradicating any possible virus contamination and raising virus contamination. Using enzymatic or monoclonal an- probability of safety assurance. Many biotechnologicalWbio- tibody column or using albumin etc. as a stabilizer, is the ex- logical products are produced from a ``substrate'' which is ample of the case, in which caution has to be taken on risk of derived from human or animal tissue, body ‰uid, etc. as an virus contamination from the source animal or cell. Further, originWraw material, and in puriˆcation or pharmaceutical there is a possibility of contamination from environment or processing of such products column materials or additives, personnel in charge of production or at handling of the which are living organism origin, are occasionally used. Ac- product. So, caution has to be taken on these respects as well. cordingly, enough safety measures should be taken against In case of protein drugs derived from cell line of human or diŠusion of the contaminant virus. Further, as mentioned in animal origin, there may be cases where latent or persistent Notice Iyakushin No. 329, any protein drug derived from cell infectious viruses (e.g., herpesvirus) or endogenous lines of human or animal origin should be carefully examined retroviruses exist in the cell. Further, adventitious viruses with respect to the risk of virus contamination through the may be introduced through the routes such as: 1) derivation cell line, the cell substrate for drug production, and through of a cell line from an infected animal; 2) use of a virus to the manufacturing process applied thereafter. drive a cell line; 3) use of a contaminated biological reagent ``Substrate for drug production'' is deˆned as a starting (e.g., animal serum components); 4) contamination during material which is at a stage where it is deemed to be in a posi- cell handling. In the manufacturing process of drug, an tion to ensure qualityWsafety of an active substance. The adventitious virus may contaminate to the ˆnal product ``substrate for drug production'' is sometimes tissue, body through the routes, such as 1) contamination through a rea- ‰uid, etc. of human or animal per se and pooled material gent of living being origin, such as serum component, which such as urine, and sometimes a material after some treat- is used for culturing, etc.; 2) use of a virus for introduction of ment. In many cases, it is considered rational that starting a speciˆc expression to code an objective protein; 3) point of full-scale test, evaluation and control should be at contamination through a reagent used for puriˆcation such the stage of ``substrate for drug production''. The more strict as a‹nity column; 4) contamination levels of test, evaluation and control achieved at the stage of through an additive used for formulation production; 5) con- ``substrate for drug production'' can more rationalize evalu- tamination at handling of cell and culture media, etc. It is ation and control of the raw material or individual level of reported that monitoring of cell culture parameter may be upper stream. On the contrary, strict evaluation and control helpful for early detection of an adventitious viral contami- of the raw material or individual level at an upper stream nation. stage can rationalize tests, evaluation or quality control at the 3.6 Basis for ensuring viral safety stage of ``substrate for drug production''. Viral safety of a biotechnologicalWbiological product pro- The measures taken for ensuring viral safety on a duced from a raw materialWsubstrate, which derived from tis- biotechnologicalWbiological product currently listed in JP can sue, body ‰uid, cell line, etc. of human or animal origin, can be assumed from the provisions of manufacturing method, be achieved by supplemental and appropriate adoption of the speciˆcation and test methods of each preparation. However, following plural methods. unitary principles or information with respect to the meas- (1) Acquaintance of possible virus contamination (source ures to be taken for ensuring viral safety, totally reviewing of contamination). the entire process up to the ˆnal product rationally and com- (2) Careful examination of eligibility of the raw material prehensively, including source Wraw material Wsubstrate, and its source, i.e., human or animal, thorough analysis puriˆcation process, etc. have not been clariˆed. The most and screening of the sample chosen as the substrate for important thing for ensuring viral safety is to take thorough drug production to determine virus contamination and measures to eliminate the risk of virus contamination at any through examination of the type of virus and its nature, stage of source animal, raw material and substrate. Although if contaminated. not the cases of a biotechnologicalWbiological product, (3) Evaluation to determine hazardous properties of the vi- known examples of a virus contamination from a raw materi- rusorvirus-likeparticletohuman,ifexists. alWsubstrate for drug production in old times are Hepatitis A (4) Choosing a product related material of living organism Virus (HAV) or Virus (HCV) contamination in origin (e.g., reagent, immune anti-body column, etc.) blood protein fraction preparations. It is also well known which is free from infectious or pathogenic virus. that Human Immunodeˆciency Virus (HIV) infection caused (5) Conduct virus free test at an appropriate stage of by blood plasma protein fraction preparations occurred in manufacturing including the ˆnal product, if necessary. 1980s. The aim of this General Information is to show con- (6) Adoption of an eŠective method to removeWinactivate crete guidelines for comprehensive viral safety assurance of the virus in the manufacturing process for viral clear- the JP listed biotechnologicalWbiological products. The ance. Combined processes sometimes achieve higher lev- pathogenic infectious viruses, currently known to con- el of viral clearance. JP XV General Information / Basic Requirements for Viral 1667

(7) Develop a deliberate viral clearance scheme. and can assure viral safety of a ˆnal product, taking into con- (8) Conduct test and evaluation to conˆrm removalWinacti- sideration the source and the properties of the raw materialW vation of the virus. substrate as well as the manufacturing process. Manufacturer is responsible for explaining rationality of 4. Raw materialWsubstrate for drug production the way of approach adopted among the comprehensive 4.1 Issues relating to animal species and its region as a strategy for viral safety on each product and its manufactur- source of raw materialWsubstrate for drug production and ing process. At the time, the approach described in this countermeasures to be taken thereto General Information shall be applicable as far as possible. For manufacturing JP listed biotechnologicalWbiological 3.7 Limit of virus test products, which require measures for viral safety, a raw Virus test has to be conducted to deˆne existence of virus, materialWsubstrate derived mainly from human, bovine, but it should be noted that virus test alone can not reach a swine or equine is used, and it is obvious that such human conclusion of inexistence of virus nor su‹cient to secure safe- and animal has to be healthy nature. A wild animal should be ty of the product. Examples of a virus not being detected are avoided, and it is recommended to use animals derived from as follows: 1) Due to statistical reason, there is an inherent a colony controlled by an appropriate SPF (Speciˆc quantitative limit, such as detection sensitivity at lower con- Pathogen-Free) condition and bred under a well deigned centration depends upon the sample size. 2) Generally, every hygienic control, including appropriate control for preven- virus test has a detection limit, and any negative result of a vi- tion of microbial contamination and contamination monitor- rus test can not completely deny existence of a virus. 3) A vi- ing system. If a standard for is available, an rus test applied is not always appropriate in terms of speciˆci- animal meeting this standard has to be used. The type of vi- ty or sensitivity for detection of a virus which exists in the tis- rus to be concerned about depend on animal species, but it sue or body ‰uid of human or animal origin. may be possible to narrow down the virus for investigation by Virus testing method is improved as science and technology means of examining the hygiene control, applicability of a progress, and it is important to apply scientiˆcally the most meat standard for food, etc. On the other hand, even with the advanced technology at the time of testing so that it can be animals of the same species, a diŠerent approach may be possible to raise the assurance level of virus detection. It necessary depending upon the region where the specimen for should be noted, however, that the limit as mentioned above arawmaterialWsubstrate is taken. For example, in case of ob- can not always be completely overcome. Further, risk of vi- taining raw materialWsubstrate from blood or other speciˆc rus contamination in a manufacturing process can not be region, it is necessary to be aware of the risk level, virus mul- completely denied, and, therefore, it is necessary to elaborate tiplication risk, etc. which may speciˆcally exists depending the countermeasure taken these eŠects into account. upon its region. Such approach may be diŠerent from those Reliable assurance of viral free ˆnal product can not be ob- applied to body waste such as urine, milk, etc. as a source of tained only by negative test results on the raw materialWsub- raw materialWsubstrate. Further, caution has to be taken on strate for drug production or on the product in general, it is transmissible spongiform encephalopathy (TSE) when pitui- also necessary to demonstrate inactivationWremoval capabil- tary gland, etc. is used as a raw material. This report does not ity of the puriˆcation process. include detailed explanation on TSE, but recommendations 3.8 Roles of viral clearance studies are to use raw material derived from 1) animals originated in Under the premises as mentioned in the preceding clause the countries (area) where incidence of TSE has not been that there is a limit of a virus test, that there is a possibility of reported; 2) animals not infected by TSE; or 3) species of existence of latent virus in a raw materialWsubstrate for drug animal which has not been reported on TSE. It is recom- production and that there is a risk of entry of a non-en- mended to discuss the matters concerned with TSE with the dogenous virus in a manufacturing process, one of the im- regulatory authority if there is any unclear point. portant measures for viral safety is how to remove or inacti- Followings are the raw materialWsubstrate used for vate the virus, which exists in a raw material, etc. and can not manufacturing biotechnological Wbiological products in be detected, or the virus, which is contingently contaminated Japan. in a manufacturing process. The purpose of viral clearance (1) Biological products derived from human study is to experimentally evaluate the viral removalWinacti- Blood plasma, placenta, urine, etc. derived from human vation capability of a step that mounted in a manufacturing are used as the sources of raw material of biotechnologicalW process. So, it is necessary to conduct an experimental scale biological product. As for these raw materials, there are 2 spike test using an appropriate virus that is selected by taking cases: 1) Appropriateness can be conˆrmed by interview or account the properties, such as particle size, shape, with or by examination of the individual who supplies each raw without envelope, type of nucleic acid (DNA type, RNA material, and 2) Such su‹cient interview or examination of type), heat and chemical treatment tolerance, etc., with an the individual can not be made due to type of raw material. aim to determine removal Winactivation capability of the virus In case that su‹cient examination of individual level is not that can not be detected in a raw material or contingently possible, it is necessary to perform test to deny virus contami- contaminated. nation at an appropriate manufacturing stage, for example, As mentioned above, the role of viral clearance study is to the stage to decide it as a substrate for drug production. speculate removalWinactivation capability of a process (2) Biological products derived from animal besides human through a model test, and it contributes to give scientiˆc basis , , etc. are manufactured from blood to assure that a biotechnologicalWbiological product of hu- plasma or from various organs of bovine, swine and equine. man or animal origin has reached an acceptable level in (3) Protein drug derived from cell line of human or animal aspect of viral safety. origin At a viral clearance study, it is necessary to adopt an ap- In the case of protein drugs derived from cell line of human propriate approach method which is deˆnitive and rational 1668 Basic Requirements for Viral / General Information JP XV or animal origin, a cell line of human or animal is the raw Table 2. Infectious viruses known to be common material per se, and the substrate for drug production is a cell between human and animal and known to bank prepared from cloned cell line (master cell bank or cause infection to each animal working cell bank). Examination at cell bank level is consi- dered enough for viral safety qualiˆcation, but it goes bovine swine sheep goat equine without saying that the more appropriate and rational qualiˆ- cation evaluation test of cell bank can be realized when more Cowpox virus w information is available on the virus of the source animal or Paravaccinia virus wwww on prehistory of driving the cell line, the base of cell bank. 4.2 Qualiˆcation evaluation test on human or animal as a Murray valley encephalitis ww source of raw materialWsubstrate for drug production virus (1) Biological products derived from human Body ‰uid etc. obtained from healthy human must be used Louping ill virus wwww for biological products production. Further, in case that in- Wesselsbron virus w terview or examination of the individual, who supplies the raw material, can be possible and is necessary, interview un- Foot-and-mouth ww der an appropriate protocol and a serologic test well evaluat- virus ed in aspects of speciˆcity, sensitivity and accuracy have to be Japanese encephalitis w performed, so that only the raw material, which is denied la- virus tent HBV, HCV and HIV, will be used. In addition to the above, it is necessary to test for gene of HBV, HCV and HIV Vesicular stomatitis virus w by a nucleic ampliˆcation test (NAT) well evaluated in Bovine papular stomatitis w aspects of speciˆcity, sensitivity and accuracy. virus In case of the raw material (e.g., urine), which can not be tested over the general medical examination of the individual Orf virus w who supplies the material, or of the raw material which is ir- Borna disease virus ww rational to conduct individual test, the pooled raw material, as the substrate for drug production, has to be conducted at Rabies virus wwwww least to deny existence of HBV, HCV and HIV, using a method well evaluated in aspects of speciˆcity, sensitivity and In‰uenza virus w accuracy, such as the antigen test or NAT. Hepatitis E virus w (2) Biological products derived from animal besides human The animal used for manufacturing biological product has Encephalomyocarditis ww to be under appropriate health control, and has to be con- virus ˆrmed of its health by various tests. Further, it is necessary Rotavirus w that the population, to which the animal belongs, has been under an appropriate breeding condition, and that no abnor- Eastern equine w mal individual has been observed in the population. Further, encephalitis virus it is necessary to demonstrate information or scientiˆc basis which can deny known causes infection or disease to human, Western equine w encephalitis virus or to deny such animal inherent latent virus by serologic test or by nucleic ampliˆcation test (NAT). The infectious virus Venezuelan equine w thatisknowntobecommonbetweenhumanandanimal,and encephalitis virus known to cause infection in each animal are tentatively listed Morbillivirus in Table 2. It is necessary that the table is completed under w careful examination, and denial of all of them, by means of Hendra virus w tests on individual animal, tissue, body ‰uid, etc. as a raw material, or on pooled raw material (as a direct substrate for Nipah virus w drug production), is not always necessary. Table 2 can be Transmissible gastroente- w used as reference information, in addition to the other infor- ritis virus mation, such as; source of animal, health condition, health and breeding control, conformity to the meat standard for Porcine respiratory w food, etc., to elaborate to which virus what kind of test has to coronavirus be performed, and for which virus it is not always necessary Porcine epidemic w to test for, etc. It is important to clarify and record the basis virus of choosing the virus and the test conducted thereof. Hemagglutinating w encephalomyelitis virus

Porcine respiratory and w reproductive syndrome virus

Hog cholera virus w JP XV General Information / Basic Requirements for Viral 1669

or more steps with diŠerent principles is recommended for vi- Parain‰uenza virus Type 3 w rus inactivationWremoval process. Further, it is important to minimize any possible virus derivation by using a reagent, TelfanWTeschen disease w virus which quality is equivalent to that of a drug. Examples of vi- rus inactivationWremoval processes are  heating (It is Reovirus w reported that almost viruses are inactivated by heating at 55 – 609C for 30 minutes with exceptions of hepatitis virus, etc. Endogenous retrovirus w and that dry heating at 609C for 10 – 24 hours is eŠective in Porcine adenovirus w case of the products of blood or urine origin.),  treatment with organic solventWsurfactant (SWD treatment),  mem- Porcine circovirus w braneˆltration(15–50nm),  acid treatment,  irradia- tion (g-irradiation, etc.),  treatment with column chro- Porcine parvovirus w matograph (e.g. a‹nity chromatography, ion-exchange chro- Porcine poxvirus w matography),  fractionation (e.g. organic solvent or am- monium fractionation),  extraction. Porcine cytomegalovirus w 5.1 Virus test conducted in advance of puriˆcation process Pseudorabies virus w (1) Biological products derived from human In many cases, samples for virus test before puriˆcation Russian spring-summer ww process are body ‰uid or tissue of individual collected as a encephalitis virus raw material, or its pooled material or extraction as a sub- strate. As mentioned in 4.2 (1), it is necessary to deny latent Rift Valley fever virus ww HBV, HCV and HIV by the test evaluated enough in aspects Crimean-Congo hemor- of speciˆcity, sensitivity and accuracy. Even in a case that a rhagic fever virus www non-puriˆed bulk before puriˆcation process is produced (Nairovirus) from a substrate, it is not always necessary to conduct virus Torovirus w test again at the stage before puriˆcation, so long as the presence of any latent virus can be denied at the stage of sub- strate by an appropriate virus test, with cases where the non- (3) Protein drug derived from cell line of human or animal puriˆed bulk is made from the substrate by adding any rea- origin gent etc. of living organisms origin are an exception. It is important to conduct thorough investigation on latent (2) Biological products derived from animal besides human endogenous and non-endogenous virus contamination in a Similar to (1) above, samples for virus test before puriˆca- master cell bank (MCB), which is the cell substrate for drug tion process are, in many cases, body ‰uid or tissue of in- production, in accordance with the Notice Iyakushin No. 329 dividual collected as a raw material, or its pooled material or entitled ``Viral safety evaluation of biotechnology products extraction as a substrate. In these cases, it is necessary to have derived from cell lines of human or animal origin''. Further, a data, which can deny latent virus of probable cause of hu- it is necessary to conduct an appropriate adventitious virus man infection or disease as mentioned in the above 4.2 (2), or test (e.g., and in vivo test) and a latent endogenous vi- to have a result of serologic test or nucleic ampliˆcation test rus test on the cell at the limit of in vitro cell age (CAL) for (NAT) evaluated enough in aspects of speciˆcity, sensitivity drug production. Each WCB as a starting cell substrate for and accuracy. The concept, which is applied to a case that drug production should be tested for adventitious virus either non-puriˆed bulk before puriˆcation process is produced by direct testing or by analysis of cells at the CAL, initiated from substrate, is the same as those provided in the above 4.2 from the WCB. When appropriate non-endogenous virus (1). tests have been performed on the MCB and cells cultured up (3) Protein drug derived from cell line of human or animal to or beyond the CAL have been derived from the WCB and origin used for testing for the presence of adventitious viruses, simi- Generally, substrate in this case is cell bank, and the sam- lar tests need not be performed on the initial WCB. ple for testing before puriˆcation process is a harvested cell 5. Points of concern with respect to manufacturing and after cell culturing or unprocessed bulk which consists of sin- virus testing gle or pooled complex culture broth. The unprocessed bulk To ensure viral safety of a biological product derived from may be sometimes culture broth without cell. Denial of latent tissue, body ‰uid etc. of human or animal origin, it is neces- virus, which is determined by virus test at a MCB or WCB sary to exclude any possibility of virus contamination from a level, does not always deny latent virus in unprocessed bulk raw material, such as tissue and body ‰uid, or a substrate, after culturing. Further, it is noted that the viral test at the paying attention to the source of virus contamination as men- CAL is meaningful as a validation but can not guarantee tioned in above 3.5, and to adopt appropriate manufacturing deˆnite assurance of latent virus denial, since the test is conditions and technologies in addition to enhancement of generally performed only once. In case of using a serum or a manufacturing environment, so that virus contamination in component of blood origin in a culture medium, deˆnite the course of process and handling and from operators, facil- denial of latent virus at the level of unprocessed bulk can not ities and environment can be minimized. be assured so long as the viral test has not been conducted on In addition to the above, eŠective virus test and viral inac- each lot at the CAL, since lot renewal can be a variable factor tivationWremoval technology, which are re‰ected by rapid on viral contamination. progress of science, have to be introduced. Adoption of two A representative sample of the unprocessed bulk, removed from the production reactor prior to further processing, 1670 Basic Requirements for Viral / General Information JP XV represents one of the most suitable levels at which the pos- possible existence of virus at window period, 3) speciˆc detec- sibility of adventitious virus contamination can be deter- tion limit of virus test, etc. and in these cases, virus contami- mined with a high probability of detection. Appropriate test- nation to the ˆnal product may occur if there is any deˆciency ing for viruses should be performed at the unprocessed bulk on the manufacturing process (e.g., damage of membrane level unless virus testing is made more sensitive by initial par- ˆlter) or any mix-up of the raw materials, etc. To avoid such tial processing (e.g., unprocessed bulk may be toxic in test accidental virus contamination, it may be recommended to cell cultures, whereas partially processed bulk may not be conduct nucleic ampliˆcation test (NAT) on the ˆnal product toxic). In certain instances it may be more appropriate to test focusing on the most risky virus among those that may possi- a mixture consisting of both intact and disrupted cells and bly to exist in the raw material. their cell culture supernatants removed from the production 6. Process evaluation on viral clearance reactor prior to further processing. 6.1 Rationale, objective and general items to be concerned In case of unprocessed bulk, it is required to conduct virus with respect to viral clearance process evaluation test on at least 3 lots obtained from pilot scale or commercial Evaluation of a viral inactivationWremoval process is im- scale production. It is recommended that manufacturers portant for ensuring safety of a biological product derived develop programs for the ongoing assessment of adventitious from tissue or body ‰uid of human or animal origin. Con- viruses in production batches. The scope, extent and fre- ducting evaluation on viral clearance is to assure, even to quency of virus testing on the unprocessed bulk should be de- some extent, elimination of the virus, which may exist in a termined by taking several points into consideration includ- raw material, etc. or may be derived to the process due to un- ing the nature of the cell lines used to produce the desired expected situation. Viral clearance studies should be made by products, the results and extent of virus tests performed dur- a carefully designed appropriate method, and has to be ra- ing the qualiˆcation of the cell lines, the cultivation method, tionally evaluated. raw material sources and results of viral clearance studies. The objective of viral clearance studies is to assess process Screening in vitro tests, using one or several cell lines, are step(s) that can be considered to be eŠective in inactivatingW generally employed to test unprocessed bulk. If appropriate, removing viruses and to estimate quantitatively the overall a NAT test or other suitable methods may be used. level of virus reduction obtained by the process. This should Generally, harvest material in which adventitious virus has be achieved by the deliberate addition (``spiking'') of sig- been detected should not be used to manufacture the niˆcant amounts of a virus at diŠerent manufacturingWpuriˆ- product. If any adventitious viruses are detected at this level, cation steps and demonstrating its removal or inactivation the process should be carefully checked to determine the during the subsequent steps. It is not necessary to evaluate or cause of the contamination, and appropriate actions taken. characterize every step of a manufacturing process if ade- 5.2 Virus test as an acceptance test of an intermediate quate clearance is demonstrated by the use of fewer steps. It material, etc. should be borne in mind that other steps in the process may When a biological product is manufactured from tissue, have an indirect eŠect on the viral inactivationWremoval body ‰uid etc. of human or animal origin, there are cases that achieved. Manufacturers should explain and justify the ap- an intermediate material, partially processed as a raw materi- proach used in studies for evaluating viral clearance. al or substrate by outside manufacturer, is purchased and The reduction of virus infectivity may be achieved by used for manufacturing. In such case, if any test to meet this removal of virus particles or by inactivation of viral infectivi- General Information has been conducted by such outside ty. For each production step assessed, the possible mechan- manufacturer, it is necessary for the manufacturer of the bio- ism of loss of viral infectivity should be described with regard logical product, who purchased the intermediate material, to to whether it is due to inactivation or removal. For examine what sort of virus test has to be conducted as accep- inactivation steps, the study should be planned in such a way tance tests, and to keep record on the basis of rationality in- that samples are taken at diŠerent times and an inactivation cluding the details of the test conducted. curve constructed. On the other hand, if no test to meet this General Informa- 6.2 Selection of virus tion has been conducted by such outside manufacturer of the To obtain broad range of information of viral inactivation raw material, all necessary virus free test has to be conducted Wremoval, it is desirable that a model virus used for viral to meet this General Information on the intermediate materi- clearance studies should be chosen from the viruses with al regarding it as the direct substrate for drug production. broad range of characteristics in aspects of DNAWRNA, with 5.3 Virus test on a ˆnal product or without envelope, particle size, signiˆcant resistance to Virus tests to be conducted on a ˆnal product (or on a physicalWchemical treatment, etc. and it is necessary to com- product to reach the ˆnal product) has to be deˆned under bine about 3 model viruses to cover these characteristics. comprehensive consideration of the type of raw material or At choice of a model virus, there are also the ways to substrate, the result of virus test conducted on raw materialW choose a virus closely related to or having the same character- substrate, the result of evaluation on viral removalWinactiva- istics of the virus known to exist in the raw material. In such tion process, any possibility of virus contamination in the case, it is in principle recommendable to choose a virus which manufacturing process, etc. Comprehensive viral safety as- demonstrates a higher resistance to inactivationWremoval surance can only be achieved by appropriate selection of the treatment if two or more candidate viruses are available for raw materialWsubstrate, an appropriate virus test conducted choice. Further, a virus which can grow at a high titer is on the raw materialWsubstrateWintermediate material, the desirable for choice, although this may not always be possi- virus test conducted at an appropriate stage of manufactur- ble. In addition to the above, choosing a virus, which will ing, an appropriate viral clearance test, etc. However, there provide eŠective and reliable assay result at each step, is are cases of having speciˆc backgrounds, such as 1) use of the necessary, since sample condition to be tested at each step of raw material derived from unspeciˆed individual human, 2) JP XV General Information / Basic Requirements for Viral 1671

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 Parain‰uenza 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 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

a production process may in‰uence the detection sensitivity. ensure the sensitivity of the method. Also, the statistics of Considerationshouldalsobegiventohealthhazardwhich sampling virus when at low concentrations (for example, may pose to the personnel performing the clearance studies. number of virus is 1-1000WL) should be considered. For the other items taken for consideration at choice of (3) Viral clearance studies are performed in a miniature virus, the Notice, Iyakushin No. 329 can be used as a refer- size system that simulates the actual production process of ence. Examples of the virus which have been used for viral the biotechnological Wbiological product used by the clearance studies are shown in Table 3 which was derived manufacturer. It is inappropriate to introduce any virus into from Iyakushin No. 329. However, the Notice, Iyakushin a production facility because of GMP constraints. Therefore, No. 329, is on viral safety of a product derived cell line of hu- viral clearance studies should be conducted in a separate man or animal origin, and a more appropriate model virus laboratory equipped for virological work and performed by has to be chosen taking into account the originWraw material staŠ with virological expertise in conjunction with produc- of biological products. tion personnel involved in designing and preparing a scaled- 6.3 Design of viral clearance studies down version of the puriˆcation process. The viral clearance The purpose of viral clearance studies is to quantitatively studies should be performed under the basic concept of GLP. evaluate removal or inactivation capability of a process, in (4) Each factor on a viral clearance study of a process, which a virus is intentionally spiked to a speciˆc step of a which is performed in miniature size, should re‰ect that of manufacturing process. actual manufacturing as far as possible, and its rationality Following are the precautions to be taken at planning viral should be clariˆed. In case of chromatograph process, length clearance studies. of column bed, linear velocity, ratio of bed volume per veloc- (1) Care should be taken in preparing the high-titer virus ity (in other words, contact time), buŠer, type of column to avoid aggregation which may enhance physical removal packing, pH, temperature, protein concentration, salt con- and decrease inactivation thus distorting the correlation with centration and concentration of the objective product are all actual production. correspondent to those of the actual production. Further, (2) Virus detection method gives great in‰uence to viral similarity of elution proˆle should be achieved. For the other clearance factor. Accordingly, it is advisable to gain detec- process, similar concept should be applied. If there is any fac- tion sensitivity of the methods available in advance, and use a tor which can not re‰ect the actual production, its eŠect to method with a detection sensitivity as high as possible. Quan- the result should be examined. titative infectivity assays should have adequate sensitivity and (5) It is desirable that two or more inactivationWremoval reproducibility in each manufacturing process, and should be processes of diŠerent principles are selected and examined. performed with su‹cient replicates to ensure adequate (6) As for the process which is expected to inactivateWre- statistical validity of the result. Quantitative assays not asso- move virus, each step should be evaluated in aspect of clear- ciated with infectivity may be used if justiˆed. Appropriate ance capability, and carefully determined if it is the stage of virus controls should be included in all infectivity assays to inactivation, removal or their combination for designing the 1672 Basic Requirements for Viral / General Information JP XV test. Generally, in viral clearance test, a virus is spiked in each omitting the product or substituting a similar protein that step which is the object of the test, and after passing through does not have anti-viral activity could aŠect the behaviour of the process in question, the reduction level of infectivity is the virus in some production steps. evaluated. But, in some case, it is accepted that a high poten- (12) Many puriˆcation schemes use the same or similar tial virus is spiked at a step of the process, and virus concen- buŠers or columns, repetitively. The eŠects of this approach tration of each succeeding step is carefully monitored. When should be taken into account when analyzing the data. The removal of virus is made by separation or fractionation, it is eŠectiveness of virus elimination by a particular process may desirable to investigate how the virus is separated or fractio- vary with the stage in manufacture at which it is used. nated (mass balance). (13) Overall reduction factors may be underestimated (7) For assessment of viral inactivation, unprocessed where production conditions or buŠers are too cytotoxic or crude material or intermediate material should be spiked with virucidal and should be discussed on a case-by-case basis. infectious virus and the reduction factor calculated. It should Overall reduction factors may also be overestimated due to be recognized that virus inactivation is not a simple, ˆrst ord- inherent limitations or inadequate design of viral clearance er reaction and is usually more complex, with a fast ``phase studies. 1'' and a slow ``phase 2''. The study should, therefore, be (14) It has to be noted that clearance capability of viral planned in such a way that samples are taken at diŠerent removalWinactivation process may vary depending upon the times and an inactivation curve constructed. It is recom- type of virus. The viral removalWinactivation process, which mended that studies for inactivation include at least one time displays viral clearance by speciˆc principle or mechanism, point less than the minimum exposure time and greater than may be quite eŠective to the virus, which meets such mechan- zero, in addition to the minimum exposure time. The ism of action, but not eŠective to the other type of viruses. reproducible clearance should be demonstrated in at least two For example, SWD treatment is generally eŠective to the virus independent studies. When there is a possibility that the virus with membrane, but not eŠective to the virus not having is a human pathogen, it is very important that the eŠective in- such membrane. Further, some virus is resistant to the gener- activation process is designed and additional data are ob- alheatingprocess(55–609C, 30 minutes). When clearance is tained. The initial virus load should be determined from the expected for such virus, introduction of a further severe con- virus which can be detected in the spiked starting material. If dition or process, which has diŠerent principle or mechan- this is not possible, the initial virus load may be calculated ism, is necessary. Virus removal membrane ˆltration, which from the titer of the spiking virus preparation. Where inacti- is diŠerent from SWD or heat treatment in aspect of principle, vation is too rapid to plot an inactivation curve using process is eŠective to a broad range of virus that can not pass through conditions, appropriate controls should be performed to the membrane. A‹nity chromatography process, which spe- demonstrate that infectivity is indeed lost by inactivation. ciˆcally absorbs the objective protein, can thoroughly wash (8) If antibody against virus exists in an unprocessed out the materials other than the objective protein including material, caution should be taken at clearance studies, since it virus etc. and is generally eŠective for viral removal. Separa- may aŠect the behavior of virus at viral removal or inactiva- tionWfractionation of a virus from an objective protein is tion process. sometimes very di‹cult, but there are not so rare that ion ex- (9) Virus spiked in unprocessed material should be change chromatography, ethanol fractionation, etc. is eŠec- su‹cient enough to evaluate viral removal or inactivation tive for clearance of a virus which can not be su‹ciently inac- capability of the process. However, the virus ``spike'' to be tivated or removed by the other process. added to the unprocessed material should be as small as pos- (15) EŠective clearance may be achieved by any of the sible in comparison with the sample volume of the unproc- following: multiple inactivation steps, multiple complemen- essed material so as not to cause characteristic change of the tary separation steps, or combinations of inactivation and 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 speciˆc physico-chemical properties of a virus (10) It is desirable that the virus in the sample is subject which in‰uence 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, eŠective 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 in- chromatographic procedures, ˆltration steps and extractions, evitable. If any manipulation, such as dilution, concentra- can be also eŠective virus removal steps provided that they tion, ˆltration, dialysis, storage, etc., is applied for prepara- are performed under appropriately controlled conditions. tion of the sample for testing, a parallel control test, which (16) An eŠective virus removal step should give passes through a similar manipulation, should be conducted reproducible reduction of virus load shown by at least two in- to assess infectivity variance at the manipulation. dependent studies. (11) BuŠers and product (desired protein or other com- (17) Over time and after repeated use, the ability of chro- ponent contained therein) should be evaluated independently matography columns and other devices used in the puriˆca- for toxicity or interference in assays used to determine the vi- tion scheme to clear virus may vary. Some estimate of the sta- rus titer, as these components may adversely aŠect the indica- bility of the viral clearance after several uses may provide tor cells. If the solutions are toxic to the indicator cells, dilu- support for repeated use of such columns. tion, adjustment of the pH, or dialysis of the buŠer contain- (18) The Notice, Iyakushin No. 329, would be used as a ing spiked virus might be necessary. If the product itself has reference when viral clearance studies on biological products anti-viral activity, the clearance study may need to be per- are designed. formed without the product in a ``mock'' run, although 6.4 Interpretation of viral clearance studies JP XV General Information / Basic Requirements for Viral 1673

6.4.1 Evaluation on viral clearance factor removal process,  sensitivity limit of virus assay method,  Viral clearance factor is a logarithm of reduction ratio of possible eŠect of the inactivationWremoval process which is viral amount (infectious titer) between each step applied for speciˆc to certain class of viruses. viral clearance of a manufacturing process. Total viral clear- Additional items to be concerned at appropriate interpreta- ance factor throughout the process is sum of the viral clear- tion and evaluation of the viral clearance data are as follows: ance factor of each step appropriately evaluated. (1) Behavior of virus used to the test Whether each and total viral clearance factor obtained are At interpretation of the vial clearance results, it is necessa- acceptable or should not be evaluated in aspects of every ry to recognize that clearance mechanism may diŠer depend- virus that can be realistically anticipated to derive into the ing upon the virus used for the test. Virus used for a test is raw material or the manufacturing process, and its rationality generally produced in tissue culture, but behavior of the virus should be recorded. prepared in the tissue culture may be diŠerent from that of In case that existence of any viral particle is recognized in a the native virus. Examples are possible diŠerences of purity substrate for drug production, e.g., a substrate of rodent ori- anddegreeofaggregationbetweenthenativeandthecul- gin for biodrug production, it is important not only to tured viruses. Further, change of surface properties of a vi- demonstrate removal or inactivation of such virus, but also rus, e.g., addition of a sucrose chain which is ascribed to to demonstrate that the puriˆcation process has enough speciˆc nature of a separation process, may give eŠect to the capability over the required level to assure safety of the ˆnal separation. These matters should be also considered at inter- product at an appropriate level. The virus amount removed pretation of the results. or inactivated in a manufacturing process should be com- (2) Design of test pared with the virus amount assumed to exist in the substrate Viral clearance test should have been designed taking into etc. used for manufacturing drug, and for this purpose, it is account variation factors of the manufacturing process and necessary to obtain the virus amount in the raw materialsW scaling down, but there still remain some variance from substrate, etc. Such ˆgure can be obtained by measuring in- actual production scale. It is necessary to consider such vari- fectious titer or by the other method such as transmission ance at the interpretation of the data and limitation of the electron microscope (TEM). For evaluation of overall proc- test. ess, a virus amount, far larger than that assumed to exist in (3) Acceptability of viral reduction data the amount of the raw materialsWsubstrate which is equiva- Total viral clearance factor is expressed as a sum of lent to single administration of the ˆnal product, has to be re- logarithmofreductionratioobtainedateachstep.Thesum- moved. It is quite rare that existence of virus can be assumed mation of the reduction factor of multiple steps, particularly in a substrate for drug production, with the exception of the of steps with little reduction (e.g., below 1 log10), may overes- substrate of rodent origin, and such suspicious raw materialW timate viral removalWinactivation capability of the overall substrate should not be used for manufacturing drug with a process. Therefore, virus titer of the order of 1 log10 or less special exceptional case that the drug in question is not avail- has to be ignored unless justiˆed. Further, viral clearance fac- able from the other process and is clinically indispensable, tor achieved by repeated use of the same or similar method and that the information including infectious properties of should be ignored for calculation unless justiˆed. the virus particle assumed to exist has been clariˆed. (4) Timedependenceofinactivation 6.4.2 Calculation of viral clearance index Inactivation of virus infectivity frequently shows biphasic Viral clearance factor, ``R'', for viral removalWinactivation curve, which consists of a rapid initial phase and subsequent process can be calculated by the following formula. slow phase. It is possible that a virus not inactivated in a step may be more resistant to the subsequent step. For example, if R＝log[(V ×T )W(V ×T )] 1 1 2 2 an inactivated virus forms , it may be resistant to In which any chemical treatment and heating. R: Logarithm of reduction ratio (5) Evaluation of viral reduction ratio shown logarithm

V1: Sample volume of the unprocessed material Viral clearance factor shown in logarithm of reduction T1: Virus amount (titer) of the unprocessed material ratio of virus titer can demonstrate drastic reduction of resid- V2: Sample volume of the processed material ual infectious virus, but there is a limit that infectious titer T2: Virus amount (titer) of the processed material can never be reduced to zero. For example, reduction in in- fectivity of a preparation containing 8 log infectious unit At the calculation of viral clearance factor, it is recom- 10 permLbyafactorof8log leaves zero log per mL or one mendable to use the virus titer detected in the sample prepa- 10 10 infectious unit per mL, taking into account the detection ration of the unprocessed material after addition of virus, not limit of assay. the viral titer added to the sample preparation wherever pos- (6) Variable factor of manufacturing process sible. If this is not possible, loaded virus amount is calculated Minor variance of a variation factor of a manufacturing from virus titer of the solution used for spike. process, e.g., contact time of a spiked sample to a buŠer or a 6.4.3 Interpretation of results and items to be concerned at column, will sometimes give in‰uence to viral removal or in- evaluation activation eŠect. In such case, it may be necessary to inves- At the interpretation and the evaluation of the data on tigate to what extent such variance of the factor has given in- eŠectiveness of viral inactivationWremoval process, there are ‰uence to the process concerned in aspect of viral inactiva- various factors to be comprehensively taken into account, tion. such as  appropriateness of the virus used for the test,  (7) Existence of anti-viral antiserum design of the viral clearance studies,  virus reduction ratio Anti-viral antiserum that exists in the sample preparation shown in logarithm,  time dependence of inactivation,  used for a test may aŠect sensitivity of distribution or inacti- factorsWitems which give in‰uence to the inactivationW vation of a virus, which may result in not only defusing the 1674 Basic Requirements for Viral / General Information JP XV virus titer but complicating interpretation of the test result. 8. Re-evaluation of viral clearance So, existence of anti-viral antiserum is one of the important Whenever signiˆcant changes in the production or puriˆca- variable factors. tion process are made, the eŠect of that change, both direct (8) Introduction of a new process for removalWinactivation and indirect, on viral clearance should be considered and the Viral clearance is an important factor for securing safety of system re-evaluated as needed. Changes in process steps may drug. In case that an achievement level of infective clearance also change the extent of viral clearance. of a process is considered insu‹cient, a process which is 9. Measurement for viral clearance studies characterized by inactivationWremoval mechanism to meet 9.1 Measurement of virus infective titer the purpose or an inactivationWremoval process which can Assay methods may be either quantal or quantitative. mutually complement to the existence process has to be in- Quantal methods include infectivity assays in animals or in troduced. cultured cell infections dose (CCID) assays, in which the (9) Limit of viral clearance studies animal or cell culture is scored as either infected or not. In- Viral clearance studies are useful for contributing to the as- fectivity titers are then measured by the proportion of surance that an acceptable level of safety in the ˆnal product animals or culture infected. In quantitative methods, the in- is achieved but do not by themselves establish safety. fectivity measured varies continuously with the virus input. However, a number of factors in the design and execution of Quantitative methods include plaque assays where each viral clearance studies may to an incorrect estimate of plaque counted corresponds to a single infectious unit. Both the ability of the process to remove virus infectivity, as de- quantal and quantitative assays are amenable to statistical scribed above. evaluation. 7. Statistics 9.2 Testing by nucleic-acid-ampliˆcation test (NAT) The viral clearance studies should include the use of NAT can detect individual or pooled raw materialWcell sub- statistical analysis of the data to evaluate the results. The strate or virus genome at a high sensitivity even in a stage that study results should be statistically valid to support the con- serum test on each virus is negative. Further, it can detect clusions reached. HBV or HCV gene, which can not be measured in culture sys- 7.1 Statistical considerations for assessing virus assays tem. Window period can be drastically shortened at the test VirustitrationssuŠertheproblemsofvariationcommon on HBV, HCV and HIV, and the method is expected to con- to all biological assay systems. Assessment of the accuracy of tribute as an eŠective measure for ensuring viral safety. the virus titrations and reduction factors derived from them However, depending upon a choice of primer, there may be a and the validity of the assays should be performed to deˆne case that not all the subtype of objective virus can be detected the reliability of a study. The objective of statistical evalua- by this method, and, therefore, it is recommendable to evalu- tion is to establish that the study has been carried out to an ate, in advance, if subtype of a broad range can be detected. acceptable level of virological competence. NAT will be an eŠective evaluation method for virus Assay removal capability at viral clearance studies. However, in 1. Assay methods may be either quantal or quantitative. case of viral inactivation process, viral inactivation obtained Both quantal and quantitative assays are amenable to statisti- by this method may be underrated, since there is a case that cal evaluation. inactivated virus still shows positive on nucleic acid. Further, 2. Variation can arise within an assay as a result of dilu- at introduction of NAT, cautions should be taken on ration- tion errors, statistical eŠects and diŠerences within the assay ality of detection sensitivity, choice of a standard which is system which are either unknown or di‹cult to control. used as run-control, quality assurance and maintenance of a These eŠects are likely to be greater when diŠerent assay runs reagent used for primer, interpretation of positive and nega- are compared (between-assay variation) than when results wi- tive results, etc. thin a single assay run are compared (within-assay variation). 10. Reporting and preservation 3. The 95z conˆdence limits for results of within-assay All the items relating to virus test and viral clearance stu- variation normally should be on the order of ±0.5 log of 10 dies should be reported and preserved. the mean. Within-assay variation can be assessed by standard textbook methods. Between-assay variation can be moni- 11. Others tored by the inclusion of a reference preparation, the estimate The Notice, Iyakushin No. 329, should be used as a refer- of whose should be within approximately 0.5 log10 of ence at virus test and viral clearance studies. the mean estimate established in the laboratory for the assay Conclusion to be acceptable. Assays with lower precision may be accepta- As mentioned at the Introduction, assurance of qualityW ble with appropriate justiˆcation. safety etc. of JP listed drugs should be achieved by state-of- 7.2 Reproducibility and conˆdence limit of viral clearance the-art methods and concepts re‰ecting the progress of studies science and accumulation of experiences. An eŠective virus inactivationWremoval step should give The basis for ensuring viral safety of JP listed biotechno- reproducible reduction of virus load shown by at least two in- logicalWbiological products is detailed in this General Infor- dependent studies. The 95z conˆdence limits for the reduc- mation.Whatisdiscussedhereisthatanalmostequallevel tion factor observed should be calculated wherever possible of measures are required for both development of new drugs in studies of viral clearance. If the 95z conˆdence limits for and for existing products as well, which means that similar the viral assays of the starting material are ±s, and for the level of concerns should be paid on both existing and new viral assays of the material after the step are ±a, the 95z products in aspect of viral safety. This document is intended conˆdence limits for the reduction factor are ± s2＋a2. to introduce a basic concept that quality and safety assurance of JP listed product should be based upon the most advanced JP XV General Information / Capillary Electrophoresis 1675 methods and concepts. This document has been written to direction to the electro-osmotic ‰ow and their velocities will cover all conceivable cases, which can be applied to all be smaller than the electro-osmotic velocity. Cations will mi- biotechnologicalWbiological products. Therefore, there may grate in the same direction as the electro-osmotic ‰ow and be cases that it is not so rational to pursue virus tests and viral their velocities will be greater than the electro-osmotic veloc- clearance studies in accordance with this document on each ity. Under conditions in which there is a fast electro-osmotic product, which has been used for a long time without any velocity with respect to the electrophoretic velocity of the so- safety issue. So, it will be necessary to elaborate the most ra- lutes, both cations and anions can be separated in the same tional ways under a case-by-case principle taking into due run. consideration source, origin, type, manufacturing process, Thetime(t) taken by the solute to migrate the distance (l) characteristics, usages at clinical stage, accumulation of the from the injection end of the capillary to the detection point past usage record, etc. relating to such biotechnologicalWbio- (capillary eŠective length) is given by the expression: logical products. l l×L t＝ ＝ nep＋neo (mep＋meo)V In general, uncoated fused-silica capillaries above pH 3 4. Capillary Electrophoresis have negative charge due to ionized silanol groups in the inner wall. Consequently, the electro-osmotic ‰ow is from This test is harmonized with the anode to cathode. The electro-osmotic ‰ow must remain con- and the U.S. Pharmacopeia. stant from run to run if good reproducibility is to be obtained General Principles in the migration velocity of the solutes. For some applica- Capillary electrophoresis is a physical method of analysis tions, it may be necessary to reduce or suppress the electro- based on the migration, inside a capillary, of charged osmotic ‰ow by modifying the inner wall of the capillary or analytes dissolved in an electrolyte solution, under the by changing the concentration, composition and/or pH of in‰uence of a direct-current electric ˆeld. the buŠer solution. The migration velocity of an analyte under an electric ˆeld After the introduction of the sample into the capillary, of intensity E, is determined by the electrophoretic mobility each analyte ion of the sample migrates within the back- of the analyte and the electro-osmotic mobility of the buŠer ground electrolyte as an independent zone, according to its inside the capillary. The electrophoretic mobility of a solute electrophoretic mobility. Zone dispersion, that is the spread-

(mep) depends on the characteristics of the solute (electric ing of each solute band, results from diŠerent phenomena. charge, molecular size and shape) and those of the buŠer in Under ideal conditions the sole contribution to the solute- which the migration takes place (type and ionic strength of zone broadening is molecular diŠusion of the solute along the the electrolyte, pH, viscosity and additives). The elec- capillary (longitudinal diŠusion). In this ideal case the trophoretic velocity (nep) of a solute, assuming a spherical e‹ciency of the zone, expressed as the number of theoretical shape, is given by the equation: plates (N), is given by: q V (m ＋m )×V×L n ＝m E＝ N＝ ep eo ep ep Ø 6phr »Ø L » 2×D×L q: eŠective charge of the solute, D: molecular diŠusion coe‹cient of the solute in the h: viscosity of the electrolyte solution, buŠer. r: Stoke's radius of the solute, In practice, other phenomena such as heat dissipation, V: applied voltage, sample adsorption onto the capillary wall, mismatched L: total length of the capillary. conductivity between sample and buŠer, length of the injec- When an electric ˆeld is applied through the capillary ˆlled tion plug, detector cell size and unlevelled buŠer reservoirs with buŠer, a ‰ow of solvent is generated inside the capillary, can also signiˆcantly contribute to band dispersion. called electro-osmotic ‰ow. The velocity of the electro-os- Separation between two bands (expressed as the resolution, motic ‰ow depends on the electro-osmotic mobility (meo) RS) can be obtained by modifying the electrophoretic mobili- which in depends on the charge on the capillary ty of the analytes, the electro-osmotic mobility induced in the internal wall and the buŠer characteristics. The electro-os- capillary and by increasing the e‹ciency for the band of each motic velocity (neo) is given by the equation: analyte, according to the equation:

ez V N(mepb－mepa) neo＝meoE＝ RS＝ Ø h »Ø L » 4(m š ep＋meo)

e: dielectric constant of the buŠer, mepa and mepb: electrophoretic mobilities of the two analytes z: zeta potential of the capillary surface. separated, mš : mean electrophoretic mobility of the two analytes The velocity of the solute (n)isgivenby: ep 1 mš ep＝ (mepb＋mepa). n＝nep＋neo 2 Apparatus The electrophoretic mobility of the analyte and the electro- An apparatus for capillary electrophoresis is composed of: osmotic mobility may act in the same direction or in opposite —a high-voltage, controllable direct-current power supply, directions, depending on the charge of the solute. In normal —two buŠer reservoirs, held at the same level, containing capillary electrophoresis, anions will migrate in the opposite the prescribed anodic and cathodic solutions, 1676 Capillary Electrophoresis / General Information JP XV

—two electrode assemblies (the cathode and the anode), main factors to be considered in the development of separa- immersed in the buŠer reservoirs and connected to the tions are instrumental and electrolytic solution parameters. power supply, Instrumental parameters —a separation capillary (usually made of fused-silica) Voltage: A Joule heating plot is useful in optimizing the which, when used with some speciˆc types of detectors, applied voltage and column temperature. Separation time is has an optical viewing window aligned with the detector. inversely proportional to applied voltage. However, an The ends of the capillary are placed in the buŠer reser- increase in the voltage used can cause excessive heat produc- voirs. The capillary is ˆlled with the solution prescribed tion, giving rise to temperature and, as a result thereof, in the monograph, viscosity gradients in the buŠer inside the capillary. This —a suitable injection system, eŠect causes band broadening and decreases resolution. —a detector able to monitor the amount of substances of Polarity: Electrode polarity can be normal (anode at the interest passing through a segment of the separation inlet and cathode at the outlet) and the electro-osmotic ‰ow capillary at a given time. It is usually based on absorp- will move toward the cathode. If the electrode polarity is tion spectrophotometry (UV and visible) or ‰uorometry, reversed, the electro-osmotic ‰ow is away from the outlet and but conductimetric, amperometric or mass spectrometric only charged analytes with electro-osmotic mobilities greater detection can be useful for speciˆc applications. Indirect than the electro-osmotic ‰ow will pass to the outlet. detection is an alternative method used to detect non- Temperature: The main eŠect of temperature is observed UV-absorbing and non-‰uorescent compounds, on buŠer viscosity and electrical conductivity, and therefore —a thermostatic system able to maintain a constant tem- on migration velocity. In some cases, an increase in capillary perature inside the capillary is recommended to obtain a temperature can cause a conformational change in proteins, good separation reproducibility, modifying their migration time and the e‹ciency of the sepa- —a recorder and a suitable integrator or a computer. ration. The deˆnition of the injection process and its automation Capillary: The dimensions of the capillary (length and in- are critical for precise quantitative analysis. Modes of ternal diameter) contribute to analysis time, e‹ciency of injection include gravity, pressure or vacuum injection and separations and load capacity. Increasing both eŠective electrokinetic injection. The amount of each sample length and total length can decrease the electric ˆelds component introduced electrokinetically depends on its (working at constant voltage) which increases migration time. electrophoretic mobility, leading to possible discrimination For a given buŠer and electric ˆeld, heat dissipation, and using this injection mode. hence sample band-broadening, depend on the internal di- Use the capillary, the buŠer solutions, the preconditioning ameter of the capillary. The latter also aŠects the detection method, the sample solution and the migration conditions limit, depending on the sample volume injected and the prescribed in the monograph of the considered substance. detection system employed. The employed electrolytic solution is ˆltered to remove Since the adsorption of the sample components on the particles and degassed to avoid bubble formation that could capillary wall limits e‹ciency, methods to avoid these interfere with the detection system or interrupt the electrical interactions should be considered in the development of a contact in the capillary during the separation run. A rigorous separation method. In the speciˆc case of proteins, several rinsing procedure should be developed for each analytical strategies have been devised to avoid adsorption on the capil- method to achieve reproducible migration times of the so- lary wall. Some of these strategies (use of extreme pH and ad- lutes. sorption of positively charged buŠer additives) only require modiˆcation of the buŠer composition to prevent protein ad- 1. Capillary Zone Electrophoresis sorption. In other strategies, the internal wall of the capillary Principle is coated with a polymer, covalently bonded to the silica, that In capillary zone electrophoresis, analytes are separated in prevents interaction between the proteins and the negatively a capillary containing only buŠer without any anticonvective charged silica surface. For this purpose, ready-to-use capilla- medium. With this technique, separation takes place because ries with coatings consisting of neutral-hydrophilic, cationic the diŠerent components of the sample migrate as discrete and anionic polymers are available. bands with diŠerent velocities. The velocity of each band Electrolytic solution parameters depends on the electrophoretic mobility of the solute and the BuŠer type and concentration: Suitable buŠers for electro-osmotic ‰ow in the capillary (see General Principles). capillary electrophoresis have an appropriate buŠer capacity Coated capillaries can be used to increase the separation in the pH range of choice and low mobility to minimize capacity of those substances adsorbing on fused-silica current generation. surfaces. Matching buŠer-ion mobility to solute mobility, whenever Using this mode of capillary electrophoresis, the analysis possible, is important for minimizing band distortion. The of both small (M º2000) and large molecules (2000ºM r r type of sample solvent used is also important to achieve º100,000) can be accomplished. Due to the high e‹ciency on-column sample focusing, which increases separation achieved in free solution capillary electrophoresis, separation e‹ciency and improves detection. of molecules having only minute diŠerences in their charge- An increase in buŠer concentration (for a given pH) to-mass ratio can be eŠected. This separation mode also al- decreases electro-osmotic ‰ow and solute velocity. lows the separation of chiral compounds by addition of chiral BuŠer pH: The pH of the buŠer can aŠect separation by selectors to the separation buŠer. modifying the charge of the analyte or additives, and by Optimization changing the electro-osmotic ‰ow. In protein and peptide Optimization of the separation is a complex process where separation, changing the pH of the buŠer from above to several separation parameters can play a major role. The below the (pI) changes the net charge of the JP XV General Information / Capillary Electrophoresis 1677 solute from negative to positive. An increase in the buŠer pH in the mobility of the solutes. Due to the rigidity of these gels, generally increases the electro-osmotic ‰ow. only electrokinetic injection can be used. Organic solvents: Organic modiˆers (methanol, acetoni- Dynamically coated gels are hydrophilic polymers, such as trile, etc.) may be added to the aqueous buŠer to increase the linear polyacrylamide, cellulose derivatives, dextran, etc., solubility of the solute or other additives and/or to aŠect the which can be dissolved in aqueous separation buŠers giving degree of ionization of the sample components. The addition rise to a separation medium that also acts as a molecular of these organic modiˆers to the buŠer generally causes a sieve. These separation media are easier to prepare than decrease in the electro-osmotic ‰ow. cross-linked polymers. They can be prepared in a vial and Additives for chiral separations:Fortheseparationof ˆlled by pressure in a wall-coated capillary (with no electro- optical isomers, a chiral selector is added to the separation osmotic ‰ow). Replacing the gel before every injection gener- buŠer. The most commonly used chiral selectors are ally improves the separation reproducibility. The porosity of cyclodextrins, but crown ethers, polysaccharides and proteins the gels can be increased by using polymers of higher molecu- may also be used. Since chiral recognition is governed by the lar mass (at a given polymer concentration) or by decreasing diŠerent interactions between the chiral selector and each of the polymer concentration (for a given polymer molecular the enantiomers, the resolution achieved for the chiral com- mass). A reduction in the gel porosity to a decrease in pounds depends largely on the type of chiral selector used. In themobilityofthesoluteforthesamebuŠer.Sincethedisso- this regard, for the development of a given separation it may lution of these polymers in the buŠer gives low viscosity solu- be useful to test cyclodextrins having a diŠerent cavity size tions, both hydrodynamic and electrokinetic injection tech- (a-, b-, or g-cyclodextrin) or modiˆed cyclodextrins with neu- niques can be used. tral (methyl, ethyl, hydroxyalkyl, etc.) or ionizable 3. Capillary Isoelectric Focusing (aminomethyl, carboxymethyl, sulfobutyl ether, etc.) groups. Principle When using modiˆed cyclodextrins, batch-to-batch varia- In isoelectric focusing, the molecules migrate under the tions in the degree of substitution of the cyclodextrins must in‰uence of the electric ˆeld, so long as they are charged, in a be taken into account since it will in‰uence the selectivity. pH gradient generated by ampholytes having pI values in a Other factors controlling the resolution in chiral separations wide range (poly-aminocarboxylic acids), dissolved in the are concentration of chiral selector, composition and pH of separation buŠer. the buŠer and temperature. The use of organic additives, The three basic steps of isoelectric focusing are loading, such as methanol or urea can also modify the resolution focusing and mobilization. achieved. Loading step: Two methods may be employed: 2. Capillary Gel Electrophoresis —loading in one step: the sample is mixed with ampholytes Principle and introduced into the capillary either by pressure or In capillary gel electrophoresis, separation takes place vacuum; inside a capillary ˆlled with a gel that acts as a molecular —sequential loading: a leading buŠer, then the ampho- sieve. Molecules with similar charge-to-mass ratios are lytes, then the sample mixed with ampholytes, again am- separated according to molecular size since smaller molecules pholytes alone and ˆnally the terminating buŠer are in- move more freely through the network of the gel and there- troduced into the capillary. The volume of the sample fore migrate faster than larger molecules. DiŠerent biological must be small enough not to modify the pH gradient. macromolecules (for example, proteins and DNA frag- Focusing step: When the voltage is applied, ampholytes ments), which often have similar charge-to-mass ratios, can migrate toward the cathode or the anode, according to their thus be separated according to their molecular mass by capil- net charge, thus creating a pH gradient from anode (lower lary gel electrophoresis. pH) to cathode (higher pH). During this step the components Characteristics of Gels to be separated migrate until they reach a pH corresponding Two types of gels are used in capillary electrophoresis: per- to their isoelectric point (pI) and the current drops to very manently coated gels and dynamically coated gels. Perma- low values. nently coated gels, such as cross-linked polyacrylamide, are Mobilization step: If mobilization is required for detec- prepared inside the capillary by polymerization of the tion, use one of the following methods. Three methods are monomers. They are usually bonded to the fused-silica wall available: and cannot be removed without destroying the capillary. If —in the ˆrst method, mobilization is accomplished during the gels are used for protein analysis under reducing condi- the focusing step under the eŠect of the electro-osmotic tions, the separation buŠer usually contains sodium dodecyl ‰ow; the electro-osmotic ‰ow must be small enough to sulfate and the samples are denatured by heating a mixture of allow the focusing of the components; sodium dodecyl sulfate and 2-mercaptoethanol or —in the second method, mobilization is accomplished by dithiothreitol before injection. When non-reducing condi- applying positive pressure after the focusing step; tions are used (for example, analysis of an intact antibody), —in the third method, mobilization is achieved after the 2-mercaptoethanol and dithiothreitol are not used. Separa- focusing step by adding salts to the cathode reservoir or tion in cross-linked gels can be optimized by modifying the the anode reservoir (depending on the direction chosen separation buŠer (as indicated in the free solution capillary for mobilization) in order to alter the pH in the capillary electrophoresis section) and controlling the gel porosity when the voltage is applied. As the pH is changed, the during the gel preparation. For cross-linked polyacrylamide proteins and ampholytes are mobilized in the direction gels, the porosity can be modiˆed by changing the concentra- of the reservoir which contains the added salts and pass tion of acrylamide and/or the proportion of cross-linker. As the detector. a rule, a decrease in the porosity of the gel leads to a decrease The separation achieved, expressed as DpI, depends on the 1678 Capillary Electrophoresis / General Information JP XV pH gradient (dpH/dx),thenumberofampholyteshaving micelle and the aqueous buŠer, and has no electrophoretic diŠerent pI values, the molecular diŠusion coe‹cient (D), the mobility, the analyte migration velocity will depend only on intensity of the electric ˆeld (E) and the variation of the elec- the partition coe‹cient between the micelle and the aqueous trophoretic mobility of the analyte with the pH (－dm/dpH): buŠer. In the electropherogram, the peaks corresponding to each uncharged solute are always between that of the electro- D(dpH/dx) DPI＝3 osmotic ‰ow marker and that of the micelle (the time elapsed E(－dm/dpH) between these two peaks is called the separation window). For electrically charged solutes, the migration velocity de- Optimization pends on both the partition coe‹cient of the solute between The main parameters to be considered in the development the micelle and the aqueous buŠer, and on the electrophoretic of separations are: mobility of the solute in the absence of micelle. Voltage: Capillary isoelectric focusing utilises very high Since the mechanism in MEKC of neutral and weakly electric ˆelds, 300 V/cm to 1000 V/cm in the focusing step. ionized solutes is essentially chromatographic, migration of Capillary: The electro-osmotic ‰ow must be reduced or the solute and resolution can be rationalized in terms of the suppressed depending on the mobilization strategy (see retention factor of the solute (k?), also referred to as mass above). Coated capillaries tend to reduce the electro-osmotic distribution ratio (D ), which is the ratio of the number of ‰ow. m moles of solute in the micelle to those in the mobile phase. Solutions: The anode buŠer reservoir is ˆlled with a For a neutral compound, k? is given by: solution with a pH lower than the pI of the most acidic ampholyte and the cathode reservoir is ˆlled with a solution t －t V k?＝ R 0 ＝K S with a pH higher than the pI of the most basic ampholyte. t V t 1－ R M Phosphoric acid for the anode and sodium hydroxide for the 0Ø t » mc cathode are frequently used. Addition of a polymer, such as methylcellulose, in the tR: migration time of the solute, ampholyte solution tends to suppress convective forces (if t0: analysis time of an unretained solute (determined by in- any) and electro-osmotic ‰ow by increasing the viscosity. jecting an electro-osmotic ‰ow marker which does not Commercial ampholytes are available covering many pH enter the micelle, for instance methanol), ranges and may be mixed if necessary to obtain an expanded tmc: micelle migration time (measured by injecting a micelle pH range. Broad pH ranges are used to estimate the isoelec- marker, such as Sudan III, which migrates while con- tric point whereas narrower ranges are employed to improve tinuously associated in the micelle), accuracy. Calibration can be done by correlating migration K: partition coe‹cient of the solute, time with isoelectric point for a series of protein markers. VS: volume of the micellar phase, During the focusing step precipitation of proteins at their VM: volume of the mobile phase. isoelectric point can be prevented, if necessary, using buŠer Likewise, the resolution between two closely-migrating additives such as , surfactants, urea or zwitterionic solutes (RS)isgivenby: buŠers. However, depending on the concentration, urea t denatures proteins. 1－ 0 N a－1 k? Ø tmc » R ＝ × × b × 4. Micellar Electrokinetic Chromatography (MEKC) S 4 a k? ＋1 t b 1＋ 0 k? Principle Ø t » a mc 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 buŠer and the pseudo-stationary phase com- (k?bÀk?a). posed of micelles, according to the partition coe‹cient of the Similar, but not identical, equations give k? and R values solute. The technique can therefore be considered as a hybrid S for electrically charged solutes. of electrophoresis and chromatography. It is a technique that Optimization can be used for the separation of both neutral and charged Themainparameterstobeconsideredinthedevelopment solutes, maintaining the e‹ciency, speed and instrumental of separations by MEKC are instrumental and electrolytic so- suitability of capillary electrophoresis. One of the most wide- lution parameters. ly used surfactants in MEKC is the anionic surfactant sodium Instrumental parameters dodecyl sulfate, although other surfactants, for example Voltage: Separation time is inversely proportional to cationic surfactants such as cetyltrimethylammonium salts, applied voltage. However, an increase in voltage can cause are also used. excessive heat production that gives rise to temperature The separation mechanism is as follows. At neutral and gradients and viscosity gradients of the buŠer in the cross- alkaline pH, a strong electro-osmotic ‰ow is generated and section of the capillary. This eŠect can be signiˆcant with moves the separation buŠer in the direction of the high conductivity buŠers such as those containing micelles. cathode. If sodium dodecyl sulfate is employed as the sur- Poor heat dissipation causes band broadening and decreases factant, the electrophoretic migration of the anionic micelle resolution. is in the opposite direction, towards the anode. As a result, Temperature: Variations in capillary temperature aŠect the overall micelle migration velocity is slowed down com- the partition coe‹cient of the solute between the buŠer and pared to the bulk ‰ow of the electrolytic solution. In the case the micelles, the critical micellar concentration and the of neutral solutes, since the analyte can partition between the JP XV General Information / Capillary Electrophoresis 1679 viscosity of the buŠer. 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 the tem improves the reproducibility of the migration time for micelle and form co-ordination complexes with the solutes. the solutes. Quantiˆcation Capillary: As in free solution capillary electrophoresis, Peak areas must be divided by the corresponding migration the dimensions of the capillary (length and internal diameter) time to give the corrected area in order to: contribute to analysis time and e‹ciency of separations. In- —compensate for the shift in migration time from run to creasing both eŠective length and total length can decrease run, thus reducing the variation of the response, the electric ˆelds (working at constant voltage), increase —compensate for the diŠerent responses of sample migration time and improve the separation e‹ciency. The in- constituents with diŠerent migration times. ternal diameter controls heat dissipation (for a given buŠer Where an internal standard is used, verify that no peak of and electric ˆeld) and consequently the sample band broaden- the substance to be examined is masked by that of the ing. internal standard. Electrolytic solution parameters Calculations Surfactant type and concentration: The type of sur- From the values obtained, calculate the content of the factant, in the same way as the stationary phase in chro- component or components being examined. When matography, aŠects the resolution since it modiˆes separa- prescribed, the percentage content of one or more compo- tion selectivity. Also, the log k? of a neutral compound nents of the sample to be examined is calculated by determin- increases linearly with the concentration of surfactant in the ing the corrected area(s) of the peak(s) as a percentage of the mobile phase. Since resolution in MEKC reaches a maximum total of the corrected areas of all peaks, excluding those due when k? approaches the value of tm/t0, modifying the con- to solvents or any added reagents (normalization procedure). centration of surfactant in the mobile phase changes the reso- The use of an automatic integration system (integrator or lution obtained. data acquisition and processing system) is recommended. BuŠer pH: Although pH does not modify the partition System Suitability coe‹cient of non-ionized solutes, it can modify the electro- In order to check the behavior of the capillary electropho- osmotic ‰ow in uncoated capillaries. A decrease in the buŠer resis system, system suitability parameters are used. The pH decreases the electro-osmotic ‰ow and therefore increases choice of these parameters depends on the mode of capillary the resolution of the neutral solutes in MEKC, resulting in a electrophoresis used. They are: retention factor (k?)(onlyfor longer analysis time. micellar electrokinetic chromatography), apparent number Organic solvents: To improve MEKC separation of of theoretical plates (N), symmetry factor (A ) and resolution hydrophobic compounds, organic modiˆers (methanol, S (R ). In previous sections, the theoretical expressions for N propanol, acetonitrile, etc.) can be added to the electrolytic S and R have been described, but more practical equations solution. The addition of these modiˆers usually decreases S that allow these parameters to be calculated from the elec- migration time and the selectivity of the separation. Since the tropherograms are given below. addition of organic modiˆers aŠects the critical micellar con- Apparent Number of Theoretical Plates centration, a given surfactant concentration can be used only The apparent number of theoretical plates (N)maybe within a certain percentage of organic modiˆer before the calculated using the expression: micellization is inhibited or adversely aŠected, resulting in 2 the absence of micelles and, therefore, in the absence of par- t N＝5.54 R tition. The dissociation of micelles in the presence of a high Ø w » h content of organic solvent does not always mean that the t : migration time or distance along the baseline from the separation will no longer be possible; in some cases the R point of injection to the perpendicular dropped from hydrophobic interaction between the ionic surfactant the maximum of the peak corresponding to the compo- monomer and the neutral solutes forms solvophobic nent, complexes that can be separated electrophoretically. w : width of the peak at half-height. Additives for chiral separations:Fortheseparationof h enantiomers using MEKC, a chiral selector is included in the Resolution micellar system, either covalently bound to the surfactant or The resolution (RS)betweenpeaksofsimilarheightoftwo added to the micellar separation electrolyte. Micelles that components may be calculated using the expression: have a moiety with chiral discrimination properties include 1.18(tR2－tR1) salts of N-dodecanoyl-L-amino acids, salts, etc. Chiral R ＝ S Ø w ＋w » resolution can also be achieved using chiral discriminators, h1 h2 such as cyclodextrins, added to the electrolytic solutions tR2ÀtR1 which contain micellized achiral surfactants. t and t : migration times or distances along the baseline Other additives: Several strategies can be carried out to R1 R2 from the point of injection to the perpendicu- modify selectivity, by adding chemicals to the buŠer. The ad- lars dropped from the maxima of two adjacent dition of several types of cyclodextrins to the buŠer can also peaks, be used to reduce the interaction of hydrophobic solutes with w and w : peak widths at half-height. the micelle, thus increasing the selectivity for this type of h1 h2 compound. When appropriate, the resolution may be calculated by

The addition of substances able to modify solute-micelle measuring the height of the valley (Hv) between two partly interactions by adsorption on the latter, is used to improve resolved peaks in a standard preparation and the height of the selectivity of the separations in MEKC. These additives the smaller peak (Hp) and calculating the peak-to-valley ratio: 1680 Decision of Limit for Bacterial / General Information JP XV

H p/n＝ p Hv Intended K (EU Wkg) Symmetry Factor Intravenous 5.0 The symmetry factor (AS)ofapeakmaybecalculated using the expression: Intravenous, for w 2.5 A ＝ 0.05 S 2d Intraspinal 0.2 w0.05: width of the peak at one-twentieth of the peak height, d: distance between the perpendicular dropped from the Notes: peak maximum and the leading edge of the peak at one- 1) Forproductstobeadministeredbymassorbyunits, twentieth of the peak height. the endotoxin limit should be decided based on the labeled amount of the principal drug. Tests for area repeatability (standard deviation of areas or 2) Sixty kg should be used as the average body mass of an of the area/migration-time ratio) and for migration time adult when calculating the maximum adult dose per kg. repeatability (standard deviation of migration time) are 3) The pediatric dose per kg body mass should be used introduced as suitability parameters. Migration time when this is higher than the adult dose. repeatability provides a test for the suitability of the capillary 4) The K values for the intravenous route are applicable washing procedures. An alternative practice to avoid the lack to drugs to be administered by any route other than those of repeatability of the migration time is to use migration time shown in the table. relative to an internal standard. A test for the veriˆcation of the signal-to-noise ratio for a standard preparation (or the determination of the limit of quantiˆcation) may also be useful for the determination of 6. Disinfection and Sterilization related substances. Signal-to-noise Ratio Methods The detection limit and quantiˆcation limit correspond to Disinfection and Sterilization Methods are applied to kill signal-to-noise ratios of 3 and 10 respectively. The signal-to- microorganisms in processing equipment utensils and areas noise ratio (S/N) is calculated using the expression: W used for drug manufacturing, as well as to perform microbio- 2H logical tests speciˆed in the monographs, and so diŠer from S/N＝ h ``Terminal Sterilization'' and ``Filtration Method'' described in ``Terminal Sterilization and Sterilization Indicators''. The H: height of the peak corresponding to the component killing eŠect on microorganisms or the estimated level of concerned, in the electropherogram obtained with the sterility assurance is greatly variable, so the conditions for prescribed reference solution, measured from the maximum disinfection and sterilization treatment must be chosen of the peak to the extrapolated baseline of the signal observed appropriately for each application. Generally, the following over a distance equal to twenty times the width at half-height, methods are to be used singly or in combination after h: range of the background in an electropherogram ob- appropriate optimization of operation procedures and condi- tained after injection of a blank, observed over a distance tions, in accordance with the kind and the degree of the equal to twenty times the width at the half-height of the peak contaminating microorganisms and the nature of the item to in the electropherogram obtained with the prescribed refer- which the methods are applied. ence solution and, if possible, situated equally around the The validation of sterilization in accordance with Terminal place where this peak would be found. Sterilization and Sterilization Indicators is required when the methods are applied to the manufacturing processes of drug products. 5. Decision of Limit for Bacterial 1. Disinfection methods Endotoxins These methods are used to reduce the number of living microorganisms, but do not always remove or kill all The endotoxin limit for injections is to be decided as fol- microorganisms present. Generally, disinfection is classiˆed lows: into chemical disinfection with the use of chemical drugs (dis- infectants) and physical disinfection with the use of moist Endotoxin limit＝K WM heat, ultraviolet light, and other agents. where K is a minimum pyrogenic dose of endotoxin per kg 1-1. Chemical disinfection body mass (EUWkg), and M is equal to the maximum dose of Microorganisms are killed with chemical drugs. The killing product per kg per hour. eŠect and mechanisms of a chemical drug diŠer depending on M isexpressedinmLWkg for products to be administered the type, applied concentration, action temperature, and by volume, in mgWkg or mEqWkg for products to be action time of the chemical drug used, the degree of contami- administered by mass, and in UnitWkg for products to be nation on the object to be disinfected, and the species and administered by biological units. Depending on the adminis- state (e.g., vegetative or spore bacteria) of microor- tration route, values for K are set as in the following table. ganisms. JP XV General Information / Disinfection and Sterilization 1681

Therefore, in applying the method, full consideration is temperature and pressure. This method is generally used for required of the sterility and permissible storage period of the heat-stable substances, such as glass, porcelain, metal, rub- prepared chemical drug, the possibility of resistance of ber, plastics, paper, and ˆber, as well as heat-stable liquids, microorganisms at the site of application, and the eŠect of such as water, culture media, reagents, test solutions, liquid residual chemical drug on the product. In selecting a suitable samples, etc. As a rule, one of the following conditions is chemical drug, the following items should be considered in used. relation to the intended use. 115–1189Cfor30minutes 1) The antimicrobial spectrum 121–1249Cfor15minutes 2) Action time for killing microorganisms 126–1299Cfor10minutes 3) Action durability (ii) Dry-heat method 4) EŠect of the presence of proteins Microorganisms are killed in dry-heated air. This method 5) In‰uence on the human body is generally used for heat-stable substances, such as glass, 6) Solubility in water porcelain, and metal, as well as heat-stable products, such as 7) In‰uence on the object to be disinfected oils, and oils, powder samples, etc. This method 8) Odor is generally conducted in the way of direct heating by gas or 9) Convenience of use electricity or circulating heated air. As a rule, one of the fol- 10) Easy disposability lowing conditions is used. 11) In‰uence on the environment at disposal 160 – 1709C for 120 minutes 12) Frequency of occurrence of resistance 170 – 1809C for 60 minutes 1-2. Physical disinfection 180 – 1909C for 30 minutes Microorganisms are killed without a chemical drug. 2-2. Irradiation methods (i) Steam ‰ow method (i) Radiation method Microorganisms are killed by direct application of steam. Microorganisms are killed by gamma-rays emitted from a This method is used for a product which may be denatured by radioisotope or electron beam and bremsstrahlung (X-ray) the moist heat method. As a rule, the product is kept in generated from an electron accelerator. This method is gener- ‰owing steam at 1009Cfor30–60minutes. ally used for radiation-resistant substances such as glass, por- (ii) Boiling method celain, metal, rubber, plastics, ˆber, etc. The dose is decided Microorganisms are killed by putting the object in boiling according to the material properties, and the degree of con- water. This method is used for a product which may be tamination of the product to be sterilized. Special considera- denatured by the moist heat method. As a rule, the product is tion is necessary of the possibility of qualitative change of the put in boiling water for 15 minutes or more. product after the application of the method. (iii) Intermittent method (ii) Microwave method Microorganisms are killed by heating for 30 – 60 minutes Microorganisms are killed by the heat generated by direct repeatedly, three to ˆve times, once a day in water at 80 – microwave irradiation. This method is generally used for 1009Corinsteam.Thismethodisusedforaproductwhich microwave-resistant products such as water, culture media, may be denatured by the moist heat method. There is another test solutions, etc. As a rule, microwave radiation with a method called the low temperature intermittent method with wavelength of around 2450±50 MHz is used. repeated heating at 60 – 809C. During the intermission 2-3. Gas methods periods between heating or warming, a suitable temperature Microorganisms are killed by a sterilizing gas. Suitable for the growth of microorganisms of 209C or higher, must be for killing microorganisms include oxide gas, 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 diŠer in accordance with the species of ultraviolet rays at a wavelength of around 254 nm. This gas used. As sterilizing gases are generally toxic to humans, method is used for products which are resistant to ultraviolet full consideration is required of the environmental control rays, such as smooth-surfaced articles, facilities, and equip- for the use of gases and the concentration of residual gas. In ment, or water and air. This method does not suŠer from the some of the gas methods, it may be di‹cult to measure or occurrence of resistance, which is observed in chemical disin- estimate quantitatively the killing of microorganisms. fection, and shows a killing eŠect on bacteria, fungi, and 2-4. Filtration method viruses. It must be taken into consideration that direct ultrav- Microorganisms are removed by ˆltration with a suitable iolet irradiation of the human body can injure the eyes and ˆltering device. This method is generally used for gas, water, skin. or culture media and test solutions containing a substance that is water-soluble and unstable to heat. As a rule, a ˆlter 2. Sterilization methods having a pore size of 0.22 mmorsmallerisusedforthesterili- 2-1. Heating methods zation. However, in this method, a ˆlter with a pore size of In these methods, the heating time before the temperature 0.45 mmorsmallerispermittedtobeused. or pressure reaches the prescribed value diŠers 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 product have reached the prescribed temperature. (i) Moist heat method Microorganisms are killed in saturated steam at a suitable 1682 Guideline for Residual Solvents, / General Information JP XV

2) Models for operating conditions for a head-space sample 7. Guideline for Residual Solvents, injection device Operating conditions (1) for the head-space sample injection Residual Solvents Test, and Models device— for the Test in Monographs Equilibration temperature for A constant tempera- inside vial ture of about 809C 1. Guideline for Residual Solvents Equilibration time for inside vial 60 minutes Refer to the Guideline for Residual Solvents in Phar- Transfer-line temperature A constant tempera- maceuticals (PABWELD Notiˆcation No.307; dated March ture of about 859C 30, 1998). Carrier gas Nitrogen The acceptable limits of residual solvents recommended in Pressurisation time 30 seconds the Guideline were estimated to keep the safety of patients. Injection volume of sample 1.0 mL The levels of residual solvents in pharmaceuticals should not Operating conditions (2) for the head-space sample injection exceed the limits, except for in a special case. Accordingly, device— pharmaceutical manufacturers should assure the quality of Equilibration temperature for A constant tempera- their products by performing the test with the products inside vial ture of about 1059C according to the Residual Solvents Test, to keep the limits Equilibration time for inside vial 45 minutes recommended in the Guideline. Transfer-line temperature A constant tempera- ture of about 1109C 2. Residual Solvents Test Carrier gas Nitrogen Method—Perform the test as directed in the Residual Sol- Pressurisation time 30 seconds vents Test under the General Tests, Processes and Apparatus. Injection volume of sample 1.0 mL International harmonization—The test may also be per- Operating conditions (3) for the head-space sample injection formed according to the Residual solvents in the EP or the device— Organic volatile impurities in the USP. Even in this case, Equilibration temperature for A constant tempera- Monographs should be described in the JP style. inside vial ture of about 809C 3. Models for the Test in Monographs Equilibration time for inside vial 45 minutes The following are typical examples for the test in Mono- Transfer-line temperature A constant tempera- graphs, but these do not necessarily imply that other suitable ture of about 1059C operating conditions can not be used. It is important to pre- Carrier gas Nitrogen pare (draft) monographs according to the Guideline for the Pressurisation time 30 seconds Preparation of the Japanese Pharmacopoeia. Injection volume of sample 1.0 mL 1) A model for test item, amounts of test sample and refer- 3) Models for operating conditions and system suitability ence standard (reference substance), preparation of the sam- In the operating conditions, generally, items required for ple solution and the standard solution, injection amount for the test such as detector, column, column temperature, carri- gas chromatography, calculation formula, and preparation er gas, ‰ow rate, and time span of measurement should be of the internal standard solution speciˆed, and in the system suitability, items such as test for Residual solvents (or name of the solvent)—Weigh ac- required detectability, system performance, and system curately about 0.200 g of △△△ (name of the substance to be repeatability should be speciˆed. tested), add exactly 5 mL of the internal standard solution to The following are several models for the operating condi- dissolve, add water to make exactly 20 mL, and use this solu- tions and the system suitability: tion as the sample solution. If necessary ˆlter or centrifuge. Operating conditions— Separately, weigh exactly 0.10 g of ◯◯◯ reference sub- Detector: Specify in the following manner: ``Hydrogen stance (name of the solvent), put in a vessel containing 50 mL ‰ame-ionization detector''. of water, and add water to make exactly 100 mL. Pipet 5 mL Column: Specify the inside diameter, length, material of of this solution, and add water to make exactly 100 mL. Pipet column, name of stationary phase liquid and thickness of sta- 2 mL of this solution, add exactly 5 mL of the internal stan- tionary phase in the following manner: ``Coat the inside wall dard solution and 20 mL of water, and use this solution as the of a fused silica tube, 0.3 mm in inside diameter and 30 m in standard solution. Perform the test with 1 mLeachofthe length, to 0.25 mm thickness with polyethylene glycol 20M.'' sample solution and standard solution as directed (in the Describe the inside diameter and length of the column, and head-space method) under Gas Chromatography according thicknessorparticlesizeofthestationaryphasebasedonthe to the following conditions, and calculate the ratios, QT and data obtained for validation of the test method. QS, of the peak area of △△△ (name of the substance to be Column temperature: Specify in the following manner: ``A tested) to that of the internal standard of each solution, constant temperature of about ×9C.'' or ``Maintain at 409C respectively. The amount of △△△ should be not more than for 20 minutes, then increase to 2409Cat109Cperminute, ×× ppm. and keep at 2409C for 20 minutes.'' Amount of ◯◯◯ (mg) Carrier gas: Specify in the following manner: ``Helium''. Q ＝amount of ◯◯◯ reference substance (mg)× T Flow rate: Specify in the following manner: ``Adjust the QS ‰ow rate so that the retention time of ▲▲▲ is about × Internal standard solution—A solution of △△△ in ▽▽▽ minutes.'' or ``35 cmWsecond''. Describe the ‰ow rate based (name of solvent) (1 in 1000). on the data obtained for validation of the test method. Time span of measurement: About × timesaslongasthe JP XV General Information / Guideline for Residual Solvents, 1683 retention time of △△△ beginning after the air peak. the relative standard deviation of the peak areas of the sub- System suitability— stance to be tested is not more than 15z. Test for required detectability: Specify in the following Test conditions (2) manner: ``Measure exactly × mL of the standard solution, Operating conditions— and add □□□ to make exactly × mL. Conˆrm that the Detector: Hydrogen ‰ame-ionization detector peak area of □□□ obtained from × mL of this solution is Column: Coat the inside wall of a fused silica tube, 0.53 equivalent to × to ××z of that of □□□ obtained from mm in inside diameter and 30 m in length, to 5 mm thickness the standard solution''. This item should be speciˆed when, with 5z phenyl-methyl silicon polymer for gas chro- in Purity, the amount of an impurity is controlled by compar- matography. If necessary use a guard column prepared by ing the area of a speciˆc peak from the sample solution with coating the inside wall of a fused silica tube, 0.53 mm in in- that of the peak of ×××× from the standard solution and side diameter and 5 m in length, to 5 mm thickness with 5z the system repeatability alone is not su‹cient to check the phenyl-methyl silicon polymer for gas chromatography. system suitability. Column temperature: Maintain at 359Cfor5minutes, System performance: Specify in the following manner: then increase to 1759Cat89C per minute, further increase to ``Dissolve × gof□□□ and × gof△△△ in × mL of ◯◯ 2609Cat359C per minute if necessary, and keep at 2609Cfor ◯. When the procedure is run with × mL of this solution un- 16 minutes. der the above operating conditions, □□□ and △△△ are Injection port temperature: A constant temperature of eluted in this order with the resolution between these peaks about 709C being not less than ×, and the number of theoretical plates Detector temperature: A constant temperature of about and the symmetry factor of the peak of □□□ are not less 2609C than ×× steps and not more than ×××, respectively.'' Carrier gas: Helium This item should be speciˆed in all of the test methods. Flow rate: 35 cmWsecond Generally, the order of elution and the resolution, and in case Split ratio: Splitless of need (such as when the peak is asymmetrical) the symmet- System suitability— ry factor, should be speciˆed. The order of elution and the System performance: When the procedure is run with the resolution may be replaced by the resolution and the number standard solution under the above operating conditions, the of theoretical plates. When no suitable reference substance is resolution between the peaks is not less than 1.0. (Note: In available to check separation, the number of theoretical the case that the number of substances to be tested is two or plates and the symmetry factor of the peak of the substance more.) to be tested may be speciˆed. System repeatability: When the test is repeated 3 times with System repeatability: Specify in the following manner: the standard solution under the above operating conditions, ``When the test is repeated × times with × mLofthesolution the relative standard deviation of the peak areas of the sub- for the system suitability test under the above operating con- stance to be tested is not more than 15z. ditions, the relative standard deviation of the peak areas of △△△ is not more than ×z.'' The system repeatability Test conditions (3) should be speciˆed in any case, except in a qualitative test. Operating conditions— Detector: Hydrogen ‰ame-ionization detector Examples of operating conditions and system suitability Column: Coat the inside wall of a fused silica tube, 0.32 Test conditions (1) mm in inside diameter and 30 m in length, to 0.25 mmthick- Operating conditions— ness with polyethylene glycol 20M for gas chromatography. Detector: Hydrogen ‰ame-ionization detector Use a guard column if necessary. Column: Coat the inside wall of a fused silica tube, 0.53 Column temperature: Maintain at 509C for 20 minutes, mm in inside diameter and 30 m in length, to 3 mm thickness then increase to 1659Cat69C per minute if necessary, and with 6z cyanopropylphenyl-methyl silicon polymer for gas keep at 1659Cfor20minutes. chromatography. Use a guard column if necessary. Injection port temperature: A constant temperature of Column temperature: Maintain at 409C for 20 minutes, about 1409C then increase to 2409Cat109C per minute if necessary, and Detector temperature: A constant temperature of about keep at 2409Cfor20minutes. 2509C Injection port temperature: A constant temperature of Carrier gas: Helium about 1409C Flow rate: 35 cmWsecond Detector temperature: A constant temperature of about Split ratio: 1:5 2509C System suitability— Carrier gas: Helium System performance: When the procedure is run with the Flow rate: 35 cmWsecond standard solution under the above operating conditions, the Split ratio: 1:5 resolution between the peaks is not less than 1.0. (Note: In System suitability— the case that the number of substances to be tested is two or System performance: When the procedure is run with the more.) standard solution under the above operating conditions, the System repeatability: When the test is repeated 3 times with resolution between the peaks is not less than 1.0. (Note: In the standard solution under the above operating conditions, the case that the number of substances to be tested is two or the relative standard deviation of the peak areas of the sub- more.) stance to be tested is not more than 15z. System repeatability: When the test is repeated 3 times with the standard solution under the above operating conditions, 1684 International Harmonization / General Information JP XV

shown in the tables below. The column headed Harmonized items shows the har- 8. International Harmonization monized items written in the Pharmacopoeial Harmonization Implemented in the Japanese Agreement Document, and the column headed JP 15 shows the items as they appear in JP 15. In the Remarks column, Pharmacopoeia Fifteenth Edition notes on any diŠerences between JP 15 and the agreement are shown as occasion demands. Items for which harmonization has been agreed among the The date on which the agreement has been signed is shown European Pharmacopoeia, the Pharmacopeia on the top pf each table. In the case where the harmonized i- and the Japanese Pharmacopoeia are implemented in the tems have been revised, this is indicated in parenthesis. Japanese Pharmacopoeia Fifteenth Edition (JP 15). They are

Oct. 2004 (Rev. 2)

Harmonized items JP 15 Remarks

Residue on IgnitionWSulphated Ash 2.44 Residue on Ignition Test Test (Introduction) (Introduction) JP's particular description: Explana- tion of the description in mongraph, etc. Procedure Procedure

Nov. 2003

Harmonized items JP 15 Remarks

Specific Surface Area 3.02 Specific Surface Area (Introduction) (Introduction) JP's particular description: Explana- tion on this test. Multi-point measurement Multi-point measurement Single-point measurement Single-point measurement Sample preparation Sample preparation Outgassing Adsorbate Quantity of sample Method 1: The dynamic flow method Method 1: The dynamic flow method Method 2: The volumetric method Method 2: The volumetric method Reference materilals Reference materilals Figure 1 Schematic diagram of the Fig. 3.02-1 Schematic diagram of the dynamic flow method apparatus dynamic flow method apparatus Figure 2 Schematic diagram of the Fig. 3.02-2 Schematic diagram of the volumetric method apparatus volumetric method apparatus JP XV General Information / Isoelectric Focusing 1685

June 2004

Harmonized items JP 15 Remarks

3.04 Particle Size Determination (Introduction) (Introduction) JP's particular description: Explana- tion on this test.

Optical microscopy Method 1. Optical microscopy JP's particular description: Explana- tion on this test. Apparatus Apparatus Adjustment Adjustment Illumination Illumination Visual characterization Visual characterization JP's particular description: Explana- tion on the method of particle size measurement. Photographic characterization Photographic characterization Preparation of the mount Preparation of the mount Crystallinity characterization Crystallinity characterization Limit test of particle size by Limit test of particle size by microscopy microscopy Particle size characterization Particle size characterization Particle shape characterization Particle shape characterization General observations General observations Figure 1 Commonly used measure- Fig. 3.04-1 Commonly used measure- ments of particle size ments of particle size Figure2Commonlyuseddescriptions Fig. 3.04-2 Commonly used descrip- of particle shape tions of particle shape Analytical sieving Method 2. Analytical sieving method JP's particular description: Explana- tion on this method. Principles of analytical sieving Principles of analytical sieving Test sieves Test sieves Test specimen Test specimen Agitation methods Agitation methods Endpoint determination Endpoint determination Sieving methods Sieving methods 1) Methanical agitation Methanical agitation JP's particular description: How to dry sieving method Dry sieving method treat the fine powder adherent on back surface of the sieve. 2) Air entrainment methods Air entrainment methods air jet and sonic sifter sieving Air jet and sonic shifter sieving Table 1 Size of standard sieve series in Table 3.04-1 Size of standard sieve range of interest series in range of interest Interpretation Interpretation 1686 Isoelectric Focusing / General Information JP XV

Jan. 2000

Harmonized items JP 15 Remarks

Bacterial Endotoxins Test 4.01 Bacterial Endotoxins Test (Introduction) (Introduction) Apparatus Apparatus Preparation of standard endotoxin Preparation of standard endotoxin JP's particular description: Endotoxin stock solution stock solution 10000 Reference Standard and En- dotoxin 100 Reference Standard. Preparation of standard endotoxin so- Preparation of standard endotoxin so- lution lution Preparation of sample solutions Preparation of sample solutions JP's particular description: Addition of the preparation of sample solutions for containers, and deletion of that for medical devices. Determination of maximum valid di- Determination of maximum valid di- JP's particular description: Addition lution lution of the concentration unit of sample so- lutions in the case where the endotoxin limit per equivalent is specified. Gel-clot technique Gel-clot technique (1) Preparatory testing (1) Preparatory testing (i) Test for confirmation of labeled (i) Test for confirmation of labeled lysate sensitivity lysate sensitivity (ii) Test for interfering factors (ii) Test for interfering factors (2) Limit test (2) Limit test (i) Procedure (i) Procedure (ii) Interpretation (ii) Interpretation (3) Assay (3) Assay (i) Procedure (i) Procedure (ii) Calculation and interpretation (ii) Calculation and interpretation Photometric techniques Photometric techniques (1) Turbidimetric techniques (1) Turbidimetric techniques (2) Chromogenic technique (2) Chromogenic technique (3) Preparatory testing (3) Preparatory testing (i) Assurance of criteria for the (i) Assurance of criteria for the JP's particular description: The test for standard curve standard curve assurance of criteria for the standard curve must be carried out for each lot of lysate reagent. (ii) Test for interfering factors (ii) Test for interfering factors JP's particular description: Two conditions which the test must meet are specified. Explanation of the test method where the interfering action is found. Explanation of the usual methods for eliminating the interference. (4) Assay (4) Assay (i) Procedure (i) Procedure (ii) Calculation (ii) Calculation JP's particular description: Addition of the alternative requirement to which solution D must meet for valid test. (iii) Interpretation (iii) Interpretation JP XV General Information / Isoelectric Focusing 1687

Regents, test solutions Deletion Amebocyte lysate Deletion Lysate TS Deletion Water for bacterial endotoxins test Deletion (BET)

Oct. 2002

Harmonized items JP 15 Remarks

Sterility 4.06 Sterility Test (Introduction) (Introduction) Precautions against microbial con- Thesamestatementappearsinthein- tamination troduction. Culture media and incubation temper- Media and rinsing ‰uids atures Fluid thioglycollate medium Fluid thioglycolate medium Note 1 – 5 Soya- digest medium -casein digest medium Sterility Sterility of media Growth promotion test of aerobes, Growth promotion test Note 6 anaerobes and fungi Validation test Validation test Membrane ˆltration Membrane ˆltration Direct inoculation Direct inoculation Test for sterility of the product to be Test for sterility of the products to be examined examined Membrane ˆltration Membrane ˆltration Note 7 Aqueous solutions a) Liquid medicines Soluble solids b) Solid medicines Oils and oily solutions c) Oils and oily solutions Ointments and creams d) Ointments and creams Direct inoculation of the culture medi- Direct inoculation of the culture medi- um um Oily liquids a) Oily liquids Ointments and creams b) Ointments and creams Catgut and other surgical sutures for The items not included in JP. veterinary use Observation and interpretation of Cultivation and observation, Observa- Note8–9 results tion and interpretation of results Application of the test to parenteral A part of this is directed in the General preparations, ophthalmic and other Test. non-injectable preparations required to comply with the test for sterility Table 2.6.1.-1. Strains of the test Table 4.06-1. Microorganisms for micro-organisms suitable for use in growth promotion test and the vali- the Growth Promotion Test and the dation test Validation Test Table 2.6.1.-2. Minimum quantity Table 4.06-3. Minimum quantity to to be used for each medium be used for each medium 1688 Isoelectric Focusing / General Information JP XV

Table 2.6.1.-3. Minimum number of Table 4.06-2. Number of items to be Note10–11 items to be tested taken from the lot

Note: 1) Non-pharmaceutical media: Not to be used. 2) Water content of agar: Not being speciˆed. 3) EŠective period of media: Unnecessary to be validated. 4) EŠective period of media stored in hermetic containers: Usable for maximum one year. 5) Medium for sterility test of the products containing a mercurial preservative: Speciˆed. 6) Periodic growth promotion test for a ready-made powder medium: Speciˆed. 7) Amount of the rinsing ‰uid each time in the membrane-ˆltration method: 100 mL per ˆlter. 8) Transferring amount from turbid medium to fresh medium: A suitable amount. 9) Requirements for the retesting in the case when the evidence of microbial growth is found: Speciˆed. 10) The table of 'Number of items to be taken from the lot': Speciˆed as a part of the General Test. 11) Number of large-volume products (more than 100 mL) to be taken from the lot: Maximum 10 containers.

Feb. 2004

Harmonized items JP 15 Remarks

Uniformity of Dosage Units 6.02 Uniformity of Dosage Units (Introduction) (Introduction) JP's particular description: Additional explanation on Liquids. Additional explanation for the part not containing drug substance. Content uniformity Content uniformity Solid dosage forms Solid dosage forms Liquid dosage forms Liquid dosage forms Calculation of acceptance value Calculation of acceptance value Mass variation Mass variation JP's particular description: Assuming that the concentration of drug sub- stance is uniform in each lot. Uncoated or film-coated tablets Uncoated or film-coated tablets Hard capsules Hard capsules Soft capsules Soft capsules Solid dosage forms other than tablets Solid dosage forms other than tablets and capsules and capsules Liquid dosage forms Liquid dosage forms The phrase ``in conditions of normal use. If necessary, compute the equiva- lent volume after determining the den- sity.'' is deleted. Calculation of acceptance value Calculation of acceptance value Criteria Criteria Solid and liquid dosage forms Solid and liquid dosage forms Table 1 Table 6.02-1 JP's particular description: Application of content uniformity Application of content uniformity Addition of ``(divided forms, lyophi- (CU) and mas variation (MV) test for (CU) and mas variation (MV) test for lized forms)'' and ``(true solution)''. dosage forms dosage forms Table 2 Table 6.02-2 The phrases ``at time of manufacture'' and ``For purposes of this Phar- macopoeia'' are deleted JP XV General Information / Isoelectric Focusing 1689

June 2004 (Rev.1)

Harmonized items JP 15 Remarks

Test for Extractable Volume of Paren- 6.05 Test for Extractable Volume of teral Preparations Parenteral Preparations (Introduction) (Introduction) JP's particular description: Explana- tion of this Test Single-dose containers (1) Single-dose containers Multi-dose containers (2) Multi-dose containers Cartridges and prefilled syringes (3) Cartridges and prefilled syringes Parenteral infusions (4) Parenteral infusions

June 2004 (Rev.1)

Harmonized items JP 15 Remarks

Particulate Matter in Injectables 6.07 Insoluble Particulate Matter Test for Injections (Introduction) (Introduction) The phrase ``other than gas bubbles'' is deleted. Method 1. Method 1. Light obscuration particle count test Light obscuration particle count test JP's particular description: Description of evaluation frequency. Apparatus Calibration JP's particular description Manual methods JP's particular description Electronic methods JP's particular description Automated methods JP's particular description Sample volume accuracy JP's particular description Sample flow rate JP's particular description Sensor JP's particular description Particle resolution JP's particular description Particle counting accuracy JP's particular description Threshold accuracy JP's particular description Reagents JP's particular description General precautions General precautions Method Method Evaluation Evaluation JP's particular description: ``equal to or more than 100 mL''. Method 2. Method 2. Microscopic particle count test Microscopic particle count test Apparatus General precautions General precautions Method Method Evaluation Evaluation JP's particular description: ``equal to or more than 100 mL''. 1. Circular diameter graticule Fig. 6.07-1 Circular diameter graticule 1690 Isoelectric Focusing / General Information JP XV

June 2004

Harmonized items JP 15 Remarks

Disintegration 6.09 Disintegration JP's particular description: This test is appliedalsotoGranulesandPills. Apparatus Apparatus Basket-rack assembly Basket-rack assembly JP's particular description: The ap- paratus may be varied somewhat provided the specifications. Disk Disk Auxiliary tube JP's particular description Procedure Procedure JP's particular description: Making use of water also as the test fluid. Setting each intervals of the immersion. Making a definition of complete disin- tegration. Setting a procedure for Granules. (1) Immediate-release preparation JP's particular description: Setting a test for Granules and Pills.

(2) Enteric coated preparations JP's particular description: Setting a test for enteric coated prepa- rations. Figure 1 Disintegration apparatus Fig. 6.09-1 Disintegration apparatus Fig. 6.09-2 Auxiliary tube JP's particular description.

June 2004

Harmonized items JP 15 Remarks

Dissolution 6.10 Dissolution Test JP's particular description: The test also aims at preventing significant bioinequivalence. Apparatus Apparatus Apparatus 1 (Basket apparatus) Apparatus for Basket Method (Apparatus 1) Apparatus 2 (Paddle apparatus) Apparatus for Paddle Method JP's particular description: The sinker (Apparatus 2) is allowed to use in case only when spe- cified in the monograph. Apparatus 3 (Reciprocating cylinder) Apparatus 4 (Flow-through cell) Apparatus for Flow-Through Cell JP's particular description: A pump Method (Apparatus 3) without the pulsation may also be used. Procedure Procedure Apparatus 1 or 2 Basket Method or Paddle Method Immediate-release dosage forms Immediate-release dosage forms Procedure Procedure Dissolution medium Dissolution medium Time Time Extended-release dosage forms Extended-release dosage forms Procedure Procedure Dissolution medium Dissolution medium Time Time JP XV General Information / Isoelectric Focusing 1691

Delayed-release dosage forms Delayed-release dosage forms Procedure Procedure In the Harmonized text: Alternative usage of Method A or B. Method A Method B Dissolution medium JP's particular description. Time Time JP's particular description: Time is specified each for the 1st and 2nd fluids. Apparatus 3 not specified Immediate-release dosage forms Procedure Dissolution medium Time Extended-release dosage forms Procedure Dissolution medium Time Delayed-release dosage forms Procedure Time Apparatus 4 Flow-Through Cell Method Immediate-release dosage forms Immediate-release dosage forms Procedure Procedure

Dissolution medium Dissolution medium Time Time Extended-release dosage forms Extended-release dosage forms Procedure Procedure Dissolution medium Dissolution medium Time Time Delayed-release dosage forms Procedure Time Interpretation Interpretation JP's particular description: Follow In- terpretation 1 when the value Q is spe- cified in the individual monograph, otherwise follow Interpretation 2. Immediate-release dosage forms Immediate-release dosage forms JP's particular description: Setting Interpretation 2. Interpretation 1 Interpretation 2 Extended-release dosage forms Extended-release dosage forms JP's particular description: Setting Interpretation 2. Interpretation 1 Interpretation 2 Delayed-release dosage forms Delayed-release dosage forms Non-harmonized items: Different dis- solution medium. Deletion of dishar- monized part on the value Q. 1692 Isoelectric Focusing / General Information JP XV

JP's particular description: Setting In- terpretation 2. Interpretation 1 The value Q is specified in the individual monograph. Interpretation 2 Acceptance Table 1 Acceptance Table 6.10-1 Acceptance Table 2 Acceptance Table 6.10-2 Acceptance Table 3 Acceptance Table 6.10-3 Acceptance Table 4 Acceptance Table 6.10-4 Figure 1 Apparatus 1. Basket stirring Fig. 6.10-1 Apparatus 1. Basket stir- element ring element Figure 2 Paddle stirring element Fig. 6.10-2 Paddle stirring element Figure 2 a Alternative sinker Fig. 6.10-2 a Alternative sinker Figure 3 Apparatus not specified Figure 4 Apparatus 4 Fig. 6.10-3 Apparatus 3 (top) large cell tablets and capsules (top) large cell tablets and capsules (bottom) holder for the large (bottom) tablet holder for the large cell cell Figure 5 Apparatus 4 Fig. 6.10-4 Apparatus 3 (top) small cell tablets and capsules (top) small cell tablets and capsules (bottom) tablet holder for the small (bottom) tablet holder for the small cell cell

Sep. 2002 (Rev. 1)

Harmonized items JP 15 Remarks

Ethanol Ethanol Identification A not specified as Identification Setting Specific gravity as specification. Identification B Identification Relative density Specific gravity Setting Specific gravity at 159C. Appearance Purity (1) Clarity and color of solu- tion Acidity or alkalinity Purity (2) Acid or alkali Volatile impurities Purity (3) Volatile impurities Absorbance Purity (4) Other impurities Residue on evaporation Purity (5) Residue on evaporation Storage Containers and storage

Sep. 2002 (Rev. 1)

Harmonized items JP 15 Remarks

Ethanol, Anhydrous Anhydrous Ethanol Definition limits of content

Identification A not specified as Identification Setting Specific gravity as specification. Identification B Identification Relative density Specific gravity Setting Specific gravity at 159C. JP XV General Information / Isoelectric Focusing 1693

Appearance Purity (1) Clarity and color of solu- tion Acidity or alkalinity Purity (2) Acid or alkali Volatile impurities Purity (3) Volatile impurities Absorbance Purity (4) Other impurities Residue on evaporation Purity (5) Residue on evaporation Storage Containers and storage

Nov. 2003 (Rev. 2)

Harmonized items JP 15 Remarks

Sodium Chloride Sodium Chloride Deˆnition limits of the content Identiˆcation A Identiˆcation (1) Identiˆcation B Identiˆcation (2) Acidity or alkalinity Purity (2) Acidity or alkalinity Sulphates Purity (3) Purity (4) Phosphates Purity (5) Bromides Iodides Purity (6) Iodides Ferrocyanides Purity (7) Ferrocyanides Purity (9) Iron Purity (10) Barium and alkaline-earth metals Purity (11) Magnesium and alkaline- earth materials not speciˆed not speciˆed not speciˆed Loss on drying Loss on drying Assay Assay

July 2003 (Rev. 1)

Harmonized items JP 15 Remarks

Carboxymethylcellulose Carmellose Calcium Deˆnition origin Identiˆcation A Identiˆcation (1) Identiˆcation B Identiˆcation (2) Identiˆcation C Identiˆcation (3) Identiˆcation D Identiˆcation (4) Alkalinity Purity (1) Alkali Loss on drying Loss on drying Residue on ignition Residue on ignition Limit of chloride Purity (2) Chloride Limit of sulfate Purity (3) Sulfate 1694 Isoelectric Focusing / General Information JP XV

Nov. 2003 (Rev. 1)

Harmonized items JP 15 Remarks

Citric Acid, Anhydrous Anhydrous Deˆnition limits of the content Appearance of solution Purity (1) Clarity and color of solu- tion Sulphates Purity (2) Sulfates Oxalic acid Purity (3) Oxalate Readily carbonisable substances Purity (5) Readily carbonizable sub- stances Aluminium not speciˆed Water Water Sulphated ash Residue on ignition Assay Assay

Nov. 2003 (Rev. 1)

Harmonized items JP 15 Remarks

Citric Acid Monohydrate Citric Acid Hydrate Deˆnition limits of the content Appearance of solution Purity (1) Clarity and color of solu- tion Sulphates Purity (2) Sulfates Oxalic acid Purity (3) Oxalate Readily carbonisable substances Purity (5) Readily carbonizable sub- stances Aluminium not speciˆed Water Water Sulphated ash Residue on ignition Assay Assay

Oct. 2001

Harmonized items JP 15 Remarks

Croscarmellose Sodium Definition Croscarmellose Sodium origin Identification A Identification (1) B Identification (2) C Identification (3) pH pH Settling volume Precipitation test Degree of substitution Degree of substitution Loss on drying Residue on ignition Loss on drying Residue on ignition Packaging and storage Containers and storate JP XV General Information / Isoelectric Focusing 1695

Oct. 2001

Harmonized items JP 15 Remarks

Wheat Starch Starch Deˆnition origin Identiˆcation A Identiˆcation (1) Identiˆcation B Identiˆcation (2) Identiˆcation C Identiˆcation (3) pH pH Iron Purity (1) Iron Total protein not speciˆed Oxidising substances Purity (2) Oxidizing substances Sulphur dioxide Purity (3) dioxide Loss on drying Loss on drying Sulphated ash Residue on ignition

Feb. 2003

Harmonized items JP 15 Remarks

Saccharin Saccharin Definition limits of content Limit of benzoate and salicylate Purity (3) Benzoate and salicylate Readily carbonisable substances Purity (5) Readily carbonizable sub- stances Loss on drying Loss on drying Residue on ignition Residue on ignition Assay Assay Packaging and storage Containers and storage

Feb. 2004 (Rev. 1)

Harmonized items JP 15 Remarks

Saccharin Sodium Saccharin Sodium Hydrate Definition limits of content Identification A, B Identification (2) Acidity or alkalinity Purity (2) Acid or alkali Limit of benzoate and salicylate Purity (4) Benzoate and salicylate Readily carbonisable substances Purity (6) Readily carbonizable sub- stances Water Water Assay Assay 1696 Isoelectric Focusing / General Information JP XV

Oct. 2001

Harmonized items JP 15 Remarks

Cellacefate Cellulose Acetate Deˆnition content of the acetyl and carboxyben- zoyl groups Identiˆcation Identiˆcation Viscosity Viscosity Limit of free acid Purity (2) Free acids Water Water Residue on ignition Residue on ignition Phthalyl content Assay (1) Carboxybenzoyl group Content of acetyl Assay (2) Packaging and storage Containers and storage

May 2005 (Rev. 1)

Harmonized items JP 15 Remarks

Microcrystalline Cellulose Microcrystalline Cellulose Definition origin Identification A Identification (1) Identification B Identification (3) pH pH Water-soluble substances Purity (2) Water- soluble substances Ether-soluble substances Purity(3) -soluble sub- stances Conductivity Conductivity Loss on drying Loss on drying Residue on ignition Residue on ignition Bulk density Bulk density

May 2005 (Rev. 1)

Harmonized items JP 15 Remarks

Powdered Cellulose Powdered Cellulose Definition origin Identification A Identification (1) Identification B Identification (3) pH pH Water-soluble substances Purity (2) Water-soluble substances Ether-soluble substances Purity (3) Diethyl ether- soluble sub- stances Loss on drying Loss on drying Residue on ignition Residue on ignition JP XV General Information / Isoelectric Focusing 1697

Feb. 2004 (Rev. 1)

Harmonized items JP 15 Remarks

Corn Starch Corn Starch Deˆnition origin Identiˆcation A Identiˆcation (1) Identiˆcation B Identiˆcation (2) Identiˆcation C Identiˆcation (3) pH pH Loss on drying Loss on drying Residue on ignition Residue on ignition Limit of iron Purity (1) Iron Limit of oxidizing substances Purity (2) Oxidizing substances Sulfur dioxide determination Purity (3) Sulfur dioxide

Feb. 2003 (Rev. 1)

Harmonized items JP 15 Remarks

Anhydrous Lactose Anhydrous Lactose Definition origin Labeling labeling of relative quantities of a and b lactose Identification Identification Specific rotation Optical rotation Clarity and color of solution Purity (1) Clarity and color of solu- tion Acidity or alkalinity Purity (2) Acid and alkali Protein and light-absorbing impurities Purity (4) Proteins and light absorb- ing substances Loss on drying Loss on drying Water Water Residue on ignition Residue on ignition Content of alpha and beta anomers Isomer ratio

Sep. 2002

Harmonized items JP 15 Remarks

Lactose Monohydrate Lactose Hydrate Definition origin Identification Identification Specific rotation Specific rotation Clarity and color of solution Purity (1) Clarity and color of solu- tion Acidity or alkalinity Purity (2) Acid and alkali Protein and light-absorbing impurities Purity (4) Protein and light absorbing substances Water Water Residue on ignition Residue on ignition 1698 Isoelectric Focusing / General Information JP XV

Feb. 2004

Harmonized items JP 15 Remarks

Ethyl Parahydroxybenzoate Ethyl Parahydroxybenzoate Definition limits of content Identification A Identification (1) Appearance of solution Purity (1) Clarity and color of solu- tion Acidity Purity (2) Acid Related substances Purity (4) Related substances System suitability is not specified. Sulphated ash Residue on ignition Assay Assay

Feb. 2004

Harmonized items JP 15 Remarks

Butyl Parahydroxybenzoate Butyl Parahydroxybenzoate Definition limits of content Identification A Identification Appearance of solution Purity (1) Clarity and color of solu- tion Acidity Purity (2) Acid Related substances Purity (4) Related substances System suitability is not specified. Sulphated ash Residue on ignition Assay Assay

Feb. 2004

Harmonized items JP 15 Remarks

Propyl Parahydroxybenzoate Propyl Parahydroxybenzoate Definition limits of content Identification A Identification (1) Appearance of solution Purity (1) Clarity and color of solu- tion Acidity Purity (2) Acid Related substances Purity (4) Related substances System suitability is not specified. Sulphated ash Residue on ignition Assay Assay

Feb. 2004

Harmonized items JP 15 Remarks

Methyl Parahydroxybenzoate Methyl Parahydroxybenzoate Definition limits of content Identification A Identification (1) Appearance of solution Purity (1) Clarity and color of solu- tion JP XV General Information / Isoelectric Focusing 1699

Acidity Purity (2) Acid Related substances Purity (4) Related substances System suitability is not specified. Sulphated ash Residue on ignition Assay Assay

Oct. 2001

Harmonized items JP 15 Remarks

Potato Starch Potato Starch Deˆnition origin Identiˆcation A Identiˆcation (1) Identiˆcation B Identiˆcation (2) Identiˆcation C Identiˆcation (3) pH pH Iron Purity (1) Iron Oxidising substances Purity (2) Oxidizing substances Sulphur dioxide Purity (3) Sulfur dioxide Loss on drying Loss on drying Sulphated ash Residue on ignition

Nov. 2003

Harmonized items JP 15 Remarks

Hydroxypropyl Methylcellulose Hypromellose Definition limits of content of methoxy group and hydroxypropoxy group Labeling labeling of viscosity Identification (1) Identification (1) Identification (2) Identification (2) Identification (3) Identification (3) Identification (4) Identification (4) Identification (5) Identification (5) Viscosity Viscosity Method 1 Method I Method 2 Method II pH pH Heavy metals Purity Heavy metals Loss on drying Loss on drying Residue on ignition Residue on ignition Assay Assay 1700 Isoelectric Focusing / General Information JP XV

July, 2000

Harmonized items JP 15 Remarks

Benzyl Alcohol Benzyl Alcohol Deˆnition limits of the content Identiˆcation Identiˆcation Appearance of solution Purity (1) Clarity and color of solu- tion Refractive index Acidity Purity (2) Acid Benzaldehyde and other related Purity (3) Benzaldehyde and other substances related substances Peroxide value Purity (4) Peroxide value Residue on evaporation Purity (5) Residue on evaporation Assay Assay

Nov. 2003

Harmonized items JP 15 Remarks

Methylcellulose Methylcellulose Definition limits of content of methoxy group Labeling labeling of viscosity Identification (1) Identification (1) Identification (2) Identification (2) Identification (3) Identification (3) Identification (4) Identification (4) Identification (5) Identification (5) Viscosity Viscosity Method 1 Method I Method 2 Method II pH pH Heavy metals Purity Heavy metals Loss on drying Loss on drying Residue on ignition Residue on ignition Assay Assay

Sep. 2002

Harmonized items JP 15 Remarks

General Information Amino Acid Analysis Amino Acid Analysis Apparatus Apparatus General Precautions General Precautions Reference Standard Material Reference Standard Material Calibration of Instrumentation Calibration of Instrumentation Repeatability Repeatability Sample Preparation Sample Preparation Internal Standards Internal Standards JP XV General Information / Isoelectric Focusing 1701

Protein Hydrolysis Protein Hydrolysis Method 1 Method 1 Hydrolysis Solution Hydrolysis Solution Procedure Procedure Method 2 Method 2 Hydrolysis Solution Hydrolysis Solution Vapor Phase Hydrolysis Vapor Phase Hydrolysis Method 3 Method 3 Hydrolysis Solution Hydrolysis Solution Vapor Phase Hydrolysis Vapor Phase Hydrolysis Method 4 Method 4 Oxidation Solution Oxidation Solution Procedure Procedure Method 5 Method 5 Hydrolysis Solution Hydrolysis Solution Liquid Phase Hydrolysis Liquid Phase Hydrolysis Method 6 Method 6 Hydrolysis Solution Hydrolysis Solution Vapor Phase Hydrolysis Vapor Phase Hydrolysis Method 7 Method 7 Reducing Solution Reducing Solution Procedure Procedure Method 8 Method 8 Stock Solutions Stock Solutions Reducing Solution Reducing Solution Procedure Procedure Method 9 Method 9 Stock Solutions Stock Solutions Carboxymethylation Solution Carboxymethylation Solution BuŠer Solution BuŠer Solution Procedure Procedure Method 10 Method 10 Reducing Solution Reducing Solution Procedure Procedure Method 11 Method 11 Reducing Solutions Reducing Solutions Procedure Procedure Methodologies of amino acid analysis Methodologies of Amino Acid Analy- general principles sis General Principles Method 1-Postcolumn ninhydrin Method 1-Postcolumn Ninhydrin detection general principle Detection General Principle Method 2-Postcolumn OPA ‰uoro- Method 2-Postcolumn OPA Fluoro- metric detection general principle metric Detection General Principle Method 3-Precolumn PITC derivati- Method 3-Precolumn PITC Derivati- zation general principle zation General Principle Method 4-Precolumn AQC derivati- Method 4-Precolumn AQC Derivati- zation general principle zation General Principle 1702 Isoelectric Focusing / General Information JP XV

Method 5-Precolumn OPA derivati- Method 5-Precolumn OPA Derivati- zation general principle zation General Principle Method 6-Precolumn DABS-Cl Method 6-Precolumn DABS-Cl derivatization general principle Derivatization General Principle Method 7-Precolumn FMOC-Cl Method 7-Precolumn FMOC-Cl derivatization general principle Derivatization General Principle Method 8-Precolumn NBD-F Method 8-Precolumn NBD-F derivatization general principle Derivatization General Principle Data calculation and analysis Data Calculation and Analysis Calculations Calculations Amino acid mole percent Amino Acid Mole Percent Unknown protein samples Unknown Protein Samples Known protein samples Known Protein Samples

Oct. 1999

Harmonized items JP 15 Remarks

General Information Sodium Dodecyl Sulphate Poly- SDS-Polyacrylamide Gel Electrophore- acrylamide Gel Electrophoresis sis (SDS-PAGE) Characteristics of polyacrylamide gels 1. Characteristics of Polyacrylamide Gels Denaturing polyacrylamide gel elec- 2. Polyacrylamide Gel Electrophore- trophoresis sis under Denaturing Conditions Reducing conditions 1) Reducing conditions Non-reducing conditions 2) Non-reducing conditions Characteristics of discontinuous 3. Characteristics of Discontinuous buŠer system gel electrophoresis BuŠer System Gel Electrophoresis Preparing vertical discontinuous 4. Preparing Vertical Discontinuous buŠer SDS polyacrylamide gels BuŠer SDS-Polyacrylamide Gels Assembling of the gel moulding cas- 1) Assembling of the gel moulding sette cassette Preparation of the gel 2) Preparation of the gel Mounting the gel in the electrophore- 3) Mounting the gel in the electro- sis apparatus and electrophoretic phoresis apparatus and electro- separation phoretic separation Detection of protein in gels 5. Detection of Proteins in Gels Coomassie staining 1) Coomassie staining Silver staining 2) Silver staining Drying of stained SDS polyacrylamide 6. Drying of Stained SDS- gels Polyacrylamide Gels Molecular-mass determination 7. Molecular-Mass Determination Validation of the test 8. Suitability of the Test Quantiˆcation of impurities 9. Quantiˆcation of Impurities Reagents, test solutions Test Solutions Blocking solution Blocking TS Coomassie staining solution Coomassie staining TS Destaining solution Destaining TS Developer solution Developer TS JP XV General Information / Isoelectric Focusing 1703

Fixing solution Fixing TS Silver nitrate reagent Silver nitrate TS for silver staining Trichloroacetic acid reagent Trichloroacetic acid TS for ˆxing Table 1 － Preparation of resolving Table 1. Preparation of resolving gel gel Table 2 － Preparation of stacking gel Table 2. Preparation of stacking gel

Sep. 2002

Harmonized items JP 15 Remarks

General Information Capillary Electrophoresis Capillary Electrophoresis Apparatus Apparatus Capillary zone electrophoresis 1. Capillary Zone Electrophoresis Optimisation Optimization Instrumental parameters Instrumental parameters Voltage Voltage Polarity Polarity Temperature Temperature Capillary Capillary Electrolytic solution parameters Electrolytic solution parameters BuŠer type and concentration BuŠer type and concentration BuŠer pH BuŠer pH Organic solvents Organic solvents Additives for chiral separations Additives for chiral separations Capillary gel electrophoresis 2. Capillary Gel Electrophoresis Characteristics of gels Characteristics of Gels Capillary isoelectric focusing 3. Capillary Isoelectric Focusing Loading step Loading step loading in one step loading in one step sequential loading sequential loading Focusing step Focusing step Mobilisation step Mobilization step Optimisation Optimization Voltage Voltage Capillary Capillary Solutions Solutions Micellar electrokinetic chro- 4. Micellar Electrokinetic Chro- matography (MEKC) matography (MEKC) Optimisation Optimization Instrumental parameters Instrumental parameters Voltage Voltage Temperature Temperature Capillary Capillary Electrolytic solution parameters Electrolytic solution parameters Surfactant type and concentration Surfactant type and concentration BuŠer pH BuŠer pH 1704 Isoelectric Focusing / General Information JP XV

Organic solvents Organic solvents Additives for chiral separations Additives for chiral separations Other additives Other additives Quantiˆcation Quantiˆcation Calculations Calculations System Suitability System Suitability Apparent number of theoretical Apparent Number of Theoretical plates Plates Resolution Resolution Symmetry factor Symmetry Factor Signal-to-noise ratio Signal-to-noise Ratio

Feb. 2004

Harmonized items JP 15 Remarks

General Information Tablet Friability Tablet Friability Test

Sep. 2002

Harmonized items JP 15 Remarks

General Information Total Protein Assay Total Protein Assay Method 1 Method 1 Standard Solution Standard Solution Test Solution Test Solution Procedure Procedure Light-scattering Light-Scattering Calculations Calculations Method 2 Method 2 Explanatory footnote ``Example: the Standard solutions Standard Solutions Minimum Requirements for Biological Products and individual monograph of Test solution Test Solution JP'' is added. Blank Blank Reagents and solutions Reagents and Solutions sulfate reagent Copper Sulfate Reagent SDS Solution 5z SDS TS Sodium Hydroxide Solution Sodium Hydroxide Solution Alkaline copper reagent Alkaline Copper Reagent Diluted Folin-Ciocalteu's Phenol Diluted Folin's TS Ragent Procedure Procedure Calculations Calculations Interfering substances Interfering Substances Sodium deoxycholate reagent Sodium Deoxycholate Reagent Trichloroacetic acid reagent Trichloroacetic Acid Reagent Procedure Procedure Method 3 Method 3 Standard solutions Standard Solutions JP XV General Information / Isoelectric Focusing 1705

Test solution Test Solution Blank Blank Coomassie reagent Coomassie Reagent Procedure Procedure Calculations Calculations Method 4 Method 4 Standard solutions Standard Solutions Test solution Test Solution Blank Blank Reagents Reagents and Solutions BCA Reagent BCA Reagent Copper sulfate reagent Copper Sulfate Reagent Copper-BCA reagent Copper-BCA Reagent Procedure Procedure Calculations Calculations Method 5 Method 5 Standard solutions Standard Solutions Test solution Test Solution Blank Blank Biuret reagent Biuret Reagent Procedure Procedure Calculations Calculations Interfering substances Interfering Substances Comments Comments Method 6 Method 6 Standard solutions Standard Solutions Test solution Test Solution Blank Blank Reagents Reagents and Solutions Borate buŠer Borate BuŠer Stock OPA reagent Stock OPA Reagent OPA reagent OPA Reagent Procedure Procedure Calculations Calculations Method 7 Method 7 Procedure A Procedure A Procedure B Procedure B Calculations Calculations

Sep. 2002

Harmonized items JP 15 Remarks

General Information Isoelectric Focusing Isoelectric Focusing Theoretical aspects Theoretical Aspects Practical aspects Practical Aspects 1706 Isoelectric Focusing / General Information JP XV

Apparatus Apparatus Isoelectric Focusing in polyacrylamide Isoelectric Focusing in Poly- gels: detailed procedure acrylamide Gels: Detailed Procedure Preparation of the gels Preparation of the Gels 1) 7.5 per cent polyacrylamide gel 7.5z Polyacrylamide gel 2) Preparation of the mould Preparation of the mould Method Method Variations to the detailed procedure Variations to the Detailed Procedure (subject to validation) (Subject to Validation) Validation of iso-electric focusing Validation of Iso-Electric Focusing procedures Procedures Speciˆed variation to the general Speciˆed Variations to the General method Method Point to consider Points to Consider Figure-Mould Figure. Mould Reagents Reagents and Solutions Coomassie staining TS and destaining Fixing solution for isoelectric focus- Fixing solution for isoelectric focus- solution are speciˆed. ing in polyacrylamide gel ing in polyacrylamide gel Coomassie staining TS Destaining solution

June 2004

Harmonized items JP 15 Remarks

General Information Powder Flow Powder Flow Angel of repose 1. Angel of repose Basic methods for angel of repose 1.1 Basic methods for angel of re- pose Variations in angel of repose 1.2 Variations in angel of repose methods methods Angel of repose general scale of 1.3 Angel of repose general scale of flowability flowability Experimental considerations for an- 1.4 Experimental considerations for gle of repose angle of repose Recommended procedure for angle 1.5 Recommended procedure for an- of repose gle of repose Compressibility index and Hausner 2. Compressibility index and Hausner ratio ratio Basic methods for compressibility in- 2.1 Basic methods for compressibili- dex and Hausner ratio ty index and Hausner ratio Experimental considerations for the 2.2 Experimental considerations for compressibility index and Hausner the compressibility index and ratio Hausner ratio Recommended procedure for com- 2.3 Recommended procedure for pressibility index and Hausner ra- compressibility index and Hausner tio ratio Flow through an orifice 3. Flow through an orifice Basic methods for flow through an 3.1 Basic methods for flow through orifice an orifice JP XV General Information / Isoelectric Focusing 1707

Variations in methods for flow 3.2 Variations in methods for flow through an orifice through an orifice General scale of flowability for flow 3.3 General scale of flowability for through an orifice flow through an orifice Experimental considerations for 3.4 Experimental considerations for flow through an orifice flow through an orifice Recommended procedure for flow 3.5 Recommended procedure for through an orifice flow through an orifice Shear cell methods 4. Shear cell methods Basic methods for shear cell 4.1 Basic methods for shear cell Recommendation for shear cell 4.2 Recommendation for shear cell Table 1 Flow properties and cor- Table 1 Flow properties and cor- responding angle repose responding angle repose Table 2 Scale of flowability Table 2 Scale of flowability

Sep. 2002

Harmonized items JP 15 Remarks

General Information Peptide Mapping Peptide Mapping Purpose and scope Purpose and Scope The peptide map The Peptide Map Isolation and puriˆcation Isolation and Puriˆcation Selective cleavage of peptide bonds Selective Cleavage of Peptide Bonds Pretreatment of sample Pretreatment of Sample Pretreatment of the cleavage agent Pretreatment of the Cleavage Agent Pretreatment of the protein Pretreatment of the Protein Establishment of optimal Establishment of Optimal Digestion conditions Conditions pH pH Temperature Temperature Time Time Amount of cleavage agent Amount of Cleavage Agent Chromatographic separation Chromatographic Separation Chromatographic column Chromatographic Column solvent Solvent Mobile phase Mobile Phase Gradient selection Gradient Selection Isocratic selection Isocratic Selection Other parameters Other Parameters Validation Validation Analysis and identiˆcation of peptides Analysis and Identiˆcation of Pep- tides Table 1. Examples of cleavage agents. Table 1. Examples of Cleavage Agents Table 2. Techniques used for the sepa- Table 2. Techniques Used for the ration of peptides. Separation of Peptides 1708 Isoelectric Focusing / General Information JP XV

lematic and it is best to prepare the sample, if possible, in deionized water or 2 per cent ampholytes, using dialysis or gel ˆltration if necessary. 9. Isoelectric Focusing The time required for completion of focusing in thin-layer polyacrylamide gels is determined by placing a colored This test is harmonized with the European Pharmacopoeia protein (e.g. hemoglobin) at diŠerent positions on the gel sur- and the U.S. Pharmacopeia. The parts of the text that are not face and by applying the electric ˆeld: the steady state is harmonized are marked with symbols ( ).  reached when all applications give an identical band pattern. General Principles In some protocols the completion of the focusing is indicated Isoelectric focusing (IEF) is a method of electrophoresis by the time elapsed after the sample application. that separates proteins according to their isoelectric point. The IEF gel can be used as an identity test when the Separation is carried out in a slab of polyacrylamide or migration pattern on the gel is compared to a suitable agarose gel that contains a mixture of amphoteric electrolytes standard preparation and IEF calibration proteins, the IEF (ampholytes). When subjected to an electric ˆeld, the ampho- gel can be used as a limit test when the density of a band on lytes migrate in the gel to create a pH gradient. In some cases IEF is compared subjectively with the density of bands gels containing an immobilized pH gradient, prepared by in- appearing in a standard preparation, or it can be used as a corporating weak acids and bases to speciˆc regions of the gel quantitative test when the density is measured using a network during the preparation of the gel, are used. When densitometer or similar instrumentation to determine the the applied proteins reach the gel fraction that has a pH that relative concentration of protein in the bands subject to is the same as their isoelectric point (pI), their charge is neu- validation. tralized and migration ceases. Gradients can be made over Apparatus various ranges of pH, according to the mixture of ampho- An apparatus for IEF consists of: lytes chosen. —a controllable generator for constant potential, current Theoretical Aspects and power. Potentials of 2500 V have been used and are When a protein is at the position of its isoelectric point, it considered optimal under a given set of operating has no net charge and cannot be moved in a gel matrix by the conditions. Supply of up to 30 W of constant power is electric ˆeld. It may, however, move from that position by recommended, diŠusion. The pH gradient forces a protein to remain in its —a rigid plastic IEF chamber that contains a cooled plate, isoelectric point position, thus concentrating it; this concen- of suitable material, to support the gel, trating eŠect is called ``focusing''. Increasing the applied —a plastic cover with platinum electrodes that are connect- voltage or reducing the sample load result in improved ed to the gel by means of paper wicks of suitable width, separation of bands. The applied voltage is limited by the length and thickness, impregnated with solutions of a- heat generated, which must be dissipated. The use of thin gels nodic and cathodic electrolytes. and an e‹cient cooling plate controlled by a thermostatic cir- Isoelectric Focusing in Polyacrylamide Gels: Detailed culator prevents the burning of the gel whilst allowing sharp Procedure focusing. The separation is estimated by determining the The following method is a detailed description of an IEF minimum pI diŠerence (DpI), which is necessary to separate 2 procedure in thick polyacrylamide slab gels, which is used un- neighboring bands: less otherwise stated in the monograph. D(dpH/dx) DpI＝3 Preparation of the Gels E(－dm/dpH) Mould The mould (see Figure) is composed of a glass D: DiŠusion coe‹cient of the protein plate (A) on which a polyester ˆlm (B) is placed to facilitate dpH/dx:pHgradient handling of the gel, one or more spacers (C), a second glass E: Intensity of the electric ˆeld, in volts per centimeter plate (D) and clamps to hold the structure together. －dm/dpH: Variation of the solute mobility with the pH in 7.5z Polyacrylamide gel Dissolve 29.1 g of acrylamide the region close to the pI and 0.9 g of N,N?-methylenebisacrylamide in 100 mL of water. To 2.5 volumes of this solution, add the mixture of Since D and －dm/dpH for a given protein cannot be ampholytes speciˆed in the monograph and dilute to 10 altered, the separation can be improved by using a narrower pH range and by increasing the intensity of the electric ˆeld. Resolution between protein bands on an IEF gel prepared with carrier ampholytes can be quite good. Improvements in resolution may be achieved by using immobilized pH gradients where the buŠering species, which are analogous to carrier ampholytes, are copolymerized within the gel matrix. Proteins exhibiting pIs diŠering by as little as 0.02 pH units may be resolved using a gel prepared with carrier ampholytes while immobilized pH gradients can resolve proteins diŠering by approximately 0.001 pH units. Practical Aspects Special attention must be paid to sample characteristics and/or preparation. Having salt in the sample can be prob- Figure. Mould JP XV General Information / Isoelectric Focusing 1709 volumes with water. Mix carefully and degas the solution. Validation of Iso-Electric Focusing Procedures Preparation of the mould Place the polyester ˆlm on the Where alternative methods to the detailed procedure are lower glass plate, apply the spacer, place the second glass employed they must be validated. The following criteria may plate and ˆt the clamps. Place 7.5z polyacrylamide gel be used to validate the separation: prepared before use on a magnetic stirrer, and add 0.25 —formation of a stable pH gradient of desired characteris- volumes of a solution of persulfate (1 in 10) and tics, assessed for example using colored pH markers of 0.25 volumes of N,N,N?,N?-tetramethylethylenediamine. known isoelectric points, Immediately ˆll the space between the glass plates of the —comparison with the electropherogram provided with the mould with the solution. chemical reference substance for the preparation to be Method examined, Dismantle the mould and, making use of the polyester ˆlm, —any other validation criteria as prescribed in the mono- transfer the gel onto the cooled support, wetted with a few graph. millilitres of a suitable liquid, taking care to avoid forming Speciˆed Variations to the General Method air bubbles. Prepare the test solutions and reference solutions Variations to the general method required for the analysis as speciˆed in the monograph. Place strips of paper for sam- of speciˆc substances may be speciˆed in detail in mono- ple application, about 10 mm×5mminsize,onthegeland graphs. These include: impregnate each with the prescribed amount of the test and —the addition of urea in the gel (3 mol/L concentration is reference solutions. Also apply the prescribed quantity of a often satisfactory to keep protein in solution but up to 8 solution of proteins with known isoelectric points as pH mar- mol/L can be used): some proteins precipitate at their isoe- kers to calibrate the gel. In some protocols the gel has pre- lectric point. In this case, urea is included in the gel formu- cast slots where a solution of the sample is applied instead of lation to keep the protein in solution. If urea is used, only using impregnated paper strips. Cut 2 strips of paper to the fresh solutions should be used to prevent carbamylation of length of the gel and impregnate them with the electrolyte so- the protein, lutions: acid for the anode and alkaline for the cathode. The —the use of alternative staining methods, compositions of the anode and cathode solutions are given in —the use of gel additives such as non-ionic detergents (e.g. the monograph. Apply these paper wicks to each side of the octylglucoside) or zwitterionic detergents (e.g., CHAPS or gel several millimetres from the edge. Fit the cover so that the CHAPSO), and the addition of ampholyte to the sample, electrodes are in contact with the wicks (respecting the anodic to prevent proteins from aggregating or precipitating. and cathodic poles). Proceed with the isoelectric focusing by applying the electrical parameters described in the mono- Points to Consider graph. Switch oŠ the current when the migration of the mix- Samples can be applied to any area on the gel, but to pro- ture of standard proteins has stabilized. Using forceps, re- tect the proteins from extreme pH environments samples move the sample application strips and the 2 electrode wicks. should not be applied close to either electrode. During Immerse the gel in ˆxing solution for isoelectric focusing in method development the analyst can try applying the protein polyacrylamide gel. Incubate with gentle shaking at room in 3 positions on the gel (i.e. middle and both ends); the temperature for 30 minutes. Drain oŠ the solution and add pattern of a protein applied at opposite ends of the gel may 200 mL of destaining solution. Incubate with shaking for 1 not be identical. hour. Drain the gel, add coomassie staining TS. Incubate for A phenomenon known as cathodic drift, where the pH 30 minutes. Destain the gel by passive diŠusion with destain- gradient decays over time, may occur if a gel is focused too ing solution until the bands are well visualized against a clear long. Although not well understood, electroendoosmosis and background. Locate the position and intensity of the bands in absorption of may be factors that lead to the electropherogram as prescribed in the monograph. cathodic drift. Cathodic drift is observed as focused protein migrating oŠ the cathode end of the gel. Immobilized pH Variations to the Detailed Procedure (Subject to Validation) gradients may be used to address this problem. Where reference to the general method on isoelectric focus- E‹cient cooling (approximately 49C) of the bed that the ing is made, variations in methodology or procedure may be gel lies on during focusing is important. High ˆeld strengths made subject to validation. These include: used during isoelectric focusing can lead to overheating and —the use of commercially available pre-cast gels and of com- aŠect the quality of the focused gel. mercial staining and destaining kits, —the use of immobilized pH gradients, Reagents and Solutions— —the use of rod gels, Fixing solution for isoelectric focusing in polyacrylamide —the use of gel cassettes of diŠerent dimensions, including gel Dissolve 35 g of 5-sulfosalicylic acid dihydrate and 100 g ultra-thin (0.2 mm) gels, of trichloroacetic acid in water to make 1000 mL. —variations in the sample application procedure, including Coomassie staining TS Dissolve 125 mg of coomassie diŠerent sample volumes or the use of sample application brilliant blue R-250 in 100 mL of a mixture of water, masks or wicks other than paper, methanol and acetic acid (100) (5:4:1), and ˆlter. —the use of alternate running conditions, including varia- Destaining solution A mixture of water, methanol and

tions in the electric ˆeld depending on gel dimensions and acetic acid (100) (5:4:1). equipment, and the use of ˆxed migration times rather than subjective interpretation of band stability, —the inclusion of a pre-focusing step, —the use of automated instrumentation, —the use of agarose gels. 1710 Laser DiŠraction Measurement / General Information JP XV

refracted light. However, since the scattering patterns of the non-spherical particles vary depending on the particle shape, the correct particle diameter can not be obtained even if the 10. Laser DiŠraction Measurement accurate physical properties (refraction index or transmit- of Particle Size tance) of the dispersing medium are given. Therefore, the Mie scattering theory can be eŠectively applied if the particles Particle size is one of the important factors concerning the are nearly spherical, and for the other cases, evaluation of the powder characteristics of solid-state drugs and pharmaceuti- data is in general di‹cult due to strongly be aŠected by the cal excipients, and therefore prompt and highly accurate de- physical properties or the particle concentration. termination method is necessary. The laser diŠraction 2. Apparatus method is considered as a potent candidate, but due to the A typical set-up for a laser diŠraction instrument is given limitofkindofparticlesandtherangeofparticlediameter in Figure 1. As the light source, the laser beam of a single that can be measured, the optical microscopy method should wavelength is generally used. A beam expander is equipped as be sometimes applied in parallel. In the pharmaceutical ˆeld, the laser beam processing unit H for irradiation to the parti- the reliable and reproducible particle size determination cle ensemble dispersed. When the sample (particle ensemble method is required for quality control of the composition of F) passes through the laser beam, a part of the laser beam is the formulation, especially, of the pharmaceutical excipients scattered by the particles, and the diŠraction images given by that are di‹cult to apply the analytical sieving method due to the scattered and transmitted beams are transcribed by the the particle diameter of not larger than 100 mm. detector J placed at the focal length of the Fourier lens D. In the Japanese Pharmacopoeia, the optical microscopy A representative sample is dispersed in a liquid or gas, and (Method 1) for the particles ranging from approximately 1 the recirculating system consisting of the optical measure- mmto100mm and the analytical sieving method (Method 2) ment cell, a dispersion bath (usually equipped with the stirrer fortheparticlesofnotsmallerthan75mm are listed as the and ultrasonic elements), a pump and tubing are commonly powder particle size determination. most used. This document describes the laser diŠraction technique for (1) Calibration of apparatus particle size determination as the method for analyzing the According to the description in ISO standard 13320-1 particles having the diameter ranging from approximately 1 (1999): Particle size analysis-Laser diŠraction methods-Part mmto1000mm by utilizing the diŠraction phenomenon 1: General principles, apparatus should be calibrated by us- against the laser beam (Fraunhofer diŠraction). There are ing the plural standard particles for particle size measurement methods utilizing the principles such as the Mie scattering having a known particle size distribution and over at least one other than the Fraunhofer diŠraction, which can be also used decade of size. for particle size determination of the particles in sub-micron (2) Particle size determination in sub-micron region range (not larger than 1 mm). In order to extend the measuring range of particle diameter 1. Principle of laser diŠraction technique to sub-micron region, apparatuses based on the Mie scatter- The laser diŠraction technique is based upon the beam ing theory have been developed and used. This type of instru- diŠraction phenomenon (Fraunhofer diŠraction), in which ment is designed so that the scattered lights toward back and particle size-dependent intensity patterns exhibit when the side are detected and analyzed, but there are large variations powder particles are exposed to the parallel rays, and ac- in the analytical results depending on the type of instrument. curate and reproducible particle size distribution image can However, this type of apparatus can produce reproducible be obtained by mathematical analysis of the diŠraction pat- data even in a sub-micron region, if the apparatus used and tern. In other words, this apparatus is designed to detect the the sample tested are restricted under the speciˆc conditions beams diŠracted to each angle by the particles when the laser in which the input number for data analysis, e.g., physical beam is radiated to the powder sample dispersed in a liquid or properties, are ˆxed for the apparatus. gas, in a way that the beam passes over the specimen and, the (3) Particle size determination of emulsion diŠraction pattern is obtained and recorded for data analysis. In case of particle size determination of emulsion, the For the analysis of data, the particles are qualiˆed as spheri- method utilizing dynamic light scattering or static light scat- cal form. In this technique, at every particle diameter interval tering is generally used. Since the emulsion particles transmit that is provided in the apparatus, distribution of the spherical the light, the laser diŠraction method is not very appropriate equivalent diameter in the standard volume is calculated by to apply. However, it is possible to evaluate emulsion particle inverse operation from the optical model to attain the highest diameter when the apparatus has been appropriately calibrat- coherence to the diŠraction intensity pattern. ed using the standard particles for particle size measurement. For the particles in sub-micron region, it is not easy to ob- 3. Measurement tain an accurate particle size distribution due to di‹culty in In case of the laser diŠraction method, the particle concen- distinction of the particle diameter-dependent scattering pat- tration aŠects the accuracy of measurement. Therefore, for tern because of weakness of the forward light scattering in- the sample having the size near the upper measuring limit of tensity, and it is generally considered that the Fraunhofer particle diameter, the particle concentration should be high diŠraction can not be applied for the evaluation of particle enough to attain the static analysis. On the contrary, for the diameter in the sub-micron range. Therefore, for the particles sample having smaller particle diameter, it is necessary for in sub-micron region, the method that applies the Mie scat- accurate measurement to adjust the particle concentration tering theory is introduced here. The particle diameter is lower to some extent to prevent the multiple scattering. measured by the analysis of the scattered lights toward back (1) Conditions of measurement and side that are easy to catch the signals of transmitted and ･Location of the apparatus: It is important that the appara- JP XV General Information / Laser DiŠraction Measurement 1711

Fig. 1 Construction of the laser diffraction apparatus

tus is not aŠected by electrical noise and mechanical vibra- ment, and the divider or the cone and quartering method, etc. tions. It is also desirable that the apparatus is located under are available for this purpose. The cone and quartering the environment where there is no variation of temperature method is used for reduction of a small amount of sample and humidity to the extent that aŠects the measurement and with poor ‰owability, and is a simple method without using is no exposure of direct sun light. any divider. ･Blank measurement: After selection of proper alignment of For sampling of the powder particles dispersed in a liquid, the optical part of the instrument, a blank measurement per- the liquid is ˆrst stirred uniformly and mixed, and it should formed in a pure dispersing medium using the same method be conˆrmed that no precipitate remains at the bottom of the as that for measurement of the sample. vessel. Then, the amount necessary for measurement is ･Preliminary observation of the sample: Prior to the meas- quickly sampled using a pipette, etc. In case of the sample urement, the powder sample should be observed in advance that contains lager particles, it is easy to cause precipitates, by visual/microscopic inspection to conˆrm the approxi- and they are precipitated at the bottom after once stopping mate particle diameter range and particle shape. agitation. Therefore, the sampling of the suspension should ･Establishment of the measurement conditions: The time for be made under agitation. measurement, the reading time of detector and the number (4) Pretreatment of the sample for measurement of repetition should be experimentally decided in accor- If the sample contains the particles or agglomerates over dance with required measurement accuracy. the upper limit of measurable particle diameter range of the ･Measurement of the sample: The sample is measured under laser diŠraction instrument, they should be removed by siev- the established measurement conditions. Data analysis is ing in advance, and the mass and percent of such particles or performed based on the pulse of dispersed particles from the agglomerates removed should be recorded. diŠerence of diŠraction intensity between the sample and (5) Liquids used as the dispersion medium the blank. A variety of liquids is available for the dispersion of the (2) Preparation of the sample powder samples, and at selection of the dispersion liquids, The dry powder sample can be dispersed in a liquid or gas, the followings should be taken into account: and the appropriate method is selected depending on the pur- —be compatible with the materials used in the instrument pose of measurement. There are two types of dispersion (O-ring, tubing, etc.) method, wet type using a liquid as the dispersion medium and —not dissolve or alter the size of particular materials, dry type using a gas as the dispersion medium. It is advisable —favour easy and stable dispersion of the particulate materi- that the powder sample for measurement is divided to an ap- als, propriate apparent volume using a sample splitting tech- —have suitable viscosity in order to enable recirculation, nique. —not be hazardous to health and should meet safety require- The result of the particle size distribution much varies de- ments. pending on the conditions of preparation of the sample. A low-foaming surfactant and dispersant may be used to Therefore, the procedures and conditions of sample prepara- facilitate the wetting of the particles, and to stabilize the dis- tion should be deˆned in advance. persion. A preliminary check on the dispersion quality can be (3) Sampling made by visual/microscopic inspection of the suspension. The amount of the sample necessary for the laser diŠrac- (6) Dispersion of the agglomerated particles by ultrasonica- tion for powder particle size measurement has deceased due tion to technical advancement of the apparatus, but the sample For the powder sample containing ˆne particles, intense for the measurement should represent the whole sample. physical power should be given to disperse the agglomerated Therefore, how to sample a small amount for actual meas- particles. The ultrasonication is generally used for this pur- urement from a sample of large volume is an important prob- pose. In this case, since the ultrasonic output, irradiation lem. It is general that a large amount of the sample is time and the volumes of both the suspension and the vessel divided/reduced to reach the amount necessary for measure- 1712 Media Fill Test / General Information JP XV used signiˆcantly aŠect the intensiˆed dispersion eŠect, the pressed as the frequency that is shared by the particles that optimum irradiation conditions should be established taking belong to the speciˆed particle diameter fraction among the account of these factors. In case that an ultrasonic bath is whole sample. used for dispersion, kinetic condition of the dispersant and ･Mathematical expression of the particle size distribution dispersion eŠect may change depending on the altitude of its The particle size distribution is expressed by a function of surface. Therefore, it is necessary to conˆrm the resonance either the normal distribution or the lognormal distribution. point where the ultrasonic wave is intensely irradiated. Fur- The lognormal distribution is characterized by distribution ther,incaseofstrongerultrasonicoutputorlongerirradia- pattern that splays out toward large particle size range, and is tion time, the particles for measurement may be destructed. generally applied for the samples having a large amount of Therefore, the change of the particle size distribution de- larger particles and a small amount of smaller particles. pending on the variation of the irradiation conditions should 5. Experimental considerations on the measurement be checked to determine the appropriate ultrasonic output Accurate particle diameter measurement can not be expect- andirradiationtime. ed for the laser diŠraction method in case of the samples hav- (7) Dispersion gases as the dispersion medium ing wide range of particle size distribution. Therefore, the An appropriate dry type dispersion apparatus using com- setting the measurement range is essential for the measure- pressed gas is used for dispersion of the powder sample in a ment of the particle diameter by the laser diŠraction method, gas. Any substance that may aŠect the measurement, such as because the scattering intensity is much in‰uenced by the low- oil, water and particulate matter, should not be contaminated er limit diameter (1 mm) and upper limit diameter (1000 mm) in the dispersion medium gas, and such substances should be of sample particles. It is desirable in this method that the removed, if necessary, by a ˆlter, etc. In case of the dry dis- measurement diameter range is conˆrmed by interpolation persion method, it is advisable to measure all the samples using the standard sample having a known particle size distri- reduced. A larger amount of sample diminishes the statistical bution considering the accuracy of measurement and the sen- error at a rough and large particle size area even if the particle sitivity of instrument used. For the particles of not larger size distribution is broad. It is also important that the parti- than 10 mm, it is di‹cult to obtain accurate particle diameter, cles are dispersed uniformly, It can be conˆrmed by compar- because the diŠerence of diŠraction pulse detected by the de- ing the results obtained by dry and wet dispersion methods. It tector is small. Particularly, for the particles in sub-micron is desirable that the equivalent results are obtained by dry and region, there is almost no diŠerence of the scattered light for- wet measurements. ward, and it is hard to distinguish them. Further, in addition 4. Data analysis to the error caused by recognizing the non-spherical particles Various softwares have been developed to analyze the data as spherical particles, decrease of the scattering intensity, utilizing some mathematical methods, and the particle di- which is much bigger than that estimated from the reduction ameter and particle size distribution can be calculated from of geometrical particle diameter, occurs as the particle di- the data to meet its object. As for the accuracy of measure- ameter decreases, and securing the accuracy of measurement ment, the reproducibility required for each apparatus is at- comes to be di‹cult. Therefore, the lower limit for measure- tained when measured on the independent triplicate sam- ment of the particle diameter by the laser diŠraction tech- plingsofthesamebatch. nique is approximately 1 mm, similar to that by the optical (1) Calculation of the particle size distribution microscopic method. In case of the laser diŠraction tech- The instrument manufacturers may use diŠerent al- nique, since the scattering intensity is related to the volume gorithms for calculation of the particle size distribution from concentration of the particles, and the maximum measure- the intensity of diŠraction (or scattering) beams obtained by ment range of particle diameter should be decided taking ac- the laser diŠraction instrument, but the principle of estimat- count of the relative variation of the scattering intensity. A ing particle diameter from the intensity of light diŠraction particle of 10 times larger in diameter has a volume of 1000 obtained by the detector under the diŠraction (or scattering) timeslarger.Thismeansthatincaseof1mm for lower limit theory is fundamentally the same. Since this method is based measurement of particle diameter, the particles of 1000 mm upon the optical model that assumes spherical particle, the have a volume of 1 billion times larger, and consequently the sphere equivalent diameter is obtained for the non-spherical sample necessary for measurement is 10 billion times larger, particles. The laser diŠraction method can not distinguish be- similar to the case of volume. Therefore, approximately 1000 tween the diŠraction of a single particle and that of the ag- mm is appropriate for the maximum particle diameter for glomerate or of the primary particle cluster that constitutes measurement. an aggregates or agglomerates. Example of the standard reference particles (2) Weaving of the results Use the standard reference particles for SAP 10-03, the For the samples having particle size distributions, the me- Speciˆcation of Association of Powder Process Industry and dian diameter or the modal diameter are used as the charac- Engineering, Japan. Either of MBP1-10 or MBP10-100 is teristic diameters, and the particle size distribution is ex- used to meet the particle size measurement range (1 – 10 mm pressed either as the cumulative size distribution or the fre- or10–100mm). quency size distribution. The cumulative size distribution is expressed as a quantitative proportion of the particles smaller than the speciˆed particle diameter among the whole sample. The analytical sieving method is generally used for the parti- 11. Media Fill Test cle size distribution determination, and this cumulative distri- bution is represented as the cumulative undersize distribu- The media ˆll test (MFT) is one of the processing valida- tion. On the other hand, the frequency size distribution is ex- tions employed to evaluate the propriety of the aseptic JP XV General Information / Media Fill Test 1713

Table 1. Alert and action levels for large numbers of Table 3. Initial performance qualification: Media fills media filled units Production Numbers of Alert level and Action level and Number of contaminated units lot size media fill runs action required action required Number of units*1 Acceptance levels Alert levels Action levels Aminimumof One contaminat- Two contaminat- 10 media fill ed unit in any ed units in single 3,000 0 not applicable ≧ 1 runs using the run. Investigate run, or one each 4,750 0 1 ≧ 2 º 500 maximum lot cause. in two runs. 6,300 0 1 – 2 ≧ 3 size of the Investigate 7,760 0 1 – 3 ≧ 4 product cause and repeat 9,160 0 1 – 4 ≧ 5 initial qualifica- 10,520 1 2 – 5 ≧ 6 tion media fills. 11,850 1 2 – 6 ≧ 7 13,150 1 – 23– 7 ≧ 8 A minimum of 3 Thesameas The same as 14,440 1 – 23– 8 ≧ 9 media fill runs above above 15,710 1 – 34– 9 ≧10 500 – 2,999 using the maxi- 16,970 1 – 34– 10 ≧11 mum lot size of the product *1 It is not necessary to relate the number of units with lot size of actual products. A minimum of 3 When any of the When any of the media fill runs media fill runs media fill runs Table 2. Upper 95z confidence limit of a Poisson variable using at least exceeds the alert exceeds the ac- for numbers of contaminated units ≧ 3,000 3,000 units level shown in tion level shown Table 1, take in Table 1, take Observed numbers of contaminated Upper 95z confidence the action set the action set units (k) limit (U) out in 2.1.1. out in 2.1.1

0 2.9957 1 4.7439 Table 4. Periodic performance requalification: Media fills 2 6.2958 3 7.7537 Production Numbers of Alert level and Action level and 4 9.1537 lot size media fill runs action required action required 5 10.5130 A minimum of 3 One contaminat- 6 11.8424 media fill runs ed unit in any 7 13.1481 º 500 using the maxi- run. Investigate 8 14.4346 mum lot size of cause and repeat 9 15.7052 the product initial qualifica- 10 16.9622 tion media fill Using the 95z confidence limit (U) in Table 2, the contamination runs. rate (P) of observed numbers of contaminated units (k) per filled One media fill One contaminat- units (n) can be calculated as P ＝ UWn (equation 1). For example: run using the ed unit. Inves- If 5,000 units were filled and two contaminated units were ob- 500 – 2,999 maximum lot tigate cause and served, n ＝ 5,000 and U ＝ 6.30 (k ＝ 2) are substituted in the equa- size of the repeat initial tion 1; P ＝ 6.30W5,000 ＝ 0.0013. The upper 95z confidence limit product qualification me- for the contamination rate would be 0.13z. dia fill runs. One media fill When the media When the media processing of pharmaceutical products using sterile media, run using fill run exceeds fill run exceeds etc. instead of actual products. Therefore, media ˆll tests at least 3,000 the alert level the action level should be conducted with the manipulations normally per- ≧ 3,000 units showninTable showninTable formed in actual processing, e.g. ˆlling and closing opera- 1, take the ac- 1, take the ac- tion, operating environment, processing operation, number tion set out in 2. tion set out in 2. 1. 1. 1. 1 of personnel involved, etc., and conducted under processing conditions that include ``worst case'' conditions. Refer to GMP (1), WHOWGMP for pharmaceutical products (2), and 1.2 Periodic performance requaliˆcation ISO 13408 (3), etc. for necessary information to conduct this 1) Conduct media ˆll requaliˆcations periodically on test. each working shift for the ˆlling line. Employees working in the aseptic processing area should be trained for aseptic 1. Frequency of media ˆlls processing operations and take part in media ˆlls. 1.1 Initial performance qualiˆcation 2) When ˆlling lines have not been used for over six Initial performance qualiˆcation should be conducted for months, conduct appropriate numbers of media ˆll runs in each new facility, item of equipment, ˆlling line, and contain- the same way as for the initial performance qualiˆcation er design (except for multiple sizes of the same container de- prior to resumption of use of the ˆlling lines. sign), etc. For production batch sizes exceeding 3,000 units, a 3) In cases of facility and equipment modiˆcation (inter- minimum of three media ˆll runs should be conducted on changing parts may not require requaliˆcation), changes in separate days. For production batch sizes of less than 3,000 personnel working in critical aseptic processing (e.g. new units, see Table 3. crews), anomalies in environmental testing results, or a 1714 Media Fill Test / General Information JP XV product sterility test showing contaminated products, con- 6) Storage conditions of sterile commodities duct appropriate numbers of media ˆll runs in the same way 7) HEPA ˆlter evaluation (airborne particulate levels, as for the initial performance qualiˆcation prior to the sched- DOP test, velocity measurements, etc.) uled media ˆlls. 8) Pre and post ˆlter integrity test data (including ˆlter housing assembly) 2. Acceptance criteria of media ˆlls 9) Room air ‰ow patterns and pressures A large number of media ˆlled units is required to detect 10) Unusual events that occurred during the media ˆll run 0.1z contamination rate. The alert level is more than 0.05z 11) Characterization of contaminants but less than 0.1z, and the action level is more than 0.1z 12) Hygienic control and training programs contamination rate at the upper 95z conˆdence level. 13) Gowning procedures and training programs Table 1 shows alert and action levels for media ˆlled units. 14) Aseptic processing technique and training programs Table 2 is to be used for the calculation of contamination rate 15) Operator's health status (especially coughing, sneez- of contaminated units found in media ˆlled units. The con- ing, etc., due to respiratory ) tamination rate of 0.05z in media ˆlls is the minimum ac- 16) Other factors that aŠect sterility ceptable level, and manufacturers should make eŠorts to achieve lower contamination rate than this. Table 3 shows the 3. Data guidance for media ˆlls alert and action levels for initial performance qualiˆcation of Each media ˆll run should be fully documented and the an aseptic processing line, and actions required for each level. following information recorded: Table 4 shows the alert and action levels for requaliˆcation of 1) Data and time of media ˆll an aseptic processing line, and actions required for each level. 2) Identiˆcation of ˆlling room and ˆlling line used The alert and action levels for production batch sizes of less 3) ContainerWclosure type and size than 3,000 units take semiautomatic or manual operation 4) Volume ˆlled per container into consideration. 5) Filling speed 2.1 Actions required for each level 6) Filter lot and catalogue number 2.1.1 Initial performance qualiˆcation 7) Type of media ˆlled 1) When the result of the media ˆll run is less than the 8) Number of units ˆlled alert level, the media ˆll run meets the requirement of the 9) Number of units not incubated and reason MFT. 10) Number of units incubated 2) When the results of any media ˆll run done with at 11) Number of units positive least three replicate runs reach the alert or the action level, an 12) Incubation time and temperature investigation regarding the cause is required, and initial 13) Procedures used to simulate any step of a normal qualiˆcation media ˆlls are to be repeated. When the result of production ˆll (e.g., mock lyophilization or substitution of each media ˆll run is less than the alert level, the initial vial headspace gas) qualiˆcation media ˆlls meet the requirement of the MFT. 14) Microbiological monitoring data obtained during the 2.1.2 Requaliˆcation media ˆll set-up and run 1) When the result of the media ˆll run is less than the 15) Listofpersonnelwhotookpartinthemediaˆll alert level, the media ˆll run meets the requirement of the 16) Growth promotion results of the media (in case of MFT. powder ˆll, an antimicrobial activity test for the powder is 2) When the result of the media ˆll run exceeds the alert necessary) level, an investigation regarding the cause is required, and 17) Characterization of the microorganisms from any one more media ˆll run is to be done. If the result is less than positive units the alert level, the media ˆll run meets the requirement of the 18) Review MFT. 4. Media ˆll procedures 3) When the result of the media ˆll run exceeds the action Methods to validate aseptic processing of liquid, powder level, a prompt review of all appropriate records relating to and freeze-dried products are described. Basically, it is possi- aseptic production between the current media ˆll and the last ble to apply media ˆll procedures for liquid products to other successful one, and an investigation regarding the cause must dosage forms and container conˆgurations. be conducted simultaneously. If necessary, appropriate ac- 4.1 Media selection and growth promotion tion to sequester stored andWor distributed products should Soybean-casein digest medium or other suitable media are be taken. After investigation regarding the cause, repeat used. As growth promotion testing microorganisms, strains three serial media ˆll runs. If the results are less than the alert listed in the Sterility Test and, if necessary, one to two level, the media ˆll runs meet the requirement of the MFT. representative microorganisms which are frequently isolated 2.2 Parameters which aŠect sterility in environmental monitoring should be used. The media in- When media ˆll alert and action levels are exceeded, an in- oculated with 10 to 100 viable microorganisms of each strain vestigation should be conducted regarding the cause, taking should show obvious growth when incubated at the predeter- into consideration the following points: mined temperature for 5 days. 1) Microbial environmental monitoring data 4.2 Sterile medium preparation 2) Particulate monitoring data The medium is sterilized according to the pre-validated 3) Personnel monitoring data (microbial monitoring data method. on gloves, gowns, etc. at the end of work) 4.3 Incubation and inspection of media ˆlled units 4) Sterilization cycles for media, commodities, equip- Leaking or damaged media ˆll evaluation units should be ment, etc. removed and recorded prior to incubation of media ˆlled 5) Calibration of sterilization equipment units. Incubate at 20 – 259C for 1 week, and then at 30 – JP XV General Information / Microbial Attributes 1715

359Cfor1week(orat30–359C for 1 week, and then at 20 – C. Lyophilized products 259C for 1 week), or at 30 – 359C for 2 weeks. Observe the Inthecaseoflyophilizedproducts,itmaybeimpossibleto media ˆlled units for growth of microorganisms at least once conduct a media ˆll run in the same way as used for actual between the third day and seventh day and on the last day of processing of lyophilized products. The process of freezing the test period, twice in total. Microorganisms present in con- and lyophilization of the solution may kill contaminant or- taminated units should be characterized. ganisms and change the characteristics of the media too. The A. Liquid products use of inert gas as a blanket gas may inhibit the growth of Media ˆll procedure aerobic bacteria and fungi. Therefore, in general, the actual Media ˆll should include normal facilityWequipment opera- freezing and lyophilization process should be avoided and air tions and clean-up routines. Containers, closures, parts of used as the blanket gas. the ˆlling machine, trays, etc. are washed and sterilized ac- Media ˆll procedures cording to the standard operating procedures. Media ˆlls Use the following method or other methods considered to should be conducted under processing conditions that include be equivalent to these methods. ``worst case'' conditions, e.g., correction of line stoppage, 1) After ˆlling of the media into containers by the ˆlling repair or replacement of ˆlling needlesWtubes, replacement of machine, cap the containers loosely and collect them in pre- on-line ˆlters, permitted interventions, duration and size of sterilized trays. run, number of personnel involved, etc. 2) After placing the trays in the lyophilizer, close the A predetermined volume of medium is ˆlled into sterilized chamber door, and conduct lyophilization according to the containers at a predetermined ˆlling speed and the containers procedures for production operation. Hold them without are sealed. The media are contacted with all product contact freezing under weak vacuum for the predetermined time. surfaces in the containers by an appropriate method, and 3) After the vacuum process, break the vacuum, and seal then incubated at the predetermined temperature. the stoppers. 4) Contact the media with all product contact surfaces in B. Powder products the containers by appropriate methods, and then cultivate B.1 Powder selection and antimicrobial activity test them at the predetermined temperature. Actual products or placebo powder are used. In general, lactose monahydrate, D-, polyethylene glycol 6,000, References carboxymethyl cellulose salts or media powder, etc. are used 1) Good manufacturing practices for pharmaceutical as placebo powders. Prior to employing any of the powders, products (WHO-GMP, 1992) evaluate whether the powder has antimicrobial activity. Me- 2) ISO 13408-1 (Aseptic processing of health care dia powders are dissolved in water and other powders in liq- products: Generals) uid medium, and the solutions are inoculated with 10 to 100 viable microorganisms of each kind, shown in 4.1, for the growth promotion test. If obvious growth appears in the 12. Microbial Attributes of medium incubated at the predetermined temperature for 5 days, the powder has no antimicrobial activity and is availa- Nonsterile Pharmaceutical ble for the media ˆll test. Products B.2 Sterilization of powders Dry powders are bagged in suitable containers (e.g. double The presence of microbial contaminants in nonsterile phar- heat-sealed polyethylene bags), and are subjected to radiation maceutical products can reduce or even inactivate the ther- sterilization. apeutic activity of the product and has the potential to aŠect B.3 Sterility of ˆlling powders adversely the health of patients. Manufacturers, therefore, The powders must pass the Sterility Test. However, if the should ensure as low as possible a contamination level for ˆn- sterilization is fully validated, sterility testing of the powders ished dosage forms, raw materials and packaging compo- can be omitted. nents to maintain appropriate quality, safety and e‹cacy of B.4 Media ˆll procedures nonsterile pharmaceutical products. This chapter provides Chose a suitable procedure from among the following guidelines for acceptable limits of viable microorganisms procedures. (bacteria and fungi) existing in raw materials and nonsterile 1) Fill sterilized liquid media into containers by suitable pharmaceutical products. Testing methods for the counting methods, and then ˆll actual products or sterilized placebo of total viable microorganisms and methods for the detection powder with the powder ˆlling machine. If sterilized powder and identiˆcation of speciˆed microorganisms (Escherichia media are used as a placebo powder, ˆll sterilized water in- coli, Salmonella, , Staphylococcus stead of sterilized liquid media. aureus, etc.) are given under the ``Microbial Limit Test''. 2) Distribute liquid media into containers, and then steri- When these tests are carried out, a microbial control program lize them in an autoclave. Remove the containers to the ˆlling must be established as an important part of the quality area, and then ˆll actual products or sterilized placebo pow- management system of the product. Personnel responsible der into the containers with the powder ˆlling machine. for conducting the tests should have specialized training in 3) Fill actual products or sterilized placebo powder into microbiology and in the interpretation of the testing results. containers with the powder ˆlling machine, and then ˆll steri- lized liquid media into the containers by appropriate 1. Deˆnitions methods. If sterilized powder media are used as a placebo 1.1 Nonsterile pharmaceutical products: Nonsterile drugs powder, ˆll sterilized water instead of sterilized liquid media. shown in monographs of the JP and nonsterile products including intermediate products and ˆnished dosage 1716 Microbial Attributes / General Information JP XV

forms. number of raw materials, etc.); 1.2 Raw materials: All materials, including raw ingredients h) Others. and excipients, used for the preparation of drugs, except In general, the tests may be performed at a high frequency for water and gases. during the initial production of a drug to get information on 1.3 Bioburden: Number and type of viable microorganisms the microbiological attributes of the product or raw materials existing in nonsterile pharmaceutical products. used for the production. However, this frequency may be 1.4 Action levels: Established bioburden levels that require reduced as bioburden data are accumulated through immediate follow-up and corrective action if they are ex- retrospective validation andWor concurrent validation. For ceeded. example, the tests may be performed at a frequency based on 1.5 Alert levels: Established bioburden levels that give early time (e.g., weekly, monthly or seasonally), or on alternate warning of a potential drift from normal bioburden level, batches. but which are not necessary grounds for deˆnitive correc- 4. Microbial control program tive action, though they may require follow-up investiga- When the ``Microbial Limit Test'' is applied to a nonsterile tion. pharmaceutical product, the methods for the recovery, culti- 1.6 Quality management system: The procedures, opera- vation and estimation of the bioburden from the product tion methods and organizational structure of a manufac- must be validated and a ``Microbial control program'' cover- turer (including responsibilities, authorities and relation- ing the items listed below must be prepared. ships between these) needed to implement quality manage- a) Subject pharmaceutical name (product name); ment. b) Frequency of sampling and testing; 2. Scope c) Sampling methods (including responsible person, quanti- In general, the tests for total viable aerobic count and for ty, environment, etc. for sampling); the detection of speciˆed microorganisms are not applied to d) Transfer methods of the samples to the testing area (in- or bacteriostatic drugs. However, the tests should cluding storage condition until the tests); be done for microorganisms that are not aŠected by the drug. e) Treatment of the samples (recovery methods of microbi- The test for total viable aerobic count is not applied to drugs al contaminants); containing viable microorganisms as an active ingredient. f) Enumeration of viable microorganisms (including testing quantity, culture media, growth-supporting test of the 3. Sampling plan and frequency of testing media, culturing methods, etc.); 3.1 Sampling methods g) Detection of speciˆed microorganisms (including testing Microbial contaminants are usually not uniformly dis- quantity, culture media, growth-supporting test of the tributed throughout the batches of non-sterile pharmaceuti- media, culturing methods, etc.); cal products or raw materials. A biased sampling plan, there- h) Estimation of the number of and characterization of fore, cannot be used to estimate the real bioburden in the microbial contaminants; product. A sampling plan which can properly re‰ect the sta- i) Establishment of ``Microbial contamination limits'' (in- tus of the product batch should be established on the basis of cluding alert level and action level); the bioburden data obtained by retrospective validation andW j) Actions to be taken when the levels exceed ``Microbial or concurrent validation. In general, a mixture of samples contamination limits''; randomly taken from at least diŠerent three portions, almost k) Persons responsible for the testing and evaluation, etc.; the same amount for each portion, is used for the tests of the l) Other necessary items product. When the sampling is di‹cult in a contamination- controlled environment, special care is required during sam- 5. Microbial contamination limits for nonsterile phar- pling to avoid introducing microbial contamination into the maceutical products product or aŠecting the nature of the product bioburden. If it By establishing ``Microbial contamination limits'', it is is conˆrmed that the product bioburden is stable for a certain possible to evaluate at the initial processing stage of the period, as in the case of nonaqueous or dried products, it is product whether the microbiological quality of the raw not necessary to do the tests for total viable aerobic counts materials is adequate or not. Furthermore, it is then possible and for the detection of speciˆed microorganisms, immedi- to implement appropriate corrective action as needed to ately after the sampling. maintain or improve the microbiological quality of the 3.2 Testing frequency product. The target limits of microbial levels for raw materi- The frequency of the tests should be established on the ba- als (systhetic compounds and minerals) are shown in Table 1. sis of a variety of factors unless otherwise speciˆed. These In general, synthetic compounds have low bioburden levels factors include: due to the high temperatures, organic solvents, etc., used in a) Dosage forms of non-sterile pharmaceutical products their manufacturing processes. Raw materials originated (dosage directions); from plants and animals in general have higher bioburdens b) Manufacturing processes; than synthetic compounds. c) Manufacturing frequency; Themicrobialqualityofthecitywaterorpuriˆedwater d) Characteristics of raw materials (natural raw material, used in the processing of active ingredients or nonsterile synthetic compound, etc.); pharmaceuticals may have a direct eŠect on the quality of the e) Batch sizes; ˆnished dosage form. This means it is necessary to keep the f) Variations in bioburden estimates (changes in batches, level of microbial contaminants in the water as low as possi- seasonal variations, etc.); ble. g) Changes aŠecting the product bioburden (changes in The microbial contamination limits for nonsterile ˆnished manufacturing process, supplier of raw materials, lot dosage forms are shown in Table 2. These microbial limits JP XV General Information / Microbiological Evaluation 1717 are based primarily on the type of dosage form, water activi- Pseudomonas ty, and so on. For oral liquids and pharmaceutical products aeruginosa having a high water activity, in general, low microbial con- tamination limits are given. In this guideline, Escherichia Varginal ≦100 ≦50 , Pseudomonas aeruginosa, Staphylococcus aureus,and coli Candida albicans are shown as speciˆed microorganisms, but Staphylococcus it is also necessary to test certain pharmaceutical products for aureus other microorganisms (for example, certain species of Clos- Candida albicans tridia, Pseudomonas, Burkholderia, Aspergillus,andEn- Otic or Topical ≦100* ≦50* Pseudomonas terobacter species) that may have the potential to present a (including aeruginosa microbial risk to patients. The selection of the speciˆed Staphylococcus microorganisms was based on following criteria; indicator patches) aureus for poor hygienic practices, pathogenic potential for route of administration, and survival proˆle of the microorganism Rectal ≦1000 ≦100 Not speciˆed and recoverability in the product. Due to the inherent preci- Oral (solid) ≦1000 ≦100 Escherichia coli sion limitations of the enumeration methods, a value exceed- ingthetargetlimitbynotmorethan2times. Oral (liquid) ≦100 ≦50 Escherichia coli 6. Microbial contamination limits for herbal drugs * For transdermal patches, the limits are expressed as cfu per Target limits of microbial contamination for herbal drugs . and herbal drug containing preparations are shown in Table 3. Category 1 indicates herbal drugs and their preparations to which boiling water is added before use, and category 2 indi- Table 3. Microbial enumeration limits for cates other herbal drugs and their preparations. In this guide- herbal drugs and their preparations line, enterobacteria and other gram-negative bacteria, Es- cherichia coli, Salmonella,andStaphylococcus aureus are Category 1 Category 2 Microorganisms mentioned as specidied microorganisms, but other microor- (cfuWgorcfuWmL) (cfuWgorcfuWmL) ganisms such as certain species of Bacillus cereus, Clostridia, Pseudomonas, Burkholderia, Asperigillus and Aerobic bacteria 107 105 species are also necessary to be tested depending on the origin Molds and 104 103 of the herbal drug raw materials or the preparation method Enterobacteria of the preparations. and other gram- * 103 negative bacteria Escherichia coli 102 not detected Table 1. Microbial enumeration limits for raw materials Salmonella not detected not detected Staphylococcus Target limit aureus ** Microorganisms (cfuWgor cfuWmL) * The limits are not speciˆed.

Total aerobic microbial count (TAMC) ≦1000

Total combined yeastsWmolds count ≦100 13. Microbiological Evaluation of (TYMC) Processing Areas for Sterile Pharmaceutical Products Table 2. Microbial enumeration limits for nonsterile ˆnished dosage forms This chapter describes the methods for the control and TAMC TYMC Examples of evaluation of microbial contamination in areas used for the Route of (cfu gor (cfu gor objectionable processing of sterile pharmaceutical products. Such process- administration W W cfuWmL) cfuWmL) microorganisms ing areas are classiˆed into critical areas and clean areas according to the required levels of air-cleanliness. A critical Inhalation ≦20 ≦20 Staphylococcus area is a deˆned space in which the airborne particulate and (liquid) aureus microorganism levels are controlled to meet grade A. The Pseudomonas cleanliness requirements for such a space extend to the sur- aeruginosa faces of the facilities and equipment which form or are locat- ed within the space, as well as to the supplied raw materials, Inhalation ≦100 ≦50 Staphylococcus chemicals, water, etc. Environmental conditions, such as (powder) aureus temperature, humidity, and air pressure, are also controlled Pseudomonas in this space when required. A clean area is a controlled space aeruginosa such that the levels of contaminants (particulates and Nasal ≦100 ≦50 Staphylococcus microorganisms) in air, gases and liquids are maintained aureus within speciˆed limits, which are less stringent than those of grade A. When sterile pharmaceutical products are manufac- 1718 Microbiological Evaluation / General Information JP XV

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

Aircleanliness Maximumnumberofairborneparticulatesperm3 at rest in operation Grade*1 ≧0.5 mm ≧0.5 mm A (Laminar-airflow zone) 3,530 3,530 B (Non laminar-airflow zone) 3,530 353,000 C 353,000 3,530,000 D 3,530,000 —*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.

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 Othercleanareas(GradeC,D) Potential productWcontainer contact areas Twice a week Non-productWcontainer 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 -heart infusion agar (or fluid) medium More than 5 days*3 agar (or fluid) medium Soybean-casein digest agar (or fluid) medium Sabouraud dextrose agar (or fluid) medium 20–259C*2 and fungi Potato-dextrose agar (or fluid) medium More than 5 days*3 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, 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 instrumentsWfacilities gloves (CFUWm3) (m ) (CFUW24–30 cm2)*3 A º10.5 º1 º1 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. JP XV General Information / Microbiological Evaluation 1719 tured, the environment, facilitiesWequipment, and personnel quirements during the preparation and ˆlling of other sterile should be routinely monitored to ensure appropriate products are generally similar to those for parenterals. microbiological control in the processing areas. The detection 2.2 Sterile products prepared aseptically after ˆltration of microorganisms should be performed under normal opera- The handling of starting materials and the preparation of tional conditions, using an appropriate sampling device, solutions should be done in a grade C environment. These ac- according to an environmental control program established tivities may be permitted in a grade D environment if addi- previously. The sampling, cultivation, counting, and evalua- tional measures are taken to minimize contamination, such as tion methods for airborne microorganisms, as well as those the use of closed vessels prior to ˆltration. After sterile ˆltra- found on surfaces, should also be chosen appropriately, tion, the product must be handled and ˆlled into containers depending on the monitoring purpose, monitoring items, and under grade A aseptic conditions. microorganisms being detected. Sampling devices, measure- 2.3 Sterile products prepared aseptically from sterile start- ment methods, media, culture conditions, frequency of ing materials monitoring, and recommended limits for environmental The handling of starting materials and all further process- microorganisms shown in this chapter are for information ing should be done under grade A conditions. only, and are not requirements. 3. Microbiological environmental monitoring program 1. Deˆnitions Environmental monitoring is especially important in steril- For the purposes of this chapter, the following deˆnitions ity assurance for sterile pharmaceutical products that are apply. manufactured by aseptic processing. The major purpose of 1) Processing areas: Areas in which actions such as culti- environmental monitoring is to predict potential deteriora- vation, extractionWpuriˆcation, weighing of raw materials, tion of the processing environment before it occurs, and to washing and drying of containers and stoppers, preparation produce high-quality, sterile pharmaceutical products under of solutions, ˆlling, sealing and packaging are performed, in- appropriate contamination control. cluding the gowning area. 3.1 Monitoring of environmental microorganisms 2) Action levels: Established microbial levels (and type of a) An environmental control program document is pre- microorganisms, if appropriate) that require immediate fol- pared for each area used for the processing of sterile phar- low-up and corrective action if they are exceeded. maceutical products. The procedures in the document in- 3) Alert levels: Established microbial levels (and type of clude: 1) items to be monitored, 2) type of microorganisms to microorganisms if appropriate) that give early warning of a be monitored, 3) frequency of monitoring, 4) methods of potential drift from normal operating conditions, but which monitoring, 5) alert and action levels, and 6) actions to be are not necessarily grounds for deˆnitive corrective action, taken when speciˆed levels are exceeded. though they may require follow-up investigation. b) The aseptic processing areas and other processing 4) Contaminants: Particulates and microorganisms caus- areas maintained under controlled conditions are monitored ing contamination by adhering to surfaces or by being incor- on a routine basis. The critical processing areas where sterile porated into materials. products are in contact with environmental air are monitored 5) Cleanliness: A quantity which indicates the condition during every operational shift. The items to be monitored in- of cleanliness of a monitored item, expressed as mass or num- clude the air, ‰oor, walls, equipment surfaces, and the gowns ber of contaminants contained in a certain volume or area. and gloves of the personnel. Table 2 shows suggested fre- 6) Contamination control: The planning, establishment quencies of the environmental monitoring. of systems and implementation activities performed in order c) The sampling devices used for monitoring environ- to maintain the required cleanliness of a speciˆed space or mental microorganisms, as well as the methods and culture surface. media, should be suitable to detect microorganisms that may 7) Shift: Scheduled period of work or production, usual- be present (aerobic bacteria, anaerobic bacteria, molds, ly less than 12 hours in length, during which operations are yeast, etc.). The cultivation conditions, such as incubation conducted by a single deˆned group of workers. temperature and time, are selected to be appropriate for the 8) Characterization of contaminants: Procedures for speciˆc growth requirements of microorganisms to be detect- classifying contaminants so that they can be diŠerentiated. In ed. Table 3 shows the culture media and cultivation condi- routine control, classiˆcation to the genus level is su‹cient; tions that are generally used in testing for environmental as required, identiˆcation to the species level is performed. microorganisms. d) The number of microorganisms in the samples is esti- 2. Air-cleanliness of processing areas for sterile pharmaceu- mated by using the Membrane Filtration, Pour Plating, tical products Spread Plating, or Serial Dilution (Most Probable Number) Airborne particulates in areas used for the processing of Methods described in the Microbial Limit Test. pharmaceutical products may act physically as a source of in- e) Table 4 shows recommended limits for environmental soluble particles in the products, and biologically as a carrier microorganisms. The alert and action levels may be adjusted of microorganisms. So, it is necessary to control strictly the if necessary after su‹cient data have been accumulated. The number of particles in the air. The air-cleanliness criteria are most important point in environmental monitoring is to con- showninTable1. ˆrm that an acceptable value of each monitoring item is 2.1 Terminally sterilized products maintained consistently. Solutions should generally be prepared in a grade C en- f) Microorganisms isolated are characterized if necessa- vironment. Solution preparation may be permitted in a grade ry. In addition, analysis of hourly or daily variation of air- D environment if additional measures are taken to minimize borne particulate numbers will provide data to assist in the contamination. For parenterals, ˆlling should be done in a control of the cleanliness of processing areas. grade A workstation in a grade B or C environment. The re- 1720 Microbiological Evaluation / General Information JP XV

3.2. Evaluation of environmental monitoring data sampler and ˆltration-type sampler. Each sampler has a) The data from the environmental monitoring are eval- speciˆc characteristics. The slit sampler is a device to trap uated on a routine basis for each area and location. The microorganisms in a known volume of air passed through a source of any discrepancy should be investigated immediately standardized slit. The air is impacted on a slowly revolving and the investigation should be documented in a report. Af- Petri dish containing a nutrient agar. The rotation rate of the ter corrective action has been taken, follow-up monitoring Petri dish and the distance from the slit to the agar surface should be done to demonstrate that the aŠected area is once are adjustable and it is possible to estimate the number of again within speciˆcation. microorganisms in the air passed through the device for a b) The report is reviewed and approved by personnel period of up to 1 hr. The Andersen sampler consists of a per- responsible for quality control and distributed to all key per- forated cover and several pieces of Petri dishes containing a sonnel associated with the aseptic processing operation. nutrient agar, and a known volume of air passed through the perforated cover impacts on the agar medium in the Petri dis- 4. Sampling devices and measuring methodology hes. The sampler is suitable for the determination of the dis- Various types of sampling devices and measurement tribution of size ranges of microorganism particulates in the methods are available for the sampling and measurement of air. The pinhole sampler resembles the slit of the slit sampler, microorganisms in the air and on surfaces, and appropriate but has pinholes in place of the slit. A known volume of air samplers and measuring methodology are selected according passed through several pinholes impacts on agar medium in a to the purpose of monitoring and the items to be monitored. slowly revolving Petri dish. The centrifugal sampler consists 4.1 Evaluation of airborne microorganisms of a propeller that pulls a known volume of air into the device a) Settle plates andthenpropelstheairoutwardtoimpactonatangentially Petri dishes of a speciˆed diameter containing a suitable placed nutrient agar strip. The sampler is portable and can be culture medium are placed at the measurement location and used anywhere, but the sampling volume of air is limited. the cover is removed there. The plates are exposed for a given See above 1) on the characteristics of the ˆltration-type time and the microorganisms deposited from the air onto the sampler. agar surface are enumerated after incubation. This method is 4.2 Measurement methods for microorganisms on surfaces not eŠective for quantitative monitoring of total airborne a) Contact plates microorganisms because it does not detect microorganisms Use a contact plate with an appropriate contact surface. that do not settle onto the surface of the culture media, and The culture medium surface should be brought into contact the settling velocity of aggregates of microorganisms is aŠect- with the sampling site for several seconds by applying ed by air currents and disturbances in air‰ow. Although the uniform pressure without circular or linear movement. After results obtained by the settle plate method are only qualita- contact and removal, the plates are covered and, as soon as tive or semi-quantitative, this method is suitable for long- possible, incubated using appropriate culture conditions. Af- term evaluation of possible contamination of products or ter a contact plate has been used, the site to which the plate devices by airborne microorganisms. was applied must be wiped aseptically to remove any adher- b) Active microbial sampling methods ent culture medium. 1) Measuring methods b) Swabs Methods in which a ˆxed volume of air is aspirated include A piece of sterilized gauze, absorbent cotton, cotton swab, ˆltration-type sampling devices and impact-type sampling or other suitable material premoistened with an appropriate devices. With the ˆlter-type sampling devices the desired rinse ‰uid is stroked in closely parallel sweeps or slowly rotat- volume of air can be collected by appropriately changing the ed over the deˆned sampling area. After sampling, the swab air intake rate or the ˆlter size. However, care must be taken is agitated in a speciˆed amount of an appropriate sterilized to ensure that sterility is maintained while the ˆlter is placed rinse ‰uid, and the rinse ‰uid is assayed for viable organisms. in and removed from the holder. When air sampling devices are used in critical areas, care must be taken to avoid distur- 5. Test methods for collection performance of a sampling bance of the air‰ow around the products. There are two types device for airborne microorganisms of ˆlters; wet-type used gelatin ˆlters and dry-type used mem- The testing of the collection performance of sampling brane ˆlters. With the dry-type ˆlters, static electricity eŠects devices for airborne microorganisms is performed in accor- can make it impossible to collect quantitatively microorgan- dance with JIS K 3836 (Testing methods for collection isms on the ˆlter. When an impact-type sampling device is e‹ciency of airborne microbe samplers) or ISO 14698 – 1 used, the following points are important: 1) The speed at (Cleanrooms and associated controlled environments. which the collected air strikes the culture medium surface Biocontamination control. General principles). must be su‹cient to capture the microorganisms, but must 6. Growth-promotion test of media and conˆrmation of an- not have an adverse eŠect on the collected microorganisms. timicrobial substances 2) A su‹cient volume of air must be sampled so that even ex- This test and conˆrmation are performed according to tremely low levels of microbiological contaminants are de- ``EŠectiveness of culture media and conˆrmation of an- tected, but the procedure must not cause a signiˆcant change timicrobial substances'' in the Microbial Limit Test. in the physical or chemical properties of the culture medium. 3) When the device is used in critical areas, care must be exer- cised to ensure that the processing of the sterile pharmaceuti- cal products is not adversely aŠected by the air disturbance. Media 2) Sampling devices Brain-heart infusion agar medium or Fluid brain-heart infu- The most commonly used samplers are as follows: Slit sion medium sampler, Andersen sampler, pinhole sampler, centrifugal Bovine brain extract powder*1 JP XV General Information / Mycoplasma Testing 1721

An amount equivalent to 200 g of calf brain Thioglycolate agar medium or thioglycolate medium I for Bovine heart extract powder*2 sterility test An amount equivalent to 250 g of the material See Sterility Test. The agar concentration of Thioglycolate Peptone 10.0 g agar medium is about 1.5z. Glucose 2.0 g Sodium chloride 5.0 g *1 Bovine brain extract powder Dried extract of bovine Disodium hydrogenphosphate fresh brain. A yellow-brown powder having a characteristic dodecahydrate 2.5 g odor. Agar 15.0 g Loss on drying:notmorethan5z. Water 1,000 mL *2 Bovine heart extract powder Dried extract of bovine Sterilize by heating in an autoclave at 1219C for 15 to 20 fresh heart. A yellow-brown powder having a characteristic min. pH after sterilization: 7.2 – 7.6. odor. Loss on drying:notmorethan5z. Fluid cooked meat medium Bovine heart extract powder*2 An amount equivalent to 450 g of the material Rinsing Liquids Peptone 20.0 g Glucose 2.0 g BuŠered sodium chloride-peptone solution (pH 7.0) Sodium chloride 5.0 g See Microbial Limit Test. Water 1,000 mL Sterilize by heating in an autoclave at 1219C for 15 to 20 LP liquid min. pH after sterilization: 7.2 – 7.6. Casein peptone 1.0 g Soybean 0.7 g Glucose peptone agar medium or Fluid glucose peptone Polysorbate 80 1.0 – 20.0 g medium Water 1,000 mL See Microbial Limit Test. Antibiotic is added if necessary. Sterilize by heating in an autoclave at 1219C for 15 to 20 min. pH after sterilization: 7.2. Nutrient agar medium or Fluid nutrient medium Meat extract 3.0 g buŠered solution (pH 7.2) Peptone 5.0 g See Microbial Limit Test. Agar 15.0 g Water 1,000 mL Ringer's solution, 1W4 concentration Sterilize by heating in an autoclave at 1219C for 15 to 20 Sodium chloride 2.25 g min. pH after sterilization: 6.6 – 7.0. Potassium chloride 0.105 g Calcium chloride dihydrate 0.16 g Potato-dextrose agar medium or Fluid potato-dextrose medi- Water 1,000 mL um Sterilize by heating in an autoclave at 1219C for 15 to 20 See Microbial Limit Test. Antibiotic is added if necessary. min. pH after sterilization: 7.0.

Reinforced clostridial agar medium or Fluid reinforced clos- -Ringer's solution tridial medium pentahydrate 0.8 g Meat extract 10.0 g Ringer's solution, 1W4 concentration 1,000 mL Peptone 10.0 g Sterilize by heating in an autoclave at 1219C for 15 to 20 3.0 g min. pH after sterilization: 6.6. Soluble starch 1.0 g Glucose 5.0 g L-Cystein hydrochloride monohydrate 0.5 g 14. Mycoplasma Testing for Sodium chloride 5.0 g Sodium acetate trihydrate 3.0 g Cell Substrates used for the Agar 15.0 g Production of Biotechnological Water 1,000 mL W For ‰uid medium, add 0.5 g of agar. Sterilize by heating Biological Products in an autoclave at 1219C for 15 to 20 min. pH after sterilization: 6.7 – 6.9. This document describes the currently available methods of mycoplasma testing that should be performed for cell sub- Sabouraud dextrose agar medium or Fluid sabouraud dex- strates that are used in the manufacture of biotechnologicalW trose medium biological products. See Microbial Limit Test. Antibiotic is added if necessary. Methods suggested for detection of mycoplasma are, A. culture method, B. indicator cell culture method, and C. Soybean-casein digest agar medium or Fluid soybean-casein polymerase chain reaction (PCR) method. digest medium Mycoplasma testing should be performed on the master See Microbial Limit Test. cell bank (MCB) and the working cell bank (WCB), as well as on the cell cultures used during the manufacturing process of 1722 Mycoplasma Testing / General Information JP XV the product. For the assessment of these cells, mycoplasma growth-inhibiting factors, such as antibiotics, these factors testing should be performed using both methods A and B. must be removed. A method such as centrifugation is recom- Method B, however, does not detect only DNA derived from mended for this purpose. mycoplasma. Therefore, if a positive result is obtained only 3) Subculture 0.2 mL of broth culture from each vessel from method B, method C can be used to determine whether on the 3rd,7th,and14th days of incubation onto two or more mycoplasma is actually present. When method C is used, it is agar plates. The plates inoculated with aerobic or anaerobic necessary to demonstrate the rationale for determining a broth cultures should be incubated aerobically or anaerobi- negative result. In such a case, the sensitivity and speciˆcity cally, respectively at 36±19C for no less than 14 days. of the method, the appropriateness of the sample prepara- 4) Examination of all plates for mycoplasma colonies tion, and the suitability of the selection of the test method, should be done microscopically on the 7th and 14th day at 100 including selection of reagents, reaction conditions and times magniˆcation or greater. primers should be taken into account. B. Indicator Cell Culture Method Prior to mycoplasma testing, the sample should be tested Using Vero cell culture substrate, pretest the suitability of to detect the presence of any factors inhibiting the growth of the method using an inoculum of no more than 100 CFU of mycoplasma. If such growth-inhibiting factors are detected M. hyorhinis DBS 1050 or M. orale CH 19299. they should be neutralized or eliminated by an appropriate An equivalent indicator cell substrate and suitable method, such as centrifugation or cell passage. mycoplasma strains may be acceptable if data demonstrate at If the test will be performed within 24 hours of obtaining least equal sensitivity for the detection of known mycoplasma thesample,thesampleshouldbestoredatatemperaturebe- contaminants. The mycoplasma strains should be obtained tween 29Cand89C. If more than 24 hours will elapse before from an o‹cial or suitably accredited agency, and handled the test is performed, the sample should be stored at －609C appropriately. The cell substrate used should be obtained or lower. from a qualiˆed cell bank and certiˆed to be mycoplasma If mycoplasma is detected, additional testing to identify free. The acquired cells should be carefully cultured and the species may be helpful in determining the source of con- propagated, and su‹cient volumes of seed stock should be tamination. prepared with the proper precautions to avoid mycoplasma A. Culture Method contamination. The stock should be tested for mycoplasma 1. Culture Medium contamination using at least one of the methods described in Both agar plates and broth are used. Each lot of agar and this document, then frozen for storage. For each test a new broth medium should be free of antibiotics except for penicil- container from the stock should be thawed and used within 6 lin. Refer to the Minimum Requirements for Biological passages. Products regarding selection of the culture media. Other cul- Indicator cell cultures should be grown on cover slips sub- ture media may be used if they fulˆll the requirements merged in culture dishes or equivalent containers for one day. described in the following section 2. Inoculate no less than 1 mL of the test sample (cell culture su- 2. Suitability of Culture Medium pernatant) into two or more of the culture dishes. Each lot of medium should be examined for mycoplasma The test should include a negative (non-infected) control growth-promoting properties. To demonstrate the capacity and two positive mycoplasma controls, such as M. hyorhinis of the media to detect known mycoplasma, each test should DBS 1050 or M. orale CH 19299. Use an inoculum of no include control cultures of at least two known species or more than 100 CFU for the positive controls. strains of mycoplasma, one of which should be a dextrose Incubate the cell cultures for 3 to 6 days at 36±19Cinan fermenter (i.e., M. pneumoniae strain FH or equivalent spe- atmosphere of air containing 5 percent carbon dioxide. cies or strains) and one of which should be an arginine Examine the cell cultures after ˆxation for the presence of hydrolyser (i.e., M. orale CH 19299 or equivalent species or mycoplasma by epi‰uorescence microscopy (400 to 600 times strains). The mycoplasma strains used for the positive control magniˆcation or greater) using a DNA-binding tests should be obtained from an o‹cial or suitably accredit- ‰uorochrome, such as bisbenzimidazole or an equivalent ed agency, and handled appropriately. Inoculate the culture stain. Compare the microscopical appearance of the test cul- medium with no more than 100 colony-forming units (CFU). tures with that of the negative and positive controls. 3. Culture and Observation Procedure 1) Inoculate no less than 0.2 mL of test sample (cell sus- 1) Aseptically place a sterilized glass cover slip into each pension) in evenly distributed amounts over the surface of cell culture dish (35 mm diameter). each of four or more agar plates. After the surfaces of the in- 2) Prepare Vero cell suspension in Eagle's minimum es- oculated plates are dried, one half of the plates should be in- sential medium containing 10 percent bovine calf serum at a cubated under aerobic conditions in an atmosphere of air concentration of 1×104 cells per 1 mL. The bovine calf containing 5 to 10 percent carbon dioxide and adequate hu- serum should be tested and conˆrmed to be free from midity, and the other half under anaerobic conditions in an mycoplasma prior to use. atmosphere of nitrogen containing 5 to 10 percent carbon di- 3) Inoculate aliquots of 2 mL of the Vero cell suspension oxide and adequate humidity at 36±19Cfornolessthan14 into each culture dish. Ensure that the cover slips are com- days. pletely submerged, and not ‰oating on the surface of the cul- 2) Inoculate no less than 10 mL of the test sample (cell ture medium. Incubate the cultures at 36±19Cinanat- suspension) into each of two vessels containing 100 mL of mosphere of air containing 5 percent carbon dioxide for one broth medium. One vessel should be incubated under aerobic day, so that the cells are attached to the glass cover slip. conditions, and the other under anaerobic conditions. 4) Replace 2 mL of the culture medium with fresh medi- If the culture medium for the sample cells contains any um, then add 0.5 mL of the test sample (cell culture super- JP XV General Information / Mycoplasma Testing 1723 natant) to each of two or more culture dishes. Perform the step PCR are nested primers from the inner portion of the same procedure for the positive (2 types of mycoplasma, such sequence. The outer and inner primers should have proven as M. hyorhinis and M. orale) and negative controls. eŠectiveness and speciˆcity as described in publications or be 5) Incubate the cultures for 3 to 6 days at 36±19Cinan validated experimentally. atmosphere of air containing 5 percent carbon dioxide. It is possible to increase the accuracy of the detection of 6) Remove the culture medium from the culture dishes, mycoplasma DNA by performing PCR tests after cultivation and add 2 mL of a mixture of acetic acid (100) and methanol of mycoplasma that may be present in samples using Vero (1:3) (ˆxative) to each dish; then, allow them to stand for 5 cells. minutes. The following is an example of a two-step PCR procedure. 7) Remove the ˆxative from each dish, then add the same The reagents and reaction conditions in this example are not amount of ˆxative again, and leave the dishes to stand for 10 exclusive. If the suitability of other reagents and conditions is minutes. veriˆed, they may be used. If another procedure is used, the 8) Remove the ˆxative and then completely air-dry all the procedure should be justiˆed and documented in detail, and dishes. the information provided should include the sensitivity and 9) Add 2 mL of bisbenzamide ‰uorochrome staining so- speciˆcity of the method. lution to each culture dish. Cover the dishes and let them Example Procedure stand at room temperature for 30 minutes. 1. Preparation of template 10) Aspirate the staining solution and rinse each dish 1) Place 600 mL of the test cell suspension (if necessary, with 2 mL of distilled water 3 times. Take out the glass cover subcultured with Vero cells) in a tube and dissolve the cells slips and dry them. with 0.1z SDS or an equivalent. Add an equal volume (600 11) Mount each cover slip with a drop of a mounting mL) of TE (10 mmolWL tris-hydrochloric acid (pH 8.0), 1 ‰uid. Blot oŠ surplus mounting ‰uid from the edges of the mmolWL EDTA) buŠer-saturated phenol, and mix. cover slips. 2) Centrifuge at 15,000 min－1 for 5 minutes at room tem- 12) Examine by epi‰uorescence microscopy at 400 to 600 perature. times magniˆcation or greater. 3) Transfer 400 mL of the supernatant to another tube, 13) Compare the microscopic appearance of the test sam- and add 10 mLof3molWL sodium acetate. ple with that of the negative and positive controls. 4) Add 1 mL (2.5 volumes) of ethanol (95) and stir 14) The test result is judged to be positive if there are thoroughly. Ice the mixture for 15 minutes, then centrifuge at more than 5 cells per 1000 (0.5z)thathaveminute‰uores- 15,000 min－1 for 10 minutes at 49C. cent spots that appear to surround, but are outside, the cell 5) Discard the supernatant and rinse the precipitate once nucleus. or twice with 200 to 300 mLof80z ethanol. Remove the rinse solution using a pipette. Centrifuge at 15,000 min－1 for 10 C. Polymerase Chain Reaction (PCR) Detection Method minutes at 49C, then remove the supernatant thoroughly and The PCR method is a highly speciˆc method that enables dry up the precipitate. the detection of trace amounts of mycoplasma DNA, and has 6) Dissolve the precipitate in 40 mL of distilled water. come to be widely used in recent years as a means of detecting 2. Perform the same procedure for the positive and nega- mycoplasma contamination. However, the sensitivity and tive controls speciˆcity depend on the procedure employed, and a positive 3. First stage of a two-step PCR result from PCR does not always indicate the presence of via- 1) Make a mixture of the heat-resistant DNA poly- ble mycoplasma. merase, dNTP solution, outer primer, and reaction buŠer so- The PCR method is based on amplifying DNA extracted lution (including Mg ions), and place 90 mLineachtube. from the cell culture with speciˆc primers so that the presence 2) Add 10 mL of the template prepared as above to each of the target DNA is detected. A two-step PCR (nested PCR) tube containing the ˆrst stage PCR solution (90 mL). is recommended in order to increase sensitivity and speciˆci- 3) Perform the DNA ampliˆcation by repeating 30 cycles ty. The tests should include both a positive control (such as of denaturation at 949C for 30 seconds, annealing at an ap- M. hyorhinis of 100 CFU or less) and a negative control. propriate temperature for the primer (559Cfortheprimerin Mycoplasma DNA from the sample of cells or cell cultures this example), and elongation at 729C for 2 minutes. Add is ampliˆed using primers which should be able to amplify drops of mineral oil or suitable equivalent as needed during some common conserved mycoplasma DNA sequence. The the reaction to prevent evaporation. ampliˆcation should be performed using an appropriate heat- 4. Second stage of a two-step PCR resistant DNA polymerase, and suitable conditions. The am- 1) Make a mixture of the heat-resistant DNA poly- pliˆed DNA can be identiˆed after agarose gel electrophore- merase, dNTP solution, inner primer, and reaction buŠer sis, followed by ethidium staining and UV irradia- solution (including Mg ions), and place 99 mLineachtube. tion of the gel. 2) Add 1 mL of the ˆrst stage PCR product from each For this method, it is important to use primers that are tube to a tube containing the second stage PCR solution (99 speciˆc to mycoplasma by choosing base sequences that are mL). well-conserved for a wide range of mycoplasma species, for 3) Perform the DNA ampliˆcation by repeating 30 cycles example, the spacer region between the 16S-23S of denaturation at 949C for 30 seconds, annealing at an ap- . propriate temperature for the primer (559Cfortheprimerin It is recommended that a two-step PCR using nested this example), and elongation at 729C for 2 minutes. Add primers should be performed to increase the sensitivity and drops of mineral oil or suitable equivalent as needed during speciˆcity, if the one-step PCR is negative. the reaction to prevent evaporation. The primers to be selected for the second stage of a two- 5. Agarose gel electrophoresis 1724 Peptide Mapping / General Information JP XV

1) Mix 10 mL of each of the ˆrst stage and second stage and the U.S. Pharmacopeia. PCR products with 2 mL of an appropriate dye as a migration Purpose and Scope marker, and perform 1z agarose gel electrophoresis. Peptide mapping is an identity test for proteins, especially 2) Stain the gel with ethidium bromide and take a photo- those obtained by r-DNA technology. It involves the chemi- graph under UV irradiation. cal or enzymatic treatment of a protein resulting in the for- 3) The test is judged to be positive if a DNA band is de- mation of peptide fragments followed by separation and tected. identiˆcation of the fragments in a reproducible manner. It is a powerful test that is capable of identifying single amino [An Example of Primer] acid changes resulting from events such as errors in the For mycoplasma detection reading of complementary DNA (cDNA) sequences or point Outer primer mutations. Peptide mapping is a comparative procedure F15?-ACACCATGGGAG(CWT)TGGTAAT-3? because the information obtained, compared to a reference R15?-CTTC(AWT)TCGACTT(CWT)CAGACCCAAGG- standard or reference material similarly treated, conˆrms the CAT-3? primary structure of the protein, is capable of detecting Inner primer whether alterations in structure have occurred, and demon- F25?-GTG(GWC)GG(AWC)TGGATCACCTCCT-3? strates process consistency and genetic stability. Each protein R25?-GCATCCACCA(AWT)A(AWT)AC(CWT)CTT-3? presents unique characteristics which must be well ( ) indicates a mixture. understood so that the scientiˆc and analytical approaches permit validated development of a peptide map that provides [PCR reaction solution] su‹cient speciˆcity. [First stage] [Second stage] This chapter provides detailed assistance in the application dNTP solution (each 1.25 mol) 16 mL16mL of peptide mapping and its validation to characterize the Primer (10 pmolWmL) F1 2 mLF22mL desired protein product, to evaluate the stability of the ex- Primer (10 pmolWmL) R1 2 mLR22mL pression construct of cells used for recombinant DNA Heat-resistant DNA poly- 2 mL2mL products and to evaluate the consistency of the overall proc- merase (1 UWmL) ess, to assess product stability as well as to ensure the identity Reaction buŠer solution 68 mL77mL of the protein product, or to detect the presence of protein 25 mmolWL magnesium chlo- 8 mL8mL variant. ride hexahydrate 10-fold buŠer solution* 10 mL10mL The Peptide Map Sterile distilled water 50 mL59mL Peptide mapping is not a general method, but involves developing speciˆc maps for each unique protein. Although *Composition of 10-fold buŠer solution the technology is evolving rapidly, there are certain methods 2-amino-2-hydroxymethyl-1,3- that are generally accepted. Variations of these methods will propanediol-hydrochloric acid be indicated, when appropriate, in speciˆc monographs. (pH 8.4) 100 mmolWL A peptide map may be viewed as a ˆngerprint of a protein Potassium chloride 500 mmolWL and is the end product of several chemical processes that pro- Magnesium chloride hexahydrate 20 mmolWL vide a comprehensive understanding of the protein being ana- Gelatin 0.1 gWL lyzed. Four major steps are necessary for the development of the procedure: isolation and puriˆcation of the protein, if the [Method of cultivating mycoplasma within Vero cells] protein is part of a formulation; selective cleavage of the pep- 1) Use at least two cell culture dishes for each of the test tide bonds; chromatographic separation of the peptides; and sample, positive control and negative control. analysis and identiˆcation of the peptides. A test sample is 2) Into each cell culture dish (diameter 35 mm), inoculate digested and assayed in parallel with a reference standard or a 2 mL of the Vero cell suspension (1×104 cells per 1 mL) in reference material. Complete cleavage of peptide bonds is Eagle's minimum essential medium containing 10 percent bo- more likely to occur when such as endoproteases vine calf serum (tested in advance using the PCR method to (e.g., ) are used, instead of chemical cleavage rea- verify that it does not contain any detectable mycoplasma gents. A map should contain enough peptides to be meaning- DNA). Incubate the cultures at 36±19Cinanatmosphereof ful. On the other hand, if there are too many fragments, the air containing 5 percent carbon dioxide for one day. map might lose its speciˆcity because many proteins will then 3) Replace the culture media with fresh media, and add havethesameproˆles. 0.5 mL of the test sample (cell culture supernatant) to each of two or more Vero cell culture dishes. Perform the same Isolation and Puriˆcation procedure for the positive (such as 100 CFU or less M. Isolation and puriˆcation are necessary for analysis of bulk hyorhinis) and negative controls. drugs or dosage forms containing interfering excipients and 4) Incubate the Vero cell culture dishes for the test sam- carrier proteins and, when required, will be speciˆed in the ple, positive and negative controls for 3 to 6 days at 36±19C monograph. Quantitative recovery of protein from the in an atmosphere of air containing 5 percent carbon dioxide. dosage form should be validated. Selective Cleavage of Peptide Bonds The selection of the approach used for the cleavage of pep- 15. Peptide Mapping tide bonds will depend on the protein under test. This selec- tion process involves determination of the type of cleavage to This test is harmonized with the European Pharmacopoeia be employed —enzymatic or chemical— and the type of JP XV General Information / Peptide Mapping 1725 cleavage agent within the chosen category. Several cleavage zation. Other pretreatments, such as the addition of agents and their speciˆcity are shown in Table 1. This list is chaotropic agents (e.g., urea) can be used to unfold the pro- not all-inclusive and will be expanded as other cleavage tein prior to mapping. To allow the to have full ac- agents are identiˆed. cess to cleavage sites and permit some unfolding of the pro- tein, it is often necessary to reduce and alkylate the disulˆde Table 1. Examples of Cleavage Agents bonds prior to digestion. Type Agent Speciˆcity Digestionwithtrypsincanintroduceambiguitiesinthe trypticmapduetosidereactionsoccurringduringthe Enzymatic Trypsin (EC 3.4.21.4) C-terminal side of digestion reaction, such as nonspeciˆc cleavage, deamida- Arg and Lys tion, disulˆde isomerization, oxidation of methionine Chymotrypsin C-terminal side of residues, or formation of pyroglutamic groups created from (EC 3.4.21.1) hydrophobic the of glutamine at the N-terminal side of a pep- residues (e.g., tide. Furthermore, peaks may be produced by autohydrolysis Leu, Met, Ala, of trypsin. Their intensities depend on the ratio of trypsin to aromatics) protein. To avoid autohydrolysis, solutions of proteases may (EC 3.4.23.1 & 2) Nonspeciˆc digest be prepared at a pH that is not optimal (e.g., at pH 5 for Lysyl endopeptidase C-terminal side of trypsin), which would mean that the enzyme would not (Lys-C Endopeptidase) Lys become active until diluted with the digest buŠer. (EC 3.4.21.50) Establishment of Optimal Digestion Conditions Factors Glutamyl endopeptidase C-terminal side of that aŠect the completeness and eŠectiveness of digestion of (from S. aureus strain V8) Glu and Asp proteins are those that could aŠect any chemical or enzymatic (EC 3.4.21.19) reactions. Peptidyl-Asp metallo N-terminal side of pH: The pH of the digestion mixture is empirically deter- endopeptidase Asp mined to ensure the optimization of the performance of the (Endoproteinase Asp-N) given cleavage agent. For example, when using cyanogen (EC 3.24.33) bromide as a cleavage agent, a highly acidic environment Clostripain (EC 3.4.22.8) C-terminal side of (e.g., pH 2, formic acid) is necessary; however, when using Arg trypsin as a cleavage agent, a slightly alkaline environment (pH 8) is optimal. As a general rule, the pH of the reaction Chemical Cyanogen bromide C-terminal side of milieu should not alter the chemical integrity of the protein Met during the digestion and should not change during the course 2-Nitro-5-thio-cyanobenzoic of the fragmentation reaction. acid N-terminal side of : A temperature between 259Cand379Cis Cys Temperature adequate for most digestions. The temperature used is o-Iodosobenzoic acid C-terminal side of intended to minimize chemical side reactions. The type of Trp and Tyr protein under test will dictate the temperature of the reaction Dilute acid Asp and Pro milieu, because some proteins are more susceptible to denatu- BNPS-skatole Trp ration as the temperature of the reaction increases. For exam- ple, digestion of recombinant bovine somatropin is conduct- Pretreatment of Sample Depending on the size or the ed at 49C, because at higher temperatures it will precipitate conˆguration of the protein, diŠerent approaches in the during digestion. pretreatment of samples can be used. For monoclonal Time: If su‹cient sample is available, a time course antibodies the heavy and light chains will need to be separat- study is considered in order to determine the optimum time to ed before mapping. If trypsin is used as a cleavage agent for obtain a reproducible map and avoid incomplete digestion. proteins with a molecular mass greater than 100,000 Da, Time of digestion varies from 2 to 30 hours. The reaction is lysine residues must be protected by citraconylation or maley- stopped by the addition of an acid which does not interfere in lation; otherwise, many peptides will be generated. the tryptic map or by freezing. Pretreatment of the Cleavage Agent Pretreatment of Amount of Cleavage Agent: Although excessive amounts cleavage agents —especially enzymatic agents— might be of cleavage agent are used to accomplish a reasonably rapid necessary for puriˆcation purposes to ensure reproducibility digestion time (i.e., 6 to 20 hours), the amount of cleavage a- of the map. For example, trypsin used as a cleavage agent will gent is minimized to avoid its contribution to the chromato- have to be treated with tosyl-L-phenylalanine chloromethyl graphic map pattern. A protein to ratio between ketone to inactivate chymotrypsin. Other methods, such as 20:1 and 200:1 is generally used. It is recommended that the puriˆcation of trypsin by HPLC or immobilization of en- cleavage agent can be added in two or more stages to op- zyme on a gel support, have been successfully used when only timize cleavage. Nonetheless, the ˆnal reaction volume a small amount of protein is available. remains small enough to facilitate the next step in peptide Pretreatment of the Protein Under certain conditions, it mapping —the separation step. To sort out digestion artifacts might be necessary to concentrate the sample or to separate that might be interfering with the subsequent analysis, a the protein from added substances and stabilizers used in for- blank determination is performed, using a digestion control mulation of the product, if these interfere with the mapping with all the reagents, except the test protein. procedure. Physical procedures used for pretreatment can in- Chromatographic Separation clude ultraˆltration, column chromatography, and lyophili- Many techniques are used to separate peptides for 1726 Peptide Mapping / General Information JP XV mapping. The selection of a technique depends on the protein mobile phase are used on the basis of their convenience of use being mapped. Techniques that have been successfully used and improved detector responses. Optimal composition of a for separation of peptides are shown in Table 2. In this sec- mobile phase to obtain clear resolution of each peak is some- tion, a most widely used reverse-phase High Performance times di‹cult to establish. Mobile phases for which slight Liquid Chromatographic (RP-HPLC) method is described as changes in component ratios or in pH signiˆcantly aŠect one of the procedures of chromatographic separation. retention times of peaks in peptide maps should not be used in isocratic HPLC systems. Table 2. Techniques Used for the Separation of Peptides Other Parameters Temperature control of the column is usually necessary to achieve good reproducibility. The ‰ow Reverse-Phase High Performance Liquid Chromatography rates for the mobile phases range from 0.1 to 2.0 mL per (RP-HPLC) minute, and the detection of peptides is performed with a UV Ion-Exchange Chromatography (IEC) detector at 200 to 230 nm. Other methods of detection have Hydrophobic Interaction Chromatography (HIC) been used (e.g., postcolumn derivatization), but they are not Polyacrylamide Gel Electrophoresis (PAGE), nondenaturat- as robust or versatile as UV detection. ing Validation This section provides an experimental SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) means for measuring the overall performance of the test Capillary Electrophoresis (CE) method. The acceptance criteria for system suitability depend Paper Chromatography-High Voltage (PCHV) on the identiˆcation of critical test parameters that aŠect data High-Voltage Paper Electrophoresis (HVPE) interpretation and acceptance. These critical parameters are also criteria that monitor peptide digestion and peptide anal- The purity of solvents and mobile phases is a critical factor ysis. An indicator that the desired digestion endpoint was in HPLC separation. HPLC-grade solvents and water that achieved is by the comparison with a Reference Standard, are commercially available, are recommended for RP- which is treated exactly as the article under test. The use of a HPLC. Dissolved gases present a problem in gradient reference standard or reference material in parallel with the systems where the solubility of the gas in a solvent may be less protein under test is critical in the development and establish- in a mixture than in a single solvent. Vacuum degassing and ment of system suitability limits. In addition a specimen agitation by sonication are often used as useful degassing chromatogram should be included with the Reference Stan- procedures. When solid particles in the solvents are drawn dard or Reference Material for additional comparison pur- into the HPLC system, they can damage the sealing of pump poses. Other indicators may include visual inspection of pro- valves or clog the top of the chromatographic column. Both tein or peptide solubility, the absence of intact protein, or pre- and post-pump ˆltration is also recommended. measurement of responses of a digestion-dependent peptide. Chromatographic Column The selection of a chromato- The critical system suitability parameters for peptide analysis graphic column is empirically determined for each protein. will depend on the particular mode of peptide separation and Columns with 100 Å or 300 Å pore size with silica support detection and on the data analysis requirements. can give optimal separation. For smaller peptides, octylsilane When peptide mapping is used as an identiˆcation test, the chemically bonded to totally porous silica articles, 3 to 10 mm system suitability requirements for the identiˆed peptides in diameter (L7) and octadecylsilane chemically bonded to covers selectivity and precision. In this case, as well as when porous silica or ceramic micro-particles, 3 to 10 mmindi- identiˆcation of variant protein is done, the identiˆcation of ameter (L1) column packings are more e‹cient than the butyl the primary structure of the peptide fragments in the peptide silane chemically bonded to totally porous silica particles, 5 map provides both a veriˆcation of the known primary struc- to 10 mmindiameter(L26)packing. ture and the identiˆcation of protein variants by comparison Solvent The most commonly used solvent is water with with the peptide map of the reference standard/reference acetonitrile as the organic modiˆer to which less than 0.1z material for the speciˆed protein. The use of a digested refer- tri‰uoroacetic acid is added. If necessary, add isopropyl ence standard or reference material for a given protein in the alcohol or n-propyl alcohol to solubilize the digest compo- determination of peptide resolution is the method of choice. nents, provided that the addition does not unduly increase For an analysis of a variant protein, a characterized mixture the viscosity of the components. of a variant and a reference standard or reference material Mobile Phase BuŠered mobile phases containing phos- can be used, especially if the variant peptide is located in a phate are used to provide some ‰exibility in the selection of less-resolved region of the map. The index of pattern con- pH conditions, since shifts of pH in the 3.0 to 5.0 range sistency can be simply the number of major peptides detect- enhance the separation of peptides containing acidic residues ed. Peptide pattern consistency can be best deˆned by the (e.g., glutamic and aspartic acids). Sodium or potassium resolution of peptide peaks. Chromatographic parameters phosphates, ammonium acetate, phosphoric acid, and a pH —such as peak-to-peak resolution, maximum peak width, between 2 and 7 (or higher for polymer-based supports) have peak area, peak tailing factors, and column e‹ciency— may also been used with acetonitrile gradients. Acetonitrile con- be used to deˆne peptide resolution. Depending on the pro- taining tri‰uoroacetic acid is used quite often. tein under test and the method of separation used, single pep- Gradient Selection Gradients can be linear, nonlinear, or tide or multiple peptide resolution requirements may be include step functions. A shallow gradient is recommended in necessary. order to separate complex mixtures. Gradients are optimized The replicate analysis of the digest of the reference to provide clear resolution of one or two peaks that will standard or reference material for the protein under test become ``marker'' peaks for the test. yields measures of precision and quantitative recovery. Isocratic Selection Isocratic HPLC systems using a single Recovery of the identiˆed peptides is generally ascertained by the use of internal or external peptide standards. The JP XV General Information / pH Test for Gastrointestinal 1727 precision is expressed as the relative standard deviation velopment of a peptide map for the speciˆed protein. (RSD). DiŠerences in the recovery and precision of the Analysis and Identiˆcation of Peptides identiˆed peptides are expected; therefore, the system This section gives guidance on the use of peptide mapping suitability limits will have to be established for both the during development in support of regulatory applications. recovery and the precision of the identiˆed peptides. These The use of a peptide map as a qualitative tool does not limits are unique for a given protein and will be speciˆed in require the complete characterization of the individual the individual monograph. peptide peaks. However, validation of peptide mapping in Visual comparison of the relative retention times, the peak support of regulatory applications requires rigorous cha- responses (the peak area or the peak height), the number of racterization of each of the individual peaks in the peptide peaks, and the overall elution pattern is completed initially. It map. Methods to characterize peaks range from N-terminal is then complemented and supported by mathematical analy- sequencing of each peak followed by amino acid analysis to sis of the peak response ratios and by the chromatographic the use of mass spectroscopy (MS). proˆle of a 1:1 (v/v) mixture of sample and reference stan- For characterization purposes, when N-terminal sequenc- dard or reference material digest. If all peaks in the sample ing and amino acids analysis are used, the analytical separa- digest and in the reference standard or reference material tion is scaled up. Since scale-up might aŠect the resolution of digest have the same relative retention times and peaks peptide peaks, it is necessary, using empirical data, to assure response ratios, then the identity of the sample under test is that there is no loss of resolution due to scale-up. Eluates cor- conˆrmed. responding to speciˆc peptide peaks are collected, vacuum- If peaks that initially eluted with signiˆcantly diŠerent rela- concentrated, and chromatographed again, if necessary. tive retention times are then observed as single peaks in the Amino acid analysis of fragments may be limited by the pep- 1:1 mixture, the initial diŠerence would be an indication of tide size. If the N-terminus is blocked, it may need to be system variability. However, if separate peaks are observed cleared before sequencing. C-terminal sequencing of proteins in the 1:1 mixture, this would be evidence of the nonequiva- in combination with carboxypeptidase and MALDITOF-MS lence of the peptides in each peak. If a peak in the 1:1 mixture can also be used for characterization purposes. is signiˆcantly broader than the corresponding peak in the The use of MS for characterization of peptide fragments is sample and reference standard or reference material digest, it by direct infusion of isolated peptides or by the use of on-line may indicate the presence of diŠerent peptides. The use of LC-MS for structure analysis. In general, it includes elec- computer-aided pattern recognition software for the analysis trospray and matrix-assisted laser desorption ionization cou- of peptide mapping data has been proposed and applied, but pled to time-of-‰ight analyzer (MALDITOF) as well as fast issues related to the validation of the computer software atom bombardment (FAB). Tandem MS has also been used preclude its use in a compendial test in the near future. Other to sequence a modiˆed protein and to determine the type of automated approaches have been used that employ mathe- amino acid modiˆcation that has occurred. The comparison matical formulas, models, and pattern recognition. Such ap- of mass spectra of the digests before and after reduction pro- proaches are, for example, the automated identiˆcation of vides a method to assign the disulˆde bonds to the various compounds by IR spectroscopy and the application of diode- sulfhydryl-containing peptides. array UV spectral analysis for identiˆcation of peptides. If regions of the primary structure are not clearly demon- These methods have limitations due to inadequate resolu- strated by the peptide map, it might be necessary to develop a tions, co-elution of fragments, or absolute peak response secondary peptide map. The goal of a validated method of diŠerences between reference standard or reference material characterization of a protein through peptide mapping is to and sample fragments. reconcile and account for at least 95z of the theoretical com- The numerical comparison of the retention times and peak position of the protein structure. areas or peak heights can be done for a selected group of relevant peaks that have been correctly identiˆed in the peptide maps. Peak areas can be calculated using one peak showing relatively small variation as an internal reference, 16. pH Test for Gastrointestinal keeping in mind that peak area integration is sensitive to baseline variation and likely to introduce error in the Medicine analysis. Alternatively, the percentage of each peptide peak In this test, medicine for the stomach and bowels, which is height relative to the sum of all peak heights can be calculated for the sample under test. The percentage is then compared to said to control stomach acid, is stirred in a ˆxed amount of that of the corresponding peak of the reference stan- the 0.1 molWL hydrochloric acid for a ˆxed duration, and the pH value of this solution is obtained. The pH value of a dard/reference material. The possibility of auto-hydrolysis of trypsin is monitored by producing a blank peptide map, stomach medicine will be based on the dose and the dosage of that is, the peptide map obtained when a blank solution is the medicine (when the dosage varies, a minimum dosage is treated with trypsin. used) and expressed in the pH value obtained from the test performed by the following procedure. The minimum requirement for the qualiˆcation of peptide mapping is an approved test procedure that includes system Preparation of Sample suitability as a test control. In general, early in the regulatory Solid medicine which conforms to the general regulations process, qualiˆcation of peptide mapping for a protein is for medicine (the powdered medicine section) can be used as su‹cient. As the regulatory approval process for the protein a sample. When the medicine is in separate packages, the progresses, additional qualiˆcations of the test can include a content of 20 or more packages is accurately weighed to cal- partial validation of the analytical procedure to provide culate the average mass for one dose and mixed evenly to assurance that the method will perform as intended in the de- 1728 Plastic Containers / General Information JP XV make a sample. For granules and similar types in separate must be su‹ciently small from the viewpoint of safety. packages, among the solid medicine which does not conform The container should have a certain level of physical to the general regulations for medicine (the powdered medi- properties such as hardness, ‰exibility, shock resistance, ten- cine section), the content of 20 or more packages is accurately sile strength, tear strength, bending strength, heat resistance weighed to calculate the average mass for one dose and is and the like, in accordance with the intended usage. then powdered to make sample. For granules and similar The quality of the pharmaceutical products contained in types not in separate packages, among solid medicine which the container must not deteriorate during storage. For exam- does not conform to the general regulations for medicine (the ple, in the case of pharmaceutical products which are unsta- powdered medicine section), 20 doses or more are powdered ble to light, the container should provide a su‹cient level of to make a sample. For capsules and tablets, 20 doses or more light shielding. In the case of pharmaceutical products which are weighed accurately to calculate the average mass for one are easily oxidized, the container material should not allow dose or average mass and then powdered to make a sample. the permeation of oxygen. In the case of aqueous pharmaceu- Liquid medicine is generously mixed to make a sample. tical products and pharmaceutical products that must be kept dry, the container material should not allow the permeation Procedure of water vapor. In addition, care should be taken that the Put50mLofthe0.1molWL hydrochloric acid with the container is impermeable to the solvent in the case of solvents molarity coe‹cient adjusted to 1.000, or equivalent 0.1 molW other than water. The concentration of the pharmaceuticals L hydrochloric acid with its volume accurately measured in a must not be decreased by more than a certain level due to the 100-mL beaker. Stir this solution with a magnetic stirrer and absorption of the pharmaceuticals on the surface of the con- a magnetic stirrer rotator (35 mm length, 8 mm diameter) at tainer, the migration of the pharmaceuticals into the inside of the speed of about 300 revolutions per minute. While stirring, the material of the container, or the loss of pharmaceuticals add the accurately weighed one-dose sample. After 10 through the container. Also, the pharmaceutical products minutes, measure the pH value of the solution using the pH contained therein must not be degraded by an interaction Determination. The solution temperature should be main- with the material of the container. tained at 37±29C throughout this operation. The container should not be deformed, should not de- teriorate and should not be degraded by the pharmaceutical products contained therein. Unacceptable loss of function of 17. Plastic Containers for the container should not result from possible high tempera- ture or low temperature or cycles thereof encountered during Pharmaceutical Products storage or transportation. The container should be of a required level of transparen- Various kinds of plastics are used in the manufacture of cy, when it is necessary to examine foreign insoluble matter containers for pharmaceutical products. Such plastics should andWor turbidity of the pharmaceutical products by visual ob- not alter the e‹cacy, safety or stability of the pharmaceutical servation. products. In selecting a suitable plastic container, it is desira- In the case of pharmaceutical products which must be steri- ble to have full information on the manufacturing processes lized, it is required to satisfy the above-mentioned essential of the plastic container including the substances added. Since requirements of the container after the sterilization if there is each plastic has speciˆc properties and a wide variety of phar- a possibility that the quality of the container may change maceutical products may be stored in containers made from after the sterilization. There should not be any residue or it, the compatibility of plastic containers with pharmaceutical generation of new toxic substances of more than certain risk products should be judged for each combination of container level after the sterilization. In addition, the container should and the speciˆc pharmaceutical product to be contained not have any inappropriate structure andWor material that therein. This judgement should be carried out by verifying might result in any bacterial contamination of the phar- that a type sample of the container for the pharmaceutical maceutical products contained therein during storage and preparation fulˆlls the essential requirements, i.e., the design transportation after sterilization. speciˆcations, according to experiments andWor scientiˆc documentation, etc. In addition, the compatibility must be Toxicity Evaluation of Container at Design Phase ensured based upon an appropriate quality assurance system. For design veriˆcation, the toxicity of the container should Furthermore, in introducing a plastic container, it is be evaluated. For the toxicity evaluation, it is desirable to desirable that proper disposal after use is taken into consider- select appropriate test methods and criteria for the evalua- ation. tion, and to clarify the rationales for the selection. The tests should be conducted using samples of the whole or a part of Essential Requirements in Designing Plastic Containers for the prototype container. If the container consists of plural Pharmaceutical Products parts of diŠerent materials, each part should be tested The plastic material for the container should be of high separately. Such materials as laminates, composites, and like quality. Therefore, recycled plastic materials, which are of are regarded as a single material. To test containers made of unknown constitution, must not be used. such materials, it is recommended to expose the inner surface The leachables or migrants from the container should not of the container, which contacts the pharmaceutical products alter the e‹cacy or stability of the pharmaceutical products containedtherein,totheextractionmediausedinthetestsas contained therein. In addition, the possible toxic hazards of far as possible. the leachables or migrants should not exceed a given level. The tests required for the toxicity evaluation of the con- Furthermore, the amounts of leachable or migratable chemi- tainer are diŠerent depending upon the tissue to which the cal substances, such as monomers and additives, from the pharmaceutical products contained therein are to be applied. containers to the pharmaceutical products contained therein JP XV General Information / Powder Flow 1729

The following tests are required for containers for 1) preparations contacting blood: Acute toxicity test, cytotoxicity test, sensitization test and hemolysis test 18. Powder Flow 2) preparations contacting skin or mucous membranes: This test is harmonized with the European Pharmacopoeia Cytotoxicity test and sensitization test and the U.S. Pharmacopeia. The parts of the text that are not 3) liquid orally administered preparations: harmonized are marked with symbols ( ). Cytotoxicity test  Four commonly reported methods for testing powder ‰ow It is recommended to conduct the tests in accordance with are (1) angle of repose, (2) compressibility index of Hausner the latest versions of the standard test methods on medical ratio, (3) ‰ow rate through an oriˆce, and (4) shear cell. In devices and materials published in Japan and other countries. addition, numerous variations of each of these basic methods Those standard test methods are listed for information: are available. (A) Selection of Tests In general, any method of measuring powder ‰ow should ・Guidelines for Basic Biological Tests of Medical Devices be practical, useful, reproducible, sensitive, and yield and Materials (PAB Notiˆcation, YAKU-KI NO.99, June 27, meaningful results. It bears repeating that no one simple 1995), Principles and selection of tests powder ‰ow method will adequately or completely character- ・ISO 10993-1: Biological evaluation of medical ize the wide range of ‰ow properties. An appropriate strategy devices—Evaluation and testing may well be the use of multiple standardized test methods to (B) Acute Toxicity Test characterize the various aspects of powder ‰ow as needed by ・ASTM F750-82: Standard practice for evaluating material the pharmaceutical scientist. extracts by systemic injection in the mice ・BS5736: Part 3 Method of test for systemic toxicity; as- 1. Angle of Repose sessment of acute toxicity of extracts from medical devices The angle of repose is the constant, three-dimensional an- ・USP 24 <88> Biological reactivity tests, in vivo gle (relative to the horizontal base) assumed by a cone-like (C) Cytotoxicity Test pile of material formed by any of several diŠerent methods. ・Guidelines for Basic Biological Tests of Medical Devices The angle of repose has been used in several branches of and Materials, I. Cytotoxicity Test 10. Cytotoxicity test using science to characterize the ‰ow properties of solids. Angle of extract of medical device or material repose is a characteristic related to interparticulate friction, ・ISO 10993-5: Biological evaluation of medical or resistance to movement between particles. Angle of repose devices—Tests for cytotoxicity : in vitro methods test results are reported to be very dependent upon the ・USP 24 <87> Biological reactivity tests, in vitro method used. Experimental di‹culties arise due to segrega- (D) Hemolysis Test tion of material and consolidation or aeration of the powder ・Guidelines for Basic Biological Tests of Medical Devices astheconeisformed. and Materials, VII. Hemolysis Test 1.1 Basic Methods for Angle of Repose ・ISO 10993-4: Biological evaluation of medical A variety of angle of repose test methods are reported in devices—Selection of tests for interaction with blood. Annex the literature. The most common methods for determining D the static angle of repose can be classiˆed based on two im- ・ASTM F756-82: Standard practice for assessment of portant experimental variables: hemolytic properties of materials (1) The height of the ``funnel'' through which the powder (E) Sensitization Test passes may be ˆxed relative to the base, or the height may be ・Guidelines for Basic Biological Tests of Medical Devices varied as the pile forms. and Materials, II. Sensitization Test (2) The base upon which the pile forms may be of ˆxed ・ISO 10993-10: Biological evaluation of medical diameter or the diameter of the powder cone may be allowed devices—Tests for irritation and sensitization to vary as the pile forms. Test Results to be Recorded per Production Unit 1.2 Variations in Angle of Repose Methods At the line production phase, it is required to establish the In addition to the above methods, variations of them have values of acceptable limits on at least the test items men- been used to some extent. tioned below and to record the test results of each production ･Drained angle of repose. This is determined by allowing an unit of plastic containers for pharmaceutical products. In ad- excess quantity of material positioned above a ˆxed di- dition, it is desirable to clarify the rationales for setting the ameter base to ``drain'' from the container. Formation of a values of limits. However, these requirements should not be cone of powder on the ˆxed diameter base allows determina- applied to orally administered preparations except liquid tion of the drained angle of repose. ones. ･Dynamic angle of repose. This is determined by ˆlling a 1) Combustion Tests: Residue of ignition, heavy metals. cylinder (with a clear, ‰at cover on one end) and rotating it If necessary, the amounts of the speciˆed metals (lead, cad- at a speciˆed speed. The dynamic angle of repose is the an- mium, etc.) gle (relative to the horizontal) formed by the ‰owing pow- 2) Extraction Tests: pH, ultraviolet absorption spectra, der. The internal angle of kinetic friction is deˆned by the -reducing substances, foaming, plane separating those particles sliding down the top layer of non-volatile residue the powder and those particles that are rotating with the 3) Cytotoxicity Test drum (with roughened surface). 4) Any other necessary tests for the speciˆc container for 1.3 Angle of Repose General Scale of Flowability aqueous infusions. 1730 Powder Flow / General Information JP XV

While there is some variation in the qualitative description powder. of powder ‰ow using the angle of repose, much of the phar- 2.1 Basic Methods for Compressibility Index and Haus- maceutical literature appears to be consistent with the classiˆ- ner Ratio cation by Carr1, which is shown in Table 1. There are exam- While there are some variations in the method of determin- ples of formulations with an angle of repose in the range of ing the compressibility index and Hausner ratio, the basic 40 to 50 degrees that manufactured satisfactorily. When the procedure is to measure (1) the unsettled apparent volume, angle of repose exceeds 50 degrees, the ‰ow is rarely accepta- V , and (2) the ˆnal tapped volume, V ,ofthepowderafter ble for manufacturing purposes. o f tapping the material until no further volume changes occur (refer to the General Test Method, 3.01 Determination of Table 1 Flow Properties and Corresponding Bulk and Tapped ). The compressibility index and Angles of Repose1) the Hausner ratio are calculated as follows:

Flow Property Angle of Repose (degrees) Compressibility Index＝100×｛(Vo－Vf)/Vo｝ Hausner Ratio＝V /V Excellent 25–30 o f Good 31–35 Alternatively, the compressibility index and Hausner ratio Fair 36–40 may be calculated using measured values for bulk density

Passable 41 – 45 (rB) and tapped density (rT)asfollows: Poor 46–55

Very poor 56 – 65 Compressibility Index＝100×｛(rT－rB)/rT｝ Very, very poor À66

Hausner Ratio＝rT/rB 1.4 Experimental Considerations for Angle of Repose Angle of repose is not an intrinsic property of the powder, In a variation of these methods, the rate of consolidation is that is to say, it is very much dependent upon the method sometimes measured rather than, or in addition to, the used to form the cone of powder. On this subject, the existing change in volume that occurs on tapping. For the compres- literature raises these important considerations: sibility index and the Hausner ratio, the generally accepted ･The peak of the cone of powder can be distorted by the im- scale of ‰owability is given in Table 2. pact of powder from above. By carefully building the pow- 1) der cone, the distortion caused by impact can be minimized. Table 2. Scale of Flowability ･The nature of the base upon which the powder cone is Compressibility Flow Character Hausner Ratio formed in‰uences the angle of repose. It is recommended Index (z) that the powder cone be formed on a ``common base'', which can be achieved by forming the cone of powder on a ≦10 Excellent 1.00 – 1.11 layer of powder. This can be done by using a base of ˆxed 11–15 Good 1.12–1.18 diameter with a protruding outer edge to retain a layer of 16–20 Fair 1.19–1.25 powder upon which the cone is formed. 21–25 Passable 1.26–1.34 26–31 Poor 1.35–1.45 1.5 Recommended Procedure for Angle of Repose 32–37 Verypoor 1.46–1.59 Form the angle of repose on a ˆxed base with a retaining »38 Very, very poor À1.60 lip to retain a layer of powder on the base. The base should  be free of vibration. Vary the height of the funnel to carefully build up a symmetrical cone of powder. Care should be taken 2.2 Experimental Considerations for the Compressibility to prevent vibration as the funnel is moved. The funnel Index and Hausner Ratio heightshouldbemaintainedapproximately2–4cmfromthe Compressibility index and Hausner ratio are not intrinsic top of the powder pile as it is being formed in order to properties of the powder, that is to say, they are dependent minimize the impact of falling powder on the tip of the cone. upon the methodology used. The existing literature points If a symmetrical cone of powder cannot be successfully or out several important considerations aŠecting the determina- reproducibly prepared, this method is not appropriate. De- tion of the (1) unsettled apparent volume, Vo,(2)theˆnal terminetheangleofreposebymeasuringtheheightofthe tapped volume, Vf, (3) the bulk density, rB,and(4)the cone of powder and calculating the angle of repose, a, from tapped density, rT: the following equation: ･Thediameterofthecylinderused tan a＝height/0.5 base ･The number of times the powder is tapped to achieve the tapped density 2. Compressibility Index and Hausner Ratio ･Themassofmaterialusedinthetest In recent years the compressibility index and the closely ･Rotation of the sample during tapping related Hausner ratio have become the simple, fast and popu- lar methods of predicting powder ‰ow characteristics. The 2.3 Recommended Procedure for Compressibility Index compressibility index bas been proposed as an indirect meas- and Hausner Ratio ure of bulk density, size and shape surface area, moisture Use a 250-mL volumetric cylinder with a test sample weight content, and cohesiveness of materials because all of these of 100 grams. Smaller weights and volumes may be used, but can in‰uence the observed compressibility index. The com- weight of the test sample and volume of the cylinder used pressibility index and the Hausner ratio are determined by should be described with the results. An average of three de- measuring both the bulk volume and tapped volume of a terminations is recommended. JP XV General Information / Powder Flow 1731

`head' of powder) is much greater than the diameter of the 3. Flow through an Oriˆce oriˆce, the ‰ow rate is virtually independent of the powder The ‰ow rate of a material depends upon many factors, head. Use a cylinder as the container because the cylinder some of which are particle-related and some related to the material should have little eŠect on ‰ow. Powder ‰ow rate process. Monitoring the rate of ‰ow of material through an often increases when the height of the powder column is less oriˆce has been proposed as a better measure of powder than two times the diameter of the column. The oriˆce should ‰owability. Of particular signiˆcance is the utility of be circular and the cylinder should be free of vibration. monitoring ‰ow continuously since pulsating ‰ow patterns General guidelines for dimensions of the cylinder are as fol- have been observed even for free ‰owing materials. Changes lows: in ‰ow rate as the container empties can also be observed. ･Diameter of opening À6 times the diameter of the particles Empirical equations relating ‰ow rate to the diameter of the ･Diameter of the cylinder À2 times the diameter of the open- opening, particle size, and particle density have been deter- ing mined. However, determining the ‰ow rate through an oriˆce Use of a hopper as the container may be appropriate and is useful only with free-‰owing materials. representative of ‰ow in a production situation. It is not ad- The ‰ow rate through an oriˆce is generally measured as visable to use a funnel, particularly one with a stem, because the mass per time ‰owing from any of a number of types of ‰ow rate will be determined by the size and length of the stem containers (cylinders, funnels, hoppers). Measurement of the as well as the friction between the stem and the powder. A ‰ow rate can be in discrete increments or continuous. truncated cone may be appropriate, but ‰ow will be in- 3.1 Basic Methods for Flow through an Oriˆce ‰uenced by the powder—wall friction coe‹cient, thus, selec- There are a variety of methods described in the literature. tion of an appropriate construction material is important. The most common for determining the ‰ow rate through an For the opening in the cylinder, use a ‰at-faced bottom oriˆce can be classiˆed based on three important experimen- plate with the option to vary oriˆce diameter to provide maxi- tal variables: mum ‰exibility and better ensure a powder-over-powder ‰ow (1) The type of container used to contain the powder. pattern. Rate measurement can be either discrete or continu- Common containers are cylinders, funnels and hoppers from ous. Continuous measurement using an electronic balance production equipment. can more eŠectively detect momentary ‰ow rate variations. (2) The size and shape of the oriˆce used. The oriˆce di- 4. Shear Cell Methods ameter and shape are critical factors in determining powder A variety of powder shear testers and methods that permit ‰ow rate. more thorough and precisely deˆned assessment of powder (3) The method of measuring powder ‰ow rate. Flow rate ‰ow properties have been developed. Shear cell methodology can be measured continuously using an electronic balance has been used extensively in the study of pharmaceutical and with some sort of recording device (strip chart recorder, materials. From these methods, a wide variety of parameters computer). It can also be measured in discrete samples (for can be obtained, including the yield loci representing the example, the time it takes for 100 grams of powder to pass shear stress-shear strain relationship, the angle of internal through the oriˆce to the nearest tenth of a second or the friction, the unconˆned yield strength, the tensile strength, amount of powder passing through the oriˆce in 10 seconds and a variety of derived parameters such as the ‰ow factor to the nearest tenth of a gram). and other ‰owability indices. Because of the ability to more 3.2 Variations in Methods for Flow through an Oriˆce precisely control experimental parameters, ‰ow properties Either mass ‰ow rate or volume ‰ow rate can be deter- can also be determined as a function of consolidation load, mined. Mass ‰ow rate is the easier of the methods, but it time, and other environmental conditions. biases the results in favor of high-density materials. Since die 4.1 Basic Methods for Shear Cell ˆll is volumetric, determining volume ‰ow rate may be One type of shear cell is the cylindrical shear cell which is preferable. split horizontally, forming a shear plane between the lower 3.3 General Scale of Flowability for Flow through an stationary base and the upper movable portion of the shear Oriˆce cell ring. After powder bed consolidation in the shear cell (us- No general scale is available because ‰ow rate is critically ing a well-deˆned procedure), the force necessary to shear the dependent on the method used to measure it. powder bed by moving the upper ring is determined. Annular shear cell designs oŠer some advantages over the cylindrical 3.4 Experimental Considerations for Flow through an shear cell design, including the need for less material. A dis- Oriˆce advantage, however, is that because of its design, the powder Flow rate through an oriˆce is not an intrinsic property of bed is not sheared as uniformly because material on the out- the powder. It is very much dependent upon the methodology side of the annulus is sheared more than material in the inner used. The existing literature points out several important con- region. A third type of shear cell (plate-type) consists of a siderations aŠecting these methods: thin sandwich of powder between a lower stationary rough ･The diameter and shape of the oriˆce surface and an upper rough surface that is moveable. ･The type of container material (metal, glass, plastic) All of the shear cell methods have their advantages and dis- ･The diameter and height of the powder bed. advantages. A signiˆcant advantage of shear cell methodolo- 3.5 Recommended Procedure for Flow through an gyingeneralisagreaterdegreeofexperimentalcontrol.The Oriˆce methodology generally is rather time-consuming and requires Flow rate through an oriˆce can be used only for materials signiˆcant amounts of material and a well-trained operator. that have some capacity to ‰ow. It is not useful for cohesive materials. Provided that the height of the powder bed (the 4.2 Recommendations for Shear Cell 1732 Preservatives-EŠectiveness / General Information JP XV

Because of the diversity of available equipment and ex- as opportunistic pathogens. In addition to these strains desig- perimental procedures, no speciˆc recommendations regard- nated as test microorganisms, it is further recommended to ing methodology are presented in this chapter. It is recom- use strains that might contaminate the product and grow on mended that the results of powder ‰ow characterization using or in it, depending on its characteristics. For the test microor- shear cell methodology include a complete description of eq- ganisms received from coordinated collections of microor- uipment and methodology used. ganisms, one passage is deˆned as the transfer of microor- ganisms from an established culture to fresh medium, and References microorganisms subjected to not more than ˆve passages 1) Carr R.L.: Evaluating ‰ow properties of solids. Chem. should be used for the tests. Single-strain challenges rather Eng., 72: 163–168(1965). than mixed cultures should be used. The test strains can be harvested by growth on solid agar or liquid media. Cultures on agar plate media: Inoculate each of the ˆve test 19. Preservatives-EŠectiveness strains on the surface of agar plates or agar slants. For growth of bacteria, use Soybean-Casein Digest Agar Medi- Tests um, and for yeasts and moulds, use Sabouraud Agar, Glucose-Peptone Agar or Potato Dextrose Agar Medium. In- The purpose of the Preservatives-EŠectiveness Tests is to cubate bacterial cultures at 309Cto359C for 18 to 24 hours, assess microbiologically the preservative e‹cacy, either due the culture of C. albicans at 209Cto259C for 40 to 48 hours to the action of product components themselves or any added and the culture of A. niger at 209Cto259C for one week or preservative(s), for multi-dose containers. The e‹cacy of the until good sporulation is obtained. Harvest these cultured preservatives is assessed by direct inoculation and mixing of cells aseptically using a platinum loop, etc. Suspend the col- the test strains in the product, and titration of survival of the lected cells in sterile physiological saline or in 0.1z peptone test strains with time. water and adjust the viable cell count to about 108 microor- Preservatives must not be used solely to comply with GMP ganisms per mL. In the case of A. niger, suspend the cultured for drugs or to reduce viable aerobic counts. In addition, cells in sterile physiological saline or 0.1z peptone water preservatives themselves are toxic substances. Therefore, containing 0.05 wWvz of polysorbate 80 and adjust the spore preservatives must not be added to products in amounts count to about 108 per mL. Use these suspensions as the inoc- which might jeopardize the safety of human beings, and con- ula. sideration must be given to minimizing the amounts of Liquid cultures: After culturing each of the four strains ex- preservative used. These tests are commonly used to verify cept for A. niger in a suitable medium, remove the medium that products maintain their preservative eŠectiveness at the by centrifugation. Wash the cells in sterile physiological sa- design phase of formulation or in the case of periodic line or 0.1z peptone water and resuspend them in the same monitoring. Although these tests are not performed for lot solution with the viable cell or spore count of the inoculum release testing, the e‹cacy of the preservative present in the adjusted to about 108 per mL. product packaged in the ˆnal containers should be veriˆed When strains other than the ˆve listed above are cultured, throughout the entire dating period. select a culture medium suitable for growth of the strain con- cerned. The cell suspension may also be prepared by a 1. Products and their Categories method suitable for that strain. Use the inoculum suspen- The products have been divided into two categories for sions within 24 hours after they have been prepared from the these tests. Category I products are those made with aqueous cultivations on agar plate media or in liquid media. Store the bases or vehicles, and Category II products are those made inoculum suspensions in a refrigerator if it is not possible to with nonaqueous bases or vehicles. Oil-in-water emulsions inoculate them into the test specimens within 2 hours. Titrate are considered Category I products, and water-in-oil emul- the viable cell count of the inocula immediately before use, sions Category II products. Category I is further divided into and then calculate the theoretical viable cell count per mL (g) three subtypes depending on the dosage forms. of the product present just after inoculation. Category IA: Injections and other sterile parenterals Category IB: Nonsterile parenterals 3. Test Procedure Category IC: Oral products made with aqueous bases (in- 3.1 Category I products cluding syrup products to be dissolved or suspended before Inject each of the cell suspensions aseptically into ˆve con- use) tainers containing the product and mix uniformly. When it is Category II: All the dosage forms listed under Category I di‹cult to inject the cell suspension into the container asepti- made with nonaqueous bases or vehicles. cally or the volume of the product in each container is too small to be tested, transfer aseptically a su‹cient 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 niger ATCC 16404, NBRC 9455 sion mixed in the product must not exceed 1W100 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 is manufactured, used or stored, and they are also recognized JP XV General Information / Preservatives-EŠectiveness 1733

105 to 106 cells per mL or per gram of the product. Incubate Table 1. Interpretation criteria by product category these inoculated containers at 209Cto259C with protection from light, and calculate the viable cell count of 1 mL or 1 g Product Interpretation criteria of the product taken at 0, 14 and 28 days subsequent to in- Microorganisms category oculation. Record any marked changes (e.g., changes in color After 14 days After 28 days or the development of a bad odor) when observed in the mix- ed samples during this time. Such changes should be consi- Bacteria 0.1z of inocu- Same or less dered when assessing the preservative e‹cacy of the product lum count or than level after concerned. Express sequential changes in the viable counts as less 14 days percentages, with the count at the start of the test taken as Category IA Yeasts moulds Same or less Same or less 100. Titration of the viable cell counts is based, in principle, W than inoculum than inoculum on the Pour Plate Methods in ``Microbial Limit Tests''. In count count this case, conˆrm whether any antimicrobial substance is present in the test specimen. If a conˆrmed antimicrobial Bacteria 1z of inoculum Same or less substance needs to be eliminated, incorporate an eŠective in- count or less than level after activator of the substance in the buŠer solution or liquid 14 days medium to be used for dilution of the test specimen, as well Category IB as in the agar plate count medium. However, it is necessary to YeastsWmoulds Same or less Same or less conˆrm that the inactivator has no eŠect on the growth of the than inoculum than inoculum microorganisms. When the occurrence of the preservative or count count the product itself aŠects titration of the viable cell count and Bacteria 10z of inocu- Same or less there is no suitable inactivator available, calculate the viable lum count or than level after cell counts by the Membrane Filtration Method in ``Microbi- less 14 days al Limit Tests''. Category IC 3.2 Category II products YeastsWmoulds Same or less Same or less The procedures are the same as those described for Catego- than inoculum than inoculum ry I products, but special procedures and considerations are count count required for both uniform dispersion of the test microorgan- ism in the product and titration of viable cell counts in the Bacteria Same or less Same or less samples. than inoculum than inoculum count count For semisolid ointment bases, heat the sample to 459Cto 509C until it becomes oily, add the cell suspension and dis- Category II YeastsWmoulds Same or less Same or less perse the inoculum uniformly with a sterile glass rod or spat- than inoculum than inoculum ula. Surfactants may also be added to achieve uniform dis- count count persion, but it is necessary to conˆrm that the surfactant added has no eŠect on survival or growth of the test microor- 5. Culture Media ganisms and that it does not potentiate the preservative e‹ca- Culture media and buŠer solution used for Preservatives- cy of the product. For titration of the viable cell count, a sur- EŠectiveness Tests are described below. Other media may be factant or emulsiˆer may be added to disperse the product used if they have similar nutritive ingredients and selective uniformly in the buŠer solution or liquid medium. Sorbitan and growth-promoting properties for the microorganisms to monooleate, polysorbate 80 or lecithin may be added to im- be tested. prove miscibility between the liquid medium and semisolid ointments or oils in which test microorganisms were inoculat- Soybean Casein Digest Agar Medium ed. These agents serve to inactivate or neutralize many of the Casein peptone 15.0 g most commonly used preservatives. Soybean peptone 5.0 g Sodium chloride 5.0 g 4. Interpretation Agar 15.0 g Interpret the preservative e‹cacy of the product according Water 1000 mL to Table 1. When the results described in Table 1 are ob- Mix all of the components and sterilize at 1219Cfor15– tained, the product examined is considered to be eŠectively 20 minutes in an autoclave. pH after sterilization: 7.1 – 7.3. preserved. There is a strong possibility of massive microbial contamination having occurred when microorganisms other Sabouraud Glucose Agar Medium than the inoculated ones are found in the sterile product to be Peptone (animal tissue and casein) 10.0 g examined, and caution is required in the test procedures andW Glucose 40.0 g or the control of the manufacturing process of the product. Agar 15.0 g When the contamination level in a nonsterile product to be Water 1000 mL examined exceeds the microbial enumeration limit speciˆed Mix all of the components and sterilize at 1219Cfor15– in ``Microbial Attributes of Nonsterile Pharmaceutical 20 minutes in an autoclave. pH after sterilization: 5.4 – 5.8. Products'' in General Information, caution is also required Glucose Peptone (GP) Agar Medium in the test procedures andWor the control of the manufactur- Glucose 20.0 g ing process of the product. Yeast extract 2.0 g Magnesium sulfate heptahydrate 0.5 g 1734 Qualiˆcation of Animals / General Information JP XV

Peptone 5.0 g mary subject that has to be considered is the absence of any Monobasic potassium phosphate 1.0 g infectious agents such as virus in the raw materials of animal Agar 15.0 g origin including human as the source of the drug. More im- Water 1000 mL portant points are whether the drugs derived from such raw Mix all of the components and sterilize at 1219Cfor15– materials are free of such infectious agents and whether there 20 minutes in an autoclave. pH after sterilization: 5.6 – 5.8. is any possibility of transmission of infectious agents when the drugs are administered to patient. The eligibility of Potato Dextrose Agar Medium animals including human, as the source of raw materials of Potato extract 4.0 g drugs, in other words ``the subject which is free from any dis- Glucose 20.0 g ease or transmission of infectious agents that is infectious to Agar 15.0 g human being at an appropriate production process and use of Water 1000 mL the drug product'' is that ``The drug should be entirely free Mix all of the components and sterilize at 1219Cfor15– from any risk of infections by means of whole procedures 20 minutes in an autoclave. pH after sterilization: 5.4 – 5.8. which include evaluation of appropriateness of the animals 0.1z Peptone Water including human as the source of their raw materials, estab- Peptone 1.0 g lishment of appropriate production processes and their ap- Sodium chloride 8.0 g propriate control, and strict adherence to the clinical indica- Water 1000 mL tions of the ˆnal product.'' Mix all of the components and sterilize at 1219Cfor15– 2. Animals including human as the source of raw materials 20 minutes in an autoclave. pH after sterilization: 7.2 – 7.4. of drugs What is the most clear and appropriate preventive meas- ures against infection to human being due to administration 20. Qualiˆcation of Animals as of drugs which are derived from animals including human is to assure the absence of any infectious agents such as virus in Origin of Animal-derived Medicinal its raw materials or an appropriate critical raw material by Products provided in the General each of the followings: (1) the use of raw materials of healthy animal origin, which are proved to be free from communica- Notices of Japanese ble disease agents to human, or (2) the use of appropriate critical raw materials (e.g., cell substrate, blood plasma, Pharmacopoeia and pooled urine after some treatments) for drug production, Other Standards which are proved to be free from communicable disease agents after certain appropriate processing on raw materials Introduction of animal origin. The O‹cial Gazette issued on March 29, 2002 announced As for raw materials of drugs of human origin, cell, tissue, that General Notices of the Japanese Pharmacopoeia and blood, placenta, urine, etc. are used. Whenever it is su‹cient other standards were amended to add a provision that and possible each donor, as the origin of such raw materials, ``When a drug product or a drug substance which is used to should be asked his (her) health condition and undergoes his manufacture a drug product, is manufactured from a raw (her) medical examination at this stage, so that the ap- material of animal origin, the animal in question should be in propriateness as a donor can be conˆrmed from the stan- principle a healthy subject, if not otherwise provided.''. dpoint of safety concerning communicable disease agents The Notice Iyaku-hatsu No. 0329001, which was issued on such as virus. the same date, provided that ``Healthy subject herein pro- For example, ``Basic concept on handling and use of a vided is the animal which does not cause any disease or any drug product, etc. which is derived from cell Wtissue'' infection to human being at an appropriate production proc- (Attachment 1 of the Notice Iyaku-Hatsu No. 1314 dated De- ess and use of the drug product, and as for the oral or exter- cember 26, 2000) and ``Guidance for quality and safety assur- nal drug for example, the animal, as its raw material of ance of a drug product, etc. which is derived from human cell animal origin, should be conˆrmed at this stage to meet the Wtissue (Attachment 2 of the Notice Iyaku-Hatsu No. 1314 Food Standard. It has to be noted that this standard of dated December 26, 2000)'' issued by the Director-General of healthy subject has to be revised timely taking into account the Medicinal Safety Bureau, Ministry of Health and Wel- the up-to-date information with respect to the amphixenosis fare, states that since the cellWtissue supplied by a human infections common between human beings and animals.''. donor comes to be applied to patients without processing This General Information describes safety assurance through any su‹cient inactivation or removal of communica- against infection of drugs, which are manufactured from raw ble disease agents, the selection and qualiˆcation criteria on materials of animal origin, to follow up the Notice as men- such donor has to be established. These criteria are to be tioned above. composed with the respect to the check items on the case history and the physical conditions as well as the test items on 1. Basic concept the various transmission of infectious agents through cellWtis- When drugs derived from raw materials of animal origin sue, and that the appropriateness of these criteria has to be including human are used, it is important to take into ac- clariˆed. Hepatitis Type-B (HBV), Hepatitis Type-C (HCV), count any possibility that communicable disease agents such Human Immune Deˆciency Viral infections (HIV), Adult as virus may cause infectious disease or any possible hazards T-Cell Leukemia and Parvovirus B19 Infections should be to patients. In such case, it goes without saying that the pri- denied through the interview to the donor and the tests (sero- JP XV General Information / Qualiˆcation of Animals 1735 logic test, nucleic-acid ampliˆcation test, etc.). Further, if derived from organ, tissue and cell, etc. of high risk of TSEs; necessary, Cytomegalovirus infection and EB Virus infection and  taking appropriate measures basing on the informa- should be denied by tests. ``Infections caused by bacteria tion collected, which includes incidence of TSEs, the results such as Treponema pallidum, , Gonococci, Tuber- of epidemiological investigation and the experimental cule bacillus, etc.'', ``septicemia and its suspicious case'', research on prion, and incidence of tardive infection on ``vicious tumor'', ``serious metabolic or endocrine-related donors after collecting raw materials, etc. disorders'', ``collagenosis and haematological disorder'', 3. Human or animal cells which are used as critical raw ``hepatic disease'' and `` (transmissible spongiform materials for drug production encephalopathies and its suspicious case)'' should be checked Cell substrates derived from humans or animals are used on the case history or by the interview, etc. and the ex- for drug production. In such case, it is desirable that the perience of being transfused orWand transplanted should be humans or the animals, which are the origins of the cell sub- checked to conˆrm eligibility as a donor. The most appropri- strates, are healthy subjects. However, it is considered practi- ate check items and test methods then available are to be cal that viral safety of the drugs derived from the cell sub- used, which need to be reconsidered at appropriate timing strates are evaluated on the cells, which are so called critical taking into account the updated knowledge and the progress raw materials for production of such drugs. In such case, the of the science and the technologies. At screening of a donor, safety should be conˆrmed through the test and analysis on reexaminations has to be made at appropriate timing using established cell bank thoroughly with respect to virus etc., as the eligible check items and the test methods taking into ac- far as possible. The items and the methods of the tests that count the window period (Initial period after infection, in have been followed in this case are described in detail in the which antibody against bacteria, fungi or virus is not detect- Notice of Japanese version on the internationally accepted ed.) ICH Guideline entitled ``Viral safety evaluation of In the case of plasma derivatives produced from the donat- biotechnology products derived from cell lines of human or ed blood in Japan, the donor should be checked by means of animal origin'' (Iyakushin No. 329 issued on February 22, self-assessed report about health conditions, and a serologic 2000 by Director, Evaluation and Licensing Division, Phar- check and a nucleic acid ampliˆcation test (NAT) on mini maceutical and Medical Safety Bureau, Ministry of Health pooled plasma should be performed at the stage of donated and Welfare). In the meantime, it is important how to handle blood. Further, the plasma material (i.e., critical raw materi- the cell in case that any virus has been detected under the cell al ) for fractionation should be stored 4 months in minimum level tests. This Notice describes how to cope with this situa- so that the arrangement could be taken based on the informa- tion as follows: ``It is recognised that some cell lines used for tion available after collection of the blood and the blood in- the manufacture of product will contain endogenous fusion to exclude the possibility of using any critical raw retroviruses, other viruses or viral sequences. In such circum- materialwhichmightcauseinfectiontopatients. stances, the action plan recommended for manufacturer is On the other hand, as for the materials such as urine which described in Section V (Rationale and action plan for viral are taken from the unspeciˆed number of the donors and clearance studies and virus tests on puriˆed bulk) of the No- come to be critical raw materials for drug production after tice. The acceptability of cell lines containing viruses other some treatments, it is unrealistic and not practical to conduct than endogenous retroviruses will be considered on an in- the tests of virus infection, etc. on the individual donor. Con- dividual basis by the regulatory authorities, by taking into ac- sequently, appropriate tests such as virus test has to be per- count a riskWbeneˆt analysis based on the beneˆt of the formed on such critical raw materials for drug production. product and its intended clinical use, the nature of the con- In the case of the animals besides human, the wild ones taminating viruses, their potential for infecting humans or should be excluded. Only the animals, which are raised under for causing disease in humans, the puriˆcation process for well sanitarily controlled conditions taken to prevent bacteri- the product (e.g., viral clearance evaluation data), and the ex- al contamination or under the eŠective bacterial pollution tent of the virus tests conducted on the puriˆed bulk.'' For monitoring systems, have to be used, and it is recommended example, it is well known that Type A-, R- and C-endogenous that the animals from a colony appropriately controlled un- particles like retrovirus are observed in the cells of the ro- der speciˆc pathogen-free (SPF) environment are to be used dents used most often for drug production. It is also known as far as possible. Further, for the animals regulated under that they are not infectious to human and is not dangerous, the Food Standard, only the animals that met this standard and CHO cells are generally used for drug production. The should be used. It should be conˆrmed by appropriate tests established cell lines (e.g., NAMALWA Cell, BALL-1 Cell, that the animals were free from pathogen, if necessary. etc.) derived from patients are sometimes used, but The concrete measures to avoid transmittance or spread of through the thorough virus tests, etc., their safety are con- infectivity of prion, which is considered to be the pathogen of ˆrmed. The established cell lines are assumed to be safer than transmissible spongiform encephalopathies (TSEs), as far as the primary cultured cells which are hard to conduct the possible are the followings:  avoidance of use of animals, thorough virus test. which are raised in the areas where high incidence or high risk of TSEs (Scrapie in sheep and goat, bovine spongiform en- 4. Establishment and control of appropriate production cephalopathies (BSE) in cattle, chronic wasting disease process and adherence to the clinical indication of ˆnal (CWD) in deer, new type of Creutzfeldt-Jacob-Disease (CJD) product for safety assurance in human, etc.) is reported, and humans, who have stayed Safety assurance against potential infections at only the long time (more than 6 months) in such areas, as raw materi- levelofanimalsthataresourceofrawmaterialsofdrugsis als or related substances of drugs;  avoidance of use of any limited. Further, ``health of animal'' can not be deˆned substances that are derived from the individual infected with univocally, and the various factors have to be taken into ac- scrapie, BSE, CJD, etc.;  avoidance of using a material count. The ˆnal goal of this subject is to protect human from 1736 Quality Control of Water / General Information JP XV any infectious disease caused by drugs. Achieving this goal, 0.05 mg/L.'' In preparing Water at various facilities from the establishment and control of appropriate production source water such as well water or industrial water, it is need- processes of each drug and the adherence to the clinical indi- ed to assure compliance with speciˆcations set forth for cations of the ˆnal product are important. Water in the Japanese Pharmacopoeia. Furthermore, when As mentioned above, the rodent cells used most often for Water is used after storage for a long period of time, microbi- the production of the drugs are known to have endogenous al proliferation should be prevented. retrovirus sometimes. The reason why such cells can be used Water is used as source water for ``Puriˆed Water'' and for the production of the drugs is that multiple measures are ``Water for Injection.'' It also is used in the production of in- applied for safety in the puriˆcation stages which include ap- termediates of active pharmaceutical ingredients (APIs) and propriate inactivation or removal processes. There are cases in pre-washing the equipments used for pharmaceutical pur- in which the production procedure involves intentional use of poses. a virus or a microorganism. In this case, relevant measures  Puriˆed Water capable of removing or inactivating of such virus or microor- The speciˆcations of Puriˆed Water are included in ``O‹- ganism are appropriately incorporated in the puriˆcation cial Monographs'' of the Japanese Pharmacopoeia. Puriˆed process, so that the risk of infection to human can be fully Water is prepared from Water by distillation, ion-exchange denied and its safety can be assured when it is used as a drug. treatment, reverse osmosis (RO), ultraˆltration (UF) capable Further, even in the case that it is di‹cult to clarify the risk of of removing substances having molecular weights of 6,000 contamination of the infectious agents or that the raw materi- and above, or a combination of these methods. Because Puri- al are contaminated by viruses etc., the raw material in ques- ˆed Water contains no components capable of inhibiting tion may be used for the production of drugs so long as ap- microbial growth, appropriate microbiological control is propriate inactivation or removal processes are introduced, needed. Particularly in the case of ion-exchange treatment, their eŠectiveness can be conˆrmed and the safety can be as- reverse osmosis or ultraˆltration, the appropriate treatment sured by appropriate control of the manufacturing processes for microbial growth inhibition or periodical sterilization under GMP, etc. should be conducted. 5. Conclusion Puriˆed Water which has been sterilized or treated with The qualiˆcation of animals including human, as the chemical agents for the purpose of microbial growth inhibi- source of raw materials of drugs, in other words ``the subject tion or maintaining the endotoxin level within an appropriate which does not cause any infectious diseases to human being control range should be appropriately controlled in order to at an appropriate production process and use of the drug maintain the quality in compliance with standards set forth product'' is that ``the drug has to be entirely free from any for the individual purposes of use. Puriˆed Water that has risk of infections by means of whole procedures which in- been sterilized is referred to as ``Sterilized Puriˆed Water.'' clude evaluation of appropriateness of the animal including  Water for Injection human as the source of their raw materials, establishment of The speciˆcations of ``Water for Injection'' are described appropriate production processes and their appropriate con- in ``O‹cial Monographs'' of the Japanese Pharmacopoeia. trol, and strict adherence to the clinical indication of the ˆnal Water for Injection is prepared from Water or Puriˆed product.'' Water by distillation, or from Puriˆed Water by reverse os- To cope with this subject, the advanced scientiˆc measures, mosis (RO), ultraˆltration (UF) capable of removing sub- which actually re‰ect the updated knowledge and progress of stances having molecular weights of 6,000 and above, or a the science and the technology about infectious diseases in combination of RO and UF. The quality of processing water human and infection of animal origin, have to be taken into by RO and/or UF should be maintained consistently at the account timely. same level as that prepared by distillation, by the validation through long-term operation of the process and elaborate routine control. Because Water for Injection contains no 21. Quality Control of Water for components that inhibit microbial growth, stringent control is needed for microorganisms and endotoxins. The standard Pharmaceutical Use for endotoxins requires a level lower than 0.25 EU/mL. Water used for producing pharmaceutical products, clean- Selection of Pharmaceutical Water ing pharmaceutical containers and equipment, and the like is Depending on the purpose of use, pharmaceutical water referred to as ``Pharmaceutical Water.'' To consistently as- can be selected from the above-described group of phar- sure the quality of pharmaceutical water, it is important to maceutical waters that allow us to assure the quality of the ˆ- verify through appropriate validation that water of required nal product without causing any trouble during the produc- quality is supplied, and to maintain that quality by routine tion process. Table 1 shows some example criteria for such control. selection (in the case of charge-in water for the preparation of drug products). Types of Pharmaceutical Water For the production of sterile drug products, for which con-  Water tamination by microorganisms or endotoxins is not permit- The speciˆcations for Water arespeciˆedinthecorre- ted, Water for Injection should be used. For ophthalmics and sponding monograph of the Japanese Pharmacopoeia. It is eye ointments, Water for Injection or Puriˆed Water should required for Water to meet the quality standards for drinking be used. For the production of non-sterile drug products, water provided by the Japanese Water Supply Law and an waterofaqualityhigherthanthatofPuriˆed Water should additional requirement for ammonium of ``no greater than be used, however for a non-sterile drug product that requires JP XV General Information / Quality Control of Water 1737

Table 1. Criteria for Selecting Pharmaceutical Water (Charge-In Water)

Class of Classiˆcation Pharmaceutical Application Remarks Water

Water for Injections, Ophthalmics, Eye Ointments Injection For ophthalmics and eye ointments for which precautions should be taken against microbial contamination, puriˆed water Ophthalmics, Eye Ointment that is subjected to microbiological con- trol by treatment with sterilization, ultraˆltration, or the like, should be used. Drug Product Aerosols, Liquids and Solutions, Extracts, Puriˆed Water Elixirs, Capsules, Granules, Pills, Suspen- sions and Emulsions, Suppositories, For liquids, solutions, ointments, suspen- Powders, Spirits, Tablets, Syrups, Infu- sions, emulsions, aerosols, and the like for sions and Decoctions, Plasters, Tinctures, which precautions should be taken against Troches, Ointments, Cataplasms, Aro- microbial contamination, select and use matic Waters, Liniments, Lemonades, puriˆed water subjected to appropriate Fluidextracts, Lotions, and Pharmaceuti- microbiological control. cal preparations of percutaneous absorp- tion type

Water for Sterile APIs, and APIs rendered sterile in Injection the formulation process

IntheproductionofAPIsthatare Active rendered sterile in the formulation process Pharmaceutical APIs, APIs rendered sterile in the formu- and have no subsequent processes capable Ingredient Puriˆed Water lation process, and API intermediates of removing endotoxins, Puriˆed Water (API) that is controlled to maintain endotoxins at a low level should be used.

Water API Intermediates

care against microbiological contamination, such as liquids, process of the drug products for which the API is used should ointments, suspensions, emulsions, suppositories, and aer- be considered, so that the quality of the ˆnal drug products is osols, water should have a quality appropriately controlled maintained at an appropriate level. Water used in the produc- from microbiological viewpoints, considering the possible tion of sterile API or to clean pharmaceutical containers or impacts of preservatives formulated in the dosage form. equipment surfaces that come in direct contact with the Water for pre-washing containers or equipment surfaces that product should have the quality controlled chemically and come in direct contact with the drug should be of a quality microbiologically at the level of Water or higher, even if the higher than that of Water. Water for ˆnal rinsing should water is to be used at an earlier stage of a synthetic or extrac- have the same quality as that of charge-in water. tion process. In the ˆnal puriˆcation process, water should For the production of non-sterile drug products, the quali- haveaqualityequaltoPuriˆed Water or higher. Water used ty of water should be higher than that of Puriˆed Water; fortheˆnalrinsingofcontainersorequipmentsurfacesthat however for the production of liquids, ointments, suspen- come in direct contact with the product should have the same sions, emulsions, suppositories, and aerosols for which care quality as that of the charge-in water. Pharmaceutical water should be taken against microbial contamination, the quality used in sterile API should be Water for Injection. Similarly, of water should be controlled appropriately from microbio- in the case of APIs for drug products, where endotoxin con- logical viewpoints in consideration of the possible eŠects of trol is required and there are no subsequent processes capable formulated preservatives. Water for pre-washing containers of removing endotoxins, Water for Injetion or Puriˆed or equipment surfaces that come in direct contact with the Water, for which endotoxins are controlled to an appropriate product should be of a quality not less than that of Water. level, should be used. The water for ˆnal rinsing should be the same quality as that Quality Control of Pharmaceutical Water of charge-in water. 3.1 Outline In selecting pharmaceutical water for Active Pharmaceuti- Routine control of pharmaceutical water is conducted on cal Ingredient (API), the characteristics and formulation 1738 Quality Control of Water / General Information JP XV the premise that the production of water of a required quality considerations for technology and product quality. Conse- has been thoroughly veriˆed by validation studies at an earli- quently, exceeding an alert or action level does not necessari- er stage of the system that produces the pharmaceutical water ly indicate that the product quality has been compromised. (pharmaceutical water system). If these requirements are 3.4 Microbiological Monitoring met, the following control methods are applicable. The main purpose of a microbiological monitoring pro- For routine control, the control of electrical conductivity gram for pharmaceutical water system is to predict any and Total Organic Carbon (TOC) is markedly useful. Items microbiological quality deterioration of the produced water to be controlled regularly should be determined according to and to prevent any adverse eŠects it may have on product the intended use of the pharmaceutical water. In addition to quality．Consequently, detecting all of the microorganisms the above, control should be made for several chemical sub- present may not be necessary; however it is required to adopt stances, viable counts, endotoxins, insoluble foreign parti- a monitoring technique capable of detecting a wide range of cles, and the like. The measurement frequency should be de- microorganisms, including slow growing microorganisms. termined in consideration of the stability of the water quality The following indicates culture-based microbiological in question. monitoring methods for pharmaceutical water systems. In The following shows microbiological control items and adopting a rapid detection system for microorganisms, it is control items for electrical conductivity and Total Organic needed to conˆrm in advance that the obtained microbial Carbon (TOC), for which special attention should be paid. counts are equivalent to or above the data obtained through Similar considerations should be made of the other control incubation. items so that the water quality meets the speciˆcations of pharmaceutical water. 3.4.1 Media and Incubation Conditions There are many mesophilic bacteria of heterotrophic type 3.2 Sampling that are adaptable to poor nutrient water environments. In Monitoring should be conducted at a su‹cient enough fre- many pharmaceutical water systems, heterotrophic bacteria quency to ensure that the pharmaceutical water system is in may form bio-ˆlms and cause water quality deterioration. It, control and that water of acceptable quality is continuously therefore, is useful to monitor the water quality by use of produced. Sampling should be made at representative loca- R2A Agar Medium, which is excellent for growing bacteria tions in the production and supply systems of the pharmaceu- of oligotrophic type. On the other hand, in routine microbial tical water, with particular care that the collected sample monitoring, an approach that identiˆes the trend in re‰ects the pharmaceutical water system. Normally, point-of- microbiological quality change is widely employed; a stan- use sites are appropriate for the sampling locations. Sampling dard agar plate is used for counting the total number of via- frequency is established on the basis of data from validation ble microorganisms capable of proliferating at 30 – 359Cina studies of the pharmaceutical water system. In making a sam- comparatively short period of time. pling plan for pharmaceutical water system, it is important to Table 2 shows examples of measurement methods, mini- cover all important points of the system by thoroughly con- mum sample sizes, media, and incubation periods for es- sidering the quality characteristics required for the water to timating viable counts. be sampled. Particularly in microbiological control, attention should be paid to the diŠerence in conditions among sam- 3.4.2 Media Growth Promotion Test pling locations. In the media growth promotion test with R2A Agar Medi- um, use the strains listed below or other strains considered to 3.3 Alert and Action Levels be equivalent to these strains. Prior to the media growth pro- In a pharmaceutical water system, monitoring is done for motion test, inoculate these strains into sterile puriˆed water microorganisms and other quality attributes that assure water and incubate at 20 – 259C for 3 days. of acceptable quality is produced during operation within the Methylobacterium extorquens: NBRC 15911 designed speciˆcations. Monitoring data thus obtained is Pseudomonas ‰uorescens: NBRC 15842, ATCC 17386, or compared against an alert level, action level, other control the like levels, and allowable limits for pharmaceutical waters. This Dilute the ‰uid containing the starved strain in puriˆed implies the alert level and action level are used for process water with sterile puriˆed water to prepare a ‰uid containing control, rather than for judging the acceptance. about 50 – 200 cfu per mL. When pipeting 1 ml of the diluted Deˆnition of Alert Level solution onto the R2A agar medium to be used for incubating An alert level indicates that, when exceeded, a process may at 20 – 259Cor30–359C for 4 – 7 days, a su‹cient number have drifted from its normal operating condition. Alert levels of recovered colonies of the microorganism must be observed constitute a warning and exceeding them does not necessarily in comparison with the inoculated cfus. require a corrective action. Alert levels are generally estab- In the media growth promotion test with standard agar lished either at a mean＋2s on the basis of past trend ana- medium, use the strains listed below or other strains consi- lysesoratalevelof70z (50z for viable counts) of the ac- dered to be equivalent to these strains. When pipeting 1 mL tion criteria, whichever is smaller. of the solution containing the microorganism for incubating at30–359C for 48 hours, a su‹cient number of recovered Deˆnition of Action Level colonies of the microorganism must be observed in compari- An action level indicates that, when exceeded, a process son with the inoculated counts. has drifted from its normal operating range. Exceeding an ac- Staphylococcus aureus: ATCC 6538, NCIMB 9518, CIP tion level indicates that corrective action must be taken to br- 4.83 or NBRC 13276 ing the process back within its normal operating range. Pseudomonas aeruginosa: ATCC 9027, NCIMB 8626, CIP Alert and action levels should be established within process 82.118 or NBRC 13275 and product speciˆcation tolerances and based on integrated JP XV General Information / Quality Control of Water 1739

Table 2. Methods for Assessment of Viable Counts in harmaceutical Water

Method Pharmaceutical Water

Water Puriˆed Water in Bulk Water for Injection in Bulk

Measurement Pour Plate Method or Pour Plate Method or Membrane Membrane Filtration Method Membrane Filtration Filtration

Minimum 1.0 mL 1.0 mL 100 mL Sample Size

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

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

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

Colon bacillus (Escherichia coli): ATCC 8739, NCIMB facility using electrical conductivity or TOC as an indicator, 8545, CIP 53.126 or NBRC 3972 appropriate alert and action levels, as well as countermeas- ures against contingency should be established for each indi- 3.4.3 Action Levels for Microorganisms in Pharmaceuti- cator. cal Water System The following action levels are considered appropriate and 3.5.1 Monitoring with an Indicator of Electrical Conduc- generally applicable to pharmaceutical water systems. Action tivity levels for Puriˆed Water and Water for Injection should be Measurement of electrical conductivity for monitoring is those obtained using R2A Agar Medium. usually conducted continuously with an in-line meter having a ‰ow-through type or pipe-insertion type cell. Alternatively, Action Levels for viable counts in various pharmaceutical batch testing may be done using a dip type cell with samples waters taken at point-of-use sites or other appropriate locations of Water: 100 cfu/mL (according to the criteria in the Water the pharmaceutical water system. The following guidelines Supply Law) for the operation control of a pharmaceutical water system Puriˆed Water:100cfu/mL address how to interpret the results of electrical conductivity Water for Injection: 10 cfu/100 mL tests, and how to approve the continuation of operation, de- (cfu: colony-forming units) pending on whether measurements are made at the standard When actual counts exceed such action levels in validation temperature (209C) or at temperatures outside the standard. studies or routine control studies, it is necessary to character- (1) When monitoring is done at the standard temperature ize the isolated microorganisms and to sterilize or disinfect (209C) the aŠected system. Tests for electrical conductivity of the Japanese Phar- 3.5 Physicochemical Monitoring macopoeia commonly require the tester to measure at the Physicochemical monitoring of a pharmaceutical water standard temperature (209C), however measurement at a system is generally performed using electrical conductivity temperature within a range of 20±59C may be acceptable on and Total Organic Carbon (TOC) as indicators. Monitoring condition that the correct equation is used. When monitoring by electrical conductivity can predict probable quantities of electrical conductivity at the standard temperature of Puri- the total inorganic salts present. Monitoring by TOC can esti- ˆed Water and Water for Injection, the recommended allow- mate the total quantity of the organic compounds present. able (action level) conductivity is as follows. Basically, the tests for electrical conductivity and total organ- ･Action Level 1.0 mS/cm (209C) ic carbon speciˆed in ``General Tests'' of the Japanese Phar- The above allowable conductivity is established on the sup- macopoeia are applied as these physicochemical monitoring position of in-line monitoring, so this standard level may be methods. However, tests for monitoring are diŠerent in pro- changed in batch testing. ˆle from those described in ``Monographs'' of the Japanese (2) When monitoring is done at temperatures other than Pharmacopoeia. The following describes the particulars for the standard temperature completing the part that cannot be worked out with the in- When the quality of water is monitored by the measure- dividual descriptions in ``General Tests'' of the Phar- ment of electrical conductivity at a temperature other than macopoeia alone. When monitoring is done at a production 1740 Quality Control of Water / General Information JP XV the standard temperature, the following three-stage approach mL of the water specimen, and determine the pH to the is applied. The method described below complies with the nearest 0.1 pH. description in ``General Chapter <645> WATER CONDUC- 2. Use Table 4 to ˆnd the allowable conductivity value TIVITY'' of the United States Pharmacopoeia (USP28, corresponding to the observed pH. If the observed conductiv- 2005), which speciˆes the establishment of allowable levels of ity is lower than the allowable level, the water specimen is electrical conductivity individually corresponding to diŠerent judged as passing the electrical conductivity test. If the ob- measurement temperatures and pH levels of specimens meas- served valued is greater than the allowable level, or if the pH ured by the three-stage approach (Stage 1 to Stage 3). of the specimen is outside of a range of pH 5.0 – 7.0, the water specimen is judged as unacceptable. Stage 1 1. When a non-temperature-compensated conductivity Table 4 Third Step Allowable Electrical Conductivity at instrument is used, measure the temperature of the water DiŠerent * specimen, in addition to the conductivity. 2. Using Table 3, ˆnd the conductivity value corre- Allowable Allowable sponding to the measured temperature or a temperature listed pH Conductivity pH Conductivity in Table 3 just below the measured temperature that can be (mS/cm) (mS/cm) used to calculate the allowable electrical conductivity at the 5.0 4.7 measured temperature. 5.1 4.1 6.1 2.4 3. If the measured conductivity is not greater than the al- 5.2 3.6 6.2 2.5 lowable conductivity value, the water specimen is judged as 5.3 3.3 6.3 2.4 passing the electrical conductivity test. If the conductivity 5.4 3.0 6.4 2.3 value exceeds the allowable conductivity value, proceed with 5.5 2.8 6.5 2.2 Stage 2. 5.6 2.6 6.6 2.1 5.7 2.5 6.7 2.6 Table 3 Stage 1 Allowable Electrical Conductivity Meas- 5.8 2.4 6.8 3.1 ured at DiŠerent Temperatures* 5.9 2.4 6.9 3.8 Allowable Allowable 6.0 2.4 7.0 4.6 Temperature Temperature Conductivity Conductivity ( C) ( C) 9 (mS/cm) 9 (mS/cm) *Applied only to a sample in equilibrium with the air at 259C. 00.6 50.8552.13.5.2 Monitoring with an Indicator of Total Organic 10 0.9 60 2.2 Carbon (TOC) 15 1.0 65 2.4 Drinking Water Standards (Prescribed under the Article 4 20 1.1 70 2.5 of the Japanese Water Supply Law) require that TOC should 25 1.3 75 2.7 be``notgreaterthan5ppm''.Howeveritispreferableforin- 30 1.4 80 2.7 dividual facilities to conduct TOC monitoring on Water with 35 1.5 85 2.7 alert and action levels separately determined for water-quali- 40 1.7 90 2.7 ty control by TOC monitoring. 45 1.8 95 2.9 The Japanese Pharmacopoeia speciˆes the Test for Total 50 1.9 100 3.1 Organic Carbon, and usually, TOC measurement is conduct- ed using an apparatus in compliance with the speciˆcations, *Applied to the conductivity value measured with a non-tem- howeveriftheapparatusconformstotheapparatussuitabil- perature-compensated conductivity instrument. ity test requirements described in ``General Chapter <643> TOTAL ORGANIC CARBON'' of the United States Phar- Stage 2 macopeia (USP28, 2005), otherwise described in ``Methods 1. Transfer a su‹cient amount of water specimen to a of Analysis 2.2.44. TOTAL ORGANIC CARBON IN suitable container, and stir the specimen. Adjust the tempera- WATER FOR PHARMACEUTICAL USE'' of the - ture (25±19C), and while vigorously agitating the specimen, an Pharmacopoeia (EP 5.0, 2005), the apparatus may be used measure the conductivity periodically. If the change in con- for monitoring a pharmaceutical water supplying system, ductivity is not greater than 0.1 mS/cm, due to the uptake of provided that sufficiently pure water not contaminated with atmospheric carbon dioxide, interpret the observed value as ionic organic substances or organic substances containing the conductivity of the specimen (in equilibrium with the at- nitrogen, sulfur or chlorine atoms is used as source water for mosphere). the system. 2. If the observed conductivity value at 259C (with the A TOC apparatus, characterized by calculating the amount specimen in equilibrium with the atmosphere) is not greater of organic carbon from the diŠerence in conductivity before than 2.1 mS/cm, the specimen is judged as passing the con- and after the decomposition of organic substances without ductivity test. If the observed value is greater than the level, separating carbon dioxide from the sample solution, may be proceed with Stage 3. in‰uenced negatively or positively, when applied to a sample Stage 3 solution containing ionic organic substances or organic sub- 1. While maintaining the sample at 25±19C, perform the stances comprised of nitrogen, sulfur, or halogens such as conductivity measurement as explained in Step 1 of Stage 2. chlorine and the like; therefore, the apparatus should be Add 0.3 mL of saturated potassium chloride solution to 100 selected appropriately depending on the purity of phar- JP XV General Information / Rapid Identiˆcation 1741 maceutical water to be measured and on the contamination 1. Preparation of template DNA risk in the case of apparatus failure. It is important to use a pure cultivated bacterium or fungus for identiˆcation. In the case of colony samples, colonies are 3.6 Storage of Water for Injection picked up with a sterilized toothpick (in the case of fungi, a In storing Water for Injection, the water should be heated small fragment of colony sample is picked up), and suspend- during circulation or other action to inhibit microbial ed in 0.3 mL of DNA releasing solution in a 1.5 mL cen- proliferation. The maximum storage time allowed should trifuge tube. In the case of culture ‰uid, a 0.5 mL portion of also be appropriately established on the basis of validation ‰uid is put in a 1.5 mL centrifuge tube and centrifuged at studies, with considerations given to risks of contamination 10,000 rpm for 10 min. After removal of the supernatant, the and quality deterioration. pellet is suspended in 0.3 mL of DNA releasing solution, and then heated at 1009C for 10 min. In general, PCR can be run for bacteria and yeasts heated in DNA releasing solution. For 22. Rapid Identiˆcation of fungi, DNA extraction after treatment with a mixer or ultra- Microorganisms Based on sonic generator may be necessary before PCR. 2. PCR Molecular Biological Method Add 2 mL of template DNA in PCR reaction solution. Use 10F/800R primers for bacteria and ITS1F/ITS1R primers for This chapter describes the methods for the identiˆcation or fungi, and then perform 30 ampliˆcation cycles at 949Cfor estimation of microorganisms (bacteria and fungi), found in 30 sec, 559C for 60 sec, and 729C for 60 sec. DNA fragments in-process control tests or lot release tests of pharmaceutical are ampliˆed about 800 bp in the case of bacteria and about products, at the species or genus level based on their DNA se- 150 – 470 bp depending on the strain in the case of fungi. In- quence homology. The identiˆcation of isolates found in the clude a negative control (water instead of the test solution) in sterility test or aseptic processing can be helpful for inves- the PCR. tigating the causes of contamination. Furthermore, informa- 3. Conˆrmation of PCR products tion on microorganisms found in raw materials used for Mix 5 mL of PCR product with 1 mL of loading buŠer pharmaceutical products, processing areas of pharmaceutical solution, place it in a 1.5 w/vz agarose gel well, and carry products, and so on is useful in designing measures to control out electrophoresis with TAE buŠer solution (1-fold concen- the microbiological quality of drugs. For the identiˆcation of tration). Carry out the electrophoresis together with microorganisms, phenotypic analysis is widely used, based on appropriate DNA size markers. After the electrophoresis, ob- morphological, physiological, and biochemical features and serve PCR products on a trans-illuminator (312 nm) and con- analysis of components. Commercial kits based on diŠer- ˆrm the presence of a single band of the targeted size. If mul- ences in phenotype patterns have been used for the identiˆca- tiple bands are observed, cut the targeted band out of the gel, tion of microorganisms, but are not always applicable to and extract DNA by using appropriate commercial DNA ex- microorganisms found in raw materials used for pharmaceu- traction . tical products and in processing areas of pharmaceutical 4. Puriˆcation of PCR products products. In general, the identiˆcation of microorganisms Remove unincorporated PCR primers and deoxynucleo- based on phenotypic analysis needs special knowledge and side triphosphates (dNTP) from PCR products by using judgment is often subjective. It is considered that the evolu- appropriate puriˆcation methods. tionary history of microorganisms (bacteria and fungi) is 5. Quantiˆcation of puriˆed DNA memorized in their ribosomal RNAs (rRNAs), so that sys- When puriˆed DNA is measured by spectrophotometer, tematic classiˆcation and identiˆcation of microorganisms in calculate 1 OD260 nm as 50 mg/mL. recent years have been based on the analysis of these se- 6. Labeling of PCR products with sequencing reagents quences. This chapter presents a rapid method to identify or Use an appropriate ‰uorescence-labeled sequencing estimate microorganisms based on partial sequences of diver- reagent suitable for the available DNA sequencer or its gent regions of the 16S rRNA gene for bacteria and of the in- program and label the PCR products according to the ternal transcribed spacer 1 (ITS1) region located between 18S instructions provided with the reagent. rRNA and 5.8S rRNA for fungi, followed by comparison of 7. Puriˆcation of sequencing reagent-labeled PCR products the sequences with those in the database. Methods described Transfer the product in 75 mL of diluted ethanol (7 in 10) in this chapter do not take the place of usual other methods into a 1.5 mL centrifuge tube, keep in an ice bath for 20 min, for the identiˆcation, and can be modiˆed based on the ex- and centrifuge at 15,000 rpm for 20 min. After removal of aminer's experience, and on the available equipment or supernatant, add 250 mL of diluted ethanol (7 in 10) to the materials. Other gene regions besides those mentioned in this precipitate and centrifuge at 15,000 rpm for 5 min. Remove chapter can be used if appropriate. the supernatant and dry the precipitate. Apparatuses 8. DNA homology analysis (1) DNA sequencer Place sequencing reagent-labeled PCR products in the Various types of sequencers used a gel board or capillary DNA sequencer and read the sequences of the can be used. PCR products. Compare the partial nucleotide sequence with (2) DNA ampliˆer those in the BLAST database. To amplify target DNA and label ampliˆed (PCR) Judgment products with sequencing reagents. If sequencing data show over 90z identity with a sequence Procedures in the database, in general, judgment may be made as fol- The following procedures are described as an example. lows. 1742 SDS-Polyacrylamide Gel / General Information JP XV

1. In the case of bacteria, compare about 300 100) and 100 mL of water. After dissolving the materials by between positions 50 to 350 in the product obtained with heating, cool the solution to about 609C, and prepare gels. the 10F primer, with the BLAST database. Higher (10) Loading buŠer solution (6-fold concentrated) ranked species are judged as identiˆed species or closely Dissolve 0.25 g of bromophenol blue, 0.25 g of related species. cyanol FF and 1.63 g of disodium dihydrogen ethylenedia- 2. In the case of fungi, compare sequencing data for the mine tetraacetate dihydrate in 50 mL of water, and add product obtained with the ITS1F primer, with the 30 mL of glycerol and water to make 100 mL. BLAST database. Higher ranked species are judged as (11) PCR primers identiˆed species or closely related species. For Primer Reagents, Test Solutions (1) 0.5 mol/L Disodium dihydrogen Bacteria 10F 5?-GTTTGATCCTGGCTCA-3? tetraacetate TS 800R 5?-TACCAGGGTATCTAATCC-3? Dissolve 18.6 g of disodium dihydrogen ethylenediamine Fungi ITS1F 5?-GTAACAAGGT(T/C)TCCGT-3? tetraacetate dihydrate in water to make 100 mL. ITS1R 5 -CGTTCTTCATCGATG-3 (2) 1 mol/L Tris buŠer solution, pH 8.0 ? ? Dissolve 24.2 g of 2-amino-2-hydroxymethyl-1,3- propanediol in a suitable amount of water, adjust the pH to 8.0 with 0.2 mol/L hydrochloric acid TS, and add water to make 200 mL. 23. SDS-Polyacrylamide Gel (3) TE buŠer solution Electrophoresis Mix 1.0 mL of 1 mol/L tris buŠer solution, pH 8.0 and 0.2 mL of 0.5 mol/L disodium dihydrogen ethylenediamine This test is harmonized with the European Pharmacopoeia tetraacetate TS, and add water to make 100 mL. and the U.S. Pharmacopeia. (4) DNA releasing solution The SDS-Polyacrylamide Gel Electrophoresis is used for Divide TE buŠer solution containing Triton X-100 (1 in the characterization of proteins in biotechnological and bio- 100) into small amounts and store frozen until use. logical products and for control of purity and quantitative (5) PCR reaction solution determinations. 10-fold buŠer solution* 5 mL This technique is a suitable analytical method with which dNTP mixture** 4 mL to identify and to assess the homogeneity of proteins in 10 mmol/L Sense primer 1 mL biotechnological and biological products. The method is also 10 mmol/L Anti-sense primer 1 mL routinely used for the estimation of protein subunit molecu- Heat-resistant DNA polymerase (1 U/mL) 1 mL lar masses and for determining the subunit compositions of Water 36 mL puriˆed proteins. * Being composed of 100 mmol/L 2-amino-2-hydrox- Ready-to-use gels and reagents are widely available on the ymethyl-1,3-propanediol hydrochloride, pH 8.4, 500 market and can be used instead of those described in this text, mmol/L potassium chloride, 20 mmol/L magnesium chlo- provided that they give equivalent results and that they meet ride and 0.1 g/L gelatin. the validity requirements given below under Validation of the ** A solution containing 2.5 mmol/L each of dGTP (sodi- Test. um 2?-deoxyguanosine 5?-triphosphate), dATP (sodium 2?- deoxyadenosine 5?-triphosphate), dCTP (sodium 2?-deox- 1. Characteristics of Polyacrylamide Gels ycytidine 5?-triphosphate) and dTTP (sodium 2?-deox- The sieving properties of polyacrylamide gels are aŠorded ythymidine 5?-triphosphate). Adequate products containing by the three-dimensional network of ˆbers and pores which is these components as described above may be used. formed as the bifunctional bisacrylamide cross-links adjacent (6) Sequencing reagent polyacrylamide chains. Polymerization is catalyzed by a free There are many kinds of sequencing methods, such as the -generating system composed of ammonium dye-primer method for labeling of primer, the dye-terminator persulfate and N,N,N?,N?-tetramethylethylenediamine methodforlabelingofdNTPterminatorandsoon.Usean (TEMED). appropriate sequencing reagent kit for the apparatus and As the acrylamide concentration of a gel increases, its program to be used. eŠective pore size decreases. The eŠective pore size of a gel is (7) 50-Fold concentrated TAE buŠer solution operationally deˆned by its sieving properties; that is, by the Dissolve 242 g of 2-amino-2-hydroxymethyl-1,3- resistance it imparts to the migration of macromolecules. propanediol in 57.1 mL of acetic acid (100) and 100 mL of There are limits on the acrylamide concentration that can be 0.5 mol/L disodium dihydrogen ethylenediamine tetraacetate used. At high acrylamide concentrations, gels break much TS, and add water to make 1000 mL. more easily and are di‹cult to handle. As the pore size of a (8) 1-Fold concentrated TAE buŠer solution gel decreases, the migration rate of a protein through the gel Diluted 50-fold concentrated TAE buŠer solution (1 in 50) decreases. By adjusting the pore size of a gel, through prepared before use is referred to as 1-fold concentrated TAE manipulating the acrylamide concentration, the resolution of buŠer solution. the method can be optimized for a given protein product. (9) Agarose gel Thus, the physical characteristics of a given gel are deter- Mix 1.5 g of agarose, 2.0 mL of 50-fold concentrated TAE mined by the relative concentrations of acrylamide and bi- buŠer solution, 10 mL of a solution of ethidium bromide (3,8- sacrylamide, used in its preparation. diamino-5-ethyl-6-phenylphenanthridinium bromide) (1 in In addition to the composition of the gel, the state of the JP XV General Information / SDS-Polyacrylamide Gel 1743 protein is an important determinant of the electrophoretic presence of suitable molecular-mass standards. mobility. In the case of proteins, the electrophoretic mobility 2) Non-reducing conditions is dependent on the pK values of the charged groups and the For some analyses, complete dissociation of the protein of size of the molecule. It is also in‰uenced by the type, concen- interest into subunit peptides is not desirable. In the absence tration and pH of the buŠer, the temperature and the ˆeld of treatment with reducing agents such as 2-mercaptoethanol strength, as well as by the nature of the support material. or DTT, disulˆde 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- The method cited as an example is limited to the analysis of pletely saturated with SDS and, hence, may not bind the de- monomeric polypeptides with a mass range of 14,000 to tergent 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 than buŠer systems, etc.), but those techniques are not discussed analyses of fully denatured polypeptides, since it is necessary in this chapter. that both standards and unknown proteins be in similar con- Analysis by electrophoresis on sodium dodecyl sulfate ˆgurations for valid comparisons. However, the staining of a (SDS) polyacrylamide gel (SDS-Polyacrylamide Gel Elec- single band in such a gel is a criterion of purity. trophoresis) under denaturing conditions is the most com- mon mode of electrophoresis used in assessing the quality of 3. Characteristics of Discontinuous BuŠer System Gel Elec- proteins in biotechnological and biological products, and will trophoresis be the focus of the example described here. Typically, analyt- The most widely used electrophoretic method for the anal- ical electrophoresis of proteins is carried out in poly- ysis of complex mixtures of proteins involves the use of a dis- acrylamide gels under conditions that ensure dissociation of continuous buŠer system consisting of two contiguous, but the proteins into their individual polypeptide subunits and distinct gels: a resolving or separating (lower) gel and a stack- that minimize aggregation. Most commonly, the strongly an- ing (upper) gel. The two gels are cast with diŠerent porosities, ionic detergent SDS is used in combination with heat to dis- pH, and ionic strengths. In addition, diŠerent mobile ions are sociate the proteins before they are loaded on the gel. The used in the gel and electrode buŠers. The buŠer discontinuity denatured polypeptides bind to SDS, become negatively acts to concentrate large-volume samples in the stacking gel, charged and exhibit a consistent charge-to-mass ratio regard- resulting in improved resolution. When power is applied, a less of protein type. Because the amount of SDS bound is voltage drop develops across the sample solution which almost always proportional to the molecular mass of the drives the proteins into the stacking gel. Glycinate ions from polypeptide and is independent of its amino acid sequence, the electrode buŠer follow the proteins into the stacking gel. SDS-polypeptide complexes migrate through polyacrylamide A moving boundary region is rapidly formed with the highly gels with mobilities that are dependent on the size of the poly- mobile chloride ions in the front and the relatively slow peptides. glycinate ions in the rear. A localized high-voltage gradient The electrophoretic mobilities of the resultant SDS- forms between the leading and trailing ion fronts, causing the polypeptide complexes all assume the same functional SDS-protein complex to form into a very thin zone (called the relationship to their molecular masses. Migration of SDS- stack)andtomigratebetweenthechlorideandglycinate complexes occurs toward the anode in a predictable manner, phases. Regardless of the height of the applied sample solu- with low-molecular-mass complexes migrating faster than tion in the wells, all SDS-protein complexes condense within larger ones. The molecular mass of a protein can therefore be broad limits and enter the resolving gel as a well-deˆned, thin estimated from its relative mobility calibrated in SDS- zone of high protein density. The large-pore stacking gel does Polyacrylamide Gel Electrophoresis and the occurrence of a not retard the migration of most proteins and serves mainly single band in such a gel is a criterion of purity. as an anticonvective medium. At the interface of the stacking However, modiˆcations to the polypeptide backbone, such and resolving gels, the proteins experience a sharp increase in as N- or O-1inked glycosylation, have a signiˆcant impact on retardation due to the smaller pore size of the resolving gel. the apparent molecular mass of a protein, since SDS does not Once the proteins are in the resolving gel, their mobility con- bind to a carbohydrate moiety in a manner similar to a poly- tinues to be slowed down by the molecular sieving eŠect of peptide. Thus, a consistent charge-to-mass ratio is not main- the matrix. The glycinate ions overtake the proteins, which tained.Theapparentmolecularmassesofproteinsthathave then move in a space of uniform pH formed by the undergone post-translational modiˆcations do not truly tris(hydroxymethyl)aminomethane and glycine. Molecular re‰ect the masses of the polypeptides. sieving causes the SDS-polypeptide complexes to separate on 1) Reducing conditions the basis of their molecular masses. Polypeptide subunits and three-dimensional structure of 4. Preparing Vertical Discontinuous BuŠer SDS- proteins are often ˆxed, at least in part, by the presence of Polyacrylamide Gels disulˆde bonds. A goal of SDS-Polyacrylamide Gel Elec- 1) Assembling of the gel moulding cassette trophoresis under reducing conditions is to disrupt this struc- Clean the two glass plates (size: e.g. 10 cm × 8cm),the ture by reducing the disulˆde bonds. Complete denaturation sample comb made of polytetra‰uoroethylene, the two and dissociation of proteins by treatment with 2-mercap- spacers and the silicone rubber tubing (diameter, e.g. 0.6 mm toethanol or dithiothreitol (DTT) will result in unfolding of × 35 cm) with mild detergent and rinse extensively with the polypeptide backbone and subsequent complexation with water. Dry all the items with a paper towel or tissue. Lubri- SDS. Under these conditions, the molecular masses of the cate the spacers and the silicone rubber tubing with non-sili- polypeptide subunits can be calculated by interpolation in the cone grease. Apply the spacers along each of the two short 1744 SDS-Polyacrylamide Gel / General Information JP XV sidesoftheglassplate2mmawayfromtheedgesand2mm electrophoretic separation away from the long side corresponding to the bottom of the After polymerization is complete (about 30 minutes), gel. Begin to lay the silicone rubber tubing on the glass plate remove the sample comb carefully. Rinse the wells immedi- by using one spacer as a guide. Carefully twist the silicone ately with water or with the running buŠer for SDS- rubber tubing at the bottom of the spacer and follow the long Polyacrylamide Gel Electrophoresis to remove any un- side of the glass plate. While holding the silicone rubber tub- polymerized acrylamide. If necessary, straighten the teeth of ing with one ˆnger along the long side again twist the tubing the sample comb of the stacking gel with a blunt hypodermic and lay it on the second short side of the glass plate, using the needle attached to a syringe. Remove the clamps on one short spacer as a guide. Place the second glass plate in perfect side, carefully pull out the silicone rubber tubing and replace alignment and hold the mould together by hand pressure. the clamps. Proceed similarly on the other short side. Re- Apply two clamps on each of the two short sides of the move the silicone rubber tubing from the bottom part of the mould. Carefully apply four clamps on the longer side of the gel. Mount the gel in the electrophoresis apparatus. Add the gel mould, thus forming the bottom of the gel mould. Verify electrophoresis buŠers to the top and bottom reservoirs. Re- that the silicone rubber tubing is running along the edge of move any bubbles that become trapped at the bottom of the the glass plates and has not been extruded while placing the gel between the glass plates. This is best done with a bent clamps. hypodermic needle attached to a syringe. Never pre-run the 2) Preparation of the gel gel before loading solutions, such as samples, since this will In a discontinuous buŠer SDS polyacrylamide gel, it is destroy the discontinuity of the buŠer systems. Before load- recommended to pour the resolving gel, let the gel set, and ing solutions, such as samples, carefully rinse the stacking gel then pour the stacking gel, since the compositions of the two wells with the running buŠer for SDS-Polyacrylamide Gel gels in acrylamide-bisacrylamide, buŠer and pH are diŠerent. Electrophoresis. Prepare the test and reference solutions in Preparation of the resolving gel: In a conical ‰ask, prepare the recommended sample buŠer and treat as speciˆed in the the appropriate volume of solution containing the desired individual monograph. Apply the appropriate volume of concentration of acrylamide for the resolving gel, using the each solution to the stacking gel wells. Start the electrophore- values given in Table 1. Mix the components in the order sis using suitable operating conditions for the electrophoresis shown. Where appropriate, before adding the ammonium equipment to be used. There are commercially available gels persulfate solution and the tetramethylethylenediamine of diŠerent surface area and thickness that are appropriate (TEMED), ˆlter the solution if necessary under vacuum for various types of electrophoresis equipment. Electropho- through a cellulose acetate membrane (pore size: 0.45 mm); resis running time and currentWvoltage may need to be altered keep the solution under vacuum by swirling the ˆltration unit depending on the type of apparatus used, in order to achieve until no more bubbles are formed in the solution. Add ap- optimum separation. Check that the dye front is moving into propriate amounts of ammonium persulfate solution and the resolving gel. When the dye is reaching the bottom of the TEMED as indicated in Table 1, swirl and pour immediately gel, stop the electrophoresis. Remove the gel assembly from into the gap between the two glass plates of the mould. Leave the apparatus and separate the glass plates. Remove the su‹cient space for the stacking gel (the length of the teeth of spacers, cut oŠ and discard the stacking gel and immediately the sample comb plus 1 cm). Using a pipette, carefully over- proceed with staining. lay the solution with water-saturated . Leave the 5. Detection of Proteins in Gels gel in a vertical position at room temperature to allow poly- Coomassie staining is the most common protein staining merization to occur. method, with a detection level of the order of 1 mgto10mgof Preparation of the stacking gel: After polymerization is protein per band. Silver staining is the most sensitive method complete (about 30 minutes), pour oŠ the isobutanol and for staining proteins in gels and a band containing l0 ng to wash the top of the gel several times with water to remove the 100 ng can be detected. All of the steps in gel staining are isobutanol overlay and any unpolymerized acrylamide. Drain done at room temperature with gentle shaking in any con- as much ‰uid as possible from the top of the gel, and then re- venient container. Gloves must be worn when staining gels, move any remaining water with the edge of a paper towel. since ˆngerprints will stain. In a conical ‰ask, prepare the appropriate volume of solu- 1) Coomassie staining tion containing the desired concentration of acrylamide, Immerse the gel in a large excess of Coomassie staining TS using the values given in Table 2. Mix the components in the and allow to stand for at least 1 hour. Remove the staining order shown. Where appropriate, before adding the ammoni- solution. um persulfate solution and the TEMED, ˆlter the solution if Destain the gel with a large excess of destaining TS. necessary under vacuum through a cellulose acetate mem- Change the destaining solution several times, until the stained brane (pore size: 0.45 mm); keep the solution under vacuum protein bands are clearly distinguishable on a clear back- by swirling the ˆltration unit until no more bubbles are ground. The more thoroughly the gel is destained, the smaller formed in the solution. Add appropriate amounts of ammo- is the amount of protein that can be detected by the method. nium persulfate solution and TEMED as indicated in Destaining can be speeded up by including 2 to 3 g of anion- Table 2, swirl and pour immediately into the gap between the exchange resin or a small sponge in the destaining TS. two glass plates of the mould directly onto the surface of the NOTE: the acid-alcohol solutions used in this procedure polymerized resolving gel. Immediately insert a clean sample do not completely ˆx proteins in the gel. This can lead to loss- comb into the stacking gel solution, taking care to avoid trap- es of some low-molecular-mass proteins during the staining ping air bubbles. Add more stacking gel solution to ˆll com- and destaining of the gel. Permanent ˆxation is obtainable by pletely the spaces of the sample comb. Leave the gel in a ver- allowing the gel to stand in trichloroacetic acid TS for ˆxing tical position and allow to polymerize at room temperature. for 1 hour before it is immersed in Coomassie staining TS. 3) Mounting the gel in the electrophoresis apparatus and JP XV General Information / SDS-Polyacrylamide Gel 1745

Table 1. Preparation of resolving gel

Component volumes (mL) per gel mould volume of Solution components 5mL 10mL 15mL 20mL 25mL 30mL 40mL 50mL

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 molWL Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5 100 gWLSDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 gWLAPS(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 molWL Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5 100 gWLSDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 gWLAPS(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 molWL Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5 100 gWLSDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 gWLAPS(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 molWL Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5 100 gWLSDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 gWLAPS(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 molWL Tris solution (pH 8.8)(2) 1.2 2.5 3.6 5.0 6.3 7.5 10.0 12.5 100 gWLSDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 gWLAPS(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 molWL Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5 100 gWLSDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5 100 gWLAPS(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 acrylamideWbisacrylamide (29:1) solution (2) 1.5 molWL Tris solution (pH 8.8): 1.5 molWL tris-hydrochloride buŠer solution, pH 8.8 (3) 100 gWL SDS: 100 gWL solution of sodium dodecyl sulfate (4) 100 gWLAPS:100gWL solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solu- tions must be prepared before use. (5) TEMED: N,N,N?,N?-tetramethylethylenediamine 1746 SDS-Polyacrylamide Gel / General Information JP XV

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 molWL Tris solution (pH 6.8)(2) 0.13 0.25 0.38 0.5 0.63 0.75 1.0 1.25 100 gWLSDS(3) 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1 100 gWLAPS(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 acrylamideWbisacrylamide (29:1) solution (2) 1.0 molWL Tris solution (pH 6.8): 1 molWL tris-hydrochloride buŠer solution, pH 6.8 (3) 100 gWL SDS: 100 gWL solution of sodium dodecyl sulfate (4) 100 gWLAPS:100gWL solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solu- tions must be prepared before use. (5) TEMED: N,N,N?,N?-tetramethylethylenediamine

2) Silver staining leading edge of the electrophoretic ion front. This can be Immerse the gel in a large excess of ˆxing TS and allow to done by cutting notches in the edges of the gel or by inserting stand for 1 hour. Remove the ˆxing solution, add fresh ˆxing a needle soaked in ink into the gel at the dye front. solution and incubate either for at least 1 hour or overnight, After staining, measure the migration distances of each pro- if convenient. Discard the ˆxing solution and wash the gel in tein band (markers and unknowns) from the top of the water for 1 hour. Soak the gel for 15 minutes in a 1 volz resolving gel. Divide the migration distance of each protein glutaraldehyde solution. Wash the gel twice for 15 minutes in by the distance traveled by the tracking dye. The normalized water. Soak the gel in fresh silver nitrate TS for silver staining migration distances so obtained are called the relative mobili- for 15 minutes, in darkness. Wash the gel three times for 5 ties of the proteins (relative to the dye front) and convention- minutes in water. Immerse the gel for about 1 minute in de- ally denoted as Rf. Construct a plot of the logarithm of the veloper TS until satisfactory staining has been obtained. Stop relative molecular masses (Mr) of the protein standards as a the development by incubation in blocking TS for 15 function of the Rf values. Note that the graphs are slightly minutes. Rinse the gel with water. sigmoid. Unknown molecular masses can be estimated by linear regression analysis or interpolation from the curves of 6. Drying of Stained SDS-Polyacrylamide Gels log M against Rfaslongasthevaluesobtainedforthe Depending on the staining method used, gels are pretreated r unknown samples are positioned along the linear part of the in a slightly diŠerent way. For Coomassie staining, after the graph. destaining step, allow the gel to stand in a diluted solution of concentrated glycerin (1 in 10) for at least 2 hours (overnight 8. Suitability of the Test (Validation) incubation is possible). For silver staining, add to the ˆnal The test is not valid unless the front end of the molecular rinsingastepof5minutesinadilutedsolutionofconcentrat- mass marker migrates 80z of the migrating distance of the ed glycerin (1 in 50). dye, and over the required separation range (e.g., the range Immerse two sheets of porous cellulose ˆlm in water and covering the product and its dimer or the product and its incubate for 5 to 10 minutes. Place one of the sheets on a related impurities) the separation obtained for the relevant drying frame. Carefully lift the gel and place it on the cellu- protein bands shows a linear relationship between the lose ˆlm. Remove any trapped air bubbles and pour 2 to 3 logarithmofthemolecularmassandtheRfasdescribedin7. mL of water around the edges of the gel. Place the second Additional requirements with respect to the solution under sheet on top and remove any trapped air bubbles. Complete test may be speciˆed in individual monographs. the assembly of the drying frame. Place in an oven or leave at 9. Quantiˆcation of Impurities room temperature until dry. Where the impurity limit is speciˆed in the individual 7. Molecular-Mass Determination monograph, a reference solution corresponding to that level Molecular masses of proteins are determined by compari- of impurity should be prepared by diluting the test solution. son of their mobilities with those of several marker proteins For example, where the limit is 5z, a reference solution of known molecular mass. Mixtures of proteins with pre- would be a 1:20 dilution of the test solution. No impurity cisely known molecular masses blended for uniform staining (any band other than the main band) in the electrophoreto- are commercially available for calibrating gels. They are ob- gram obtained with the test solution may be more intense tainable in various molecular mass ranges. Concentrated than the main band obtained with the reference solution. stock solutions of proteins of known molecular mass are Under validated conditions, impurities may be quantiˆed diluted in the appropriate sample buŠer and loaded on the by normalization to the main band, using an integrating den- same gel as the protein sample to be studied. sitometer. In this case, the responses must be validated for Immediately after the gel has been run, the position of the linearity. bromophenol blue tracking dye is marked to identify the JP XV General Information / Solid and Particle Densities 1747

g/cm3＝1000 kg/m3). Test solutions: Coomassie staining TS Dissolve 125 mg of coomassie Crystal Density brilliant blue R-250 in 100 mL of a mixture of water, The crystal density of a substance is the average mass per methanol and acetic acid (100) (5:4:1), and ˆlter. unit volume, exclusive of all voids that are not a fundamental Developer TS Dissolve 2 g of citric acid monohydrate in part of the molecular packing arrangement. It is an intrinsic water to make 100 mL. To 2.5 mL of this solution add 0.27 property concerning the speciˆc crystal structure of the sub- mL of formaldehyde solution and water to make 500 mL. stance, and is not aŠected by the method of determination. Fixing TS To 250 mL of methanol add 0.27 mL of for- The crystal density can be determined either by calculation or maldehyde solution and water to make 500 mL. by simple measurement. Silver nitrate TS for silver staining To 40 mL of sodium A. The calculated crystal density is obtained using: hydroxide TS add 3 mL of solution (28), then add 1) For example, the crystallographic data (volume and dropwise 8 mL of a solution of silver nitrate (1 in 5) while composition of the unit cell) obtained by indexing stirring, and add water to make 200 mL. the perfect crystal X-ray diŠraction data from single Destaining TS A mixture of water, methanol and acetic crystal or the powder X-ray diŠraction data. acid (100) (5:4:1). 2) Molecularmassofthesubstance. Blocking TS To 10 mL of acetic acid (100) add water to B. The measured crystal density is obtained as the mass to make 100 mL. volume ratio after measuring the single crystal mass and Trichloroacetic acid TS for ˆxing Dissolve 10 g of volume. trichloroacetic acid in a mixture of water and methanol (5:4) Particle Density to make 100 mL. The particle density takes account both the crystal density and the intraparticulate porosity (sealed and/or experimen- tally non-accessible open pores) as a part of the particle 24. Solid and Particle Densities volume. The particle density depends on the value of the volume determined, and the volume in turn depends on the Density of a solid or a powder as a state of aggregation has method of measurement. Particle density can be determined diŠerent deˆnitions depending on the way of including of the either by gas displacement pycnometry or mercury porosi- interparticulate and intraparticulate voids that exist between metry, but the Japanese Pharmacopoeia speciˆes the the particles or inside the powder. DiŠerent ˆgures are ob- pycnometry as the ``Powder Particle Density Determina- tained in each case, and there are diŠerent practical mean- tion''. ings. Generally, there are three levels of deˆnitions of the A. The pycnometric density is obtained by assuming that solid or powder density. the volume of the gas displaced, which is measured with (1) Crystal density: It is assumed that the system is the gas displacement pycnometer, is equivalent to that homogeneous with no intraparticulate void. Crystal density is of a known mass of the powder. In pycnometric density also called true density. measurements, any volume with the open pores accessi- (2) Particle density: The sealed pores or the experimental- ble to the gas is not included as a part of volume of the ly non-accessible open pores is also included as a part of the powder, but the sealed pores or pores inaccessible to the volumesofthesolidorthepowder. gas is included as a part of the volume of the powder. (3) Bulk density: The interparticulate void formed in the Due to the high diŠusivity of helium which can penetrate powder bed is also included as a part of the volumes of the to most open pores, it is recommendable as the measure- solid or the powder. Bulk density is also called apparent ment gas of particle density. Therefore, the pycnometric density. Generally, the powder densities at loose packing and particle density of a ˆnely milled powder is generally not at tapping are deˆned as the bulk density and the tapped den- very diŠerent from the crystal density. Hence, the parti- sity, respectively. cle density by this method is the best estimate of the true Generally, the densities of liquid or gas are aŠected only by density of an amorphous or partially crystalline sample, temperature and pressure, but the solid or powder density is and can be widely used for manufacturing control of the aŠected by the state of aggregation of the molecules or the processed pharmaceutical powder samples. particles. Therefore, the solid or powder densities naturally B. The mercury porosimetric density is also called granular vary depending on crystal structure or crystallinity of the sub- density. This method also includes the sealed pores as a stance concerned, and also varies depending on the method part of the volumes of the solid or the powder, but of preparation or handling if the sample is amorphous form excludes the volume only from the open pores larger or partially amorphous. Consequently, even in a case that than some size limit. This pore size limit or minimal two solids or powders are chemically identical, it may be pos- access diameter depends on the maximal mercury sible that the diŠerent ˆgures of density are obtained if their intrusion pressure applied during the measurement and crystal structures are diŠerent. As the solid or powder parti- under normal operating pressure, the mercury does not cle densities are important physical properties for the pow- penetrate the ˆnenest pores accessible to helium. Since dered pharmaceutical drugs or the powdered raw materials of this method is capable of measuring the density which drugs, the Japanese Pharmacopoeia speciˆes each density de- corresponds to the pore size limit at each mercury termination as ``Powder Particle Density Determination'' for intrusion pressure, the various granular densities can be the particle density and as ``Determination of Bulk and obtained from one sample. Tapped Densities'' for the bulk density. Bulk Density and Tapped Density The solid or powder densities are expressed in mass per The bulk density of a powder includes the contribution of unit volume (kg/m3), and generally expressed in g/cm3 (1 1748 Sterility Assurance / General Information JP XV interparticulate void volume as a part of the volume of the 1.2 Validation powder. Therefore, the bulk density depends on both the A documented procedure for obtaining, recording and in- powder particle density and the space arrangement of terpreting the results needed to show that a process will con- particles in the power bed. Further, since the slightest sistently yield a product complying with predetermined disturbance of the bed may result in variation of the space ar- speciˆcations. rangement, it is often very di‹cult to determine the bulk den- 1.3 Periodic re-validation sity with good reproducibility. Therefore, it is essential to Validation that is regularly performed to reconˆrm that a specify how the determination was made upon reporting the process is consistently yielding a product complying with bulk density. predetermined speciˆcations. It should conˆrm that variables The Japanese Pharmacopoeia speciˆes ``Determination of and the acceptable ranges are permissible to yield a product Bulk and Tapped Densities''. consistently of the required quality. A. The bulk density is determined by measuring the 1.4 FacilityWequipment qualiˆcation apparent volume of a known mass of powder sample This is to provide evidence that the manufacturing facilities that has been passed through a screen in a graduated Wequipment, measuring equipment, and manufacturing en- cylinder (constant mass method). Separately, the Phar- vironment control facilities, etc. have been properly selected, macopoeia speciˆes the method of determining bulk correctly installed, and are operated in conformity with the density by measuring the mass of powder in a vessel hav- speciˆcations at the time of installation and during opera- ing a known volume (constant volume method). tion. B. The tapped density is obtained by mechanically tapping 1.5 Operation qualiˆcation a measuring cylinder containing a powder sample. After This is to provide evidence to conˆrm physically, chemical- determining the initial bulk volume, carry out tapping ly and microbiologically that equipment, operated in accor- under a ˆxed measurement condition (tapping rate and dance with its operational instructions, operates as speciˆed drop height), and the measurement is carried out repeat- and aŠords a product meeting the speciˆcations. edly until the bulk volume variation obtained at consecu- 1.6 Support system for sterilization process tive two measurements is within an acceptable range This refers to the facilityWequipment that is associated with (constant mass method). Separately, the Pharmacopoeia the sterilization devices, such as the preconditioning and aer- speciˆes the method of determining the tapped density ation for ethylene oxide sterilization, the steam supply equip- by measuring the mass of a ˆxed volume of the tapped ment for moist heat sterilization, and the loading devices for powder (constant volume method). radiation sterilization. 1.7 Quality system The procedures, resources and organizational structure of a manufacturer (responsibilities, authorities and relation- 25. Sterility Assurance for ships between these) required to implement quality manage- Terminally Sterilized ment. 1.8 Change control system Pharmaceutical Products A system designed to evaluate all of the changes that may aŠect the quality of the pharmaceutical product, in order to As indicated in the ``Terminal Sterilization and Steriliza- ensure that the process is continuously controlled. tion Indicators'', the pharmaceuticals to which terminal 1.9 F0 value sterilization can be applied, generally must be sterilized so Assume a value of 109CfortheZ value deˆned as the num- －6 that a sterility assurance level of 10 or less is obtained. The ber of degrees of temperature required for a 10-fold change sterility assurance level of 10－6 or less can be proven by using in the D value. The F0 value indicates the time (minutes) re- a sterilization process validation based on physical and quired to give the equivalent lethality at Tb of the sterilization microbiological methods, but cannot be proven by sterility heat obtained by integrating the lethality rate (L)overanen- tests of the sterilized products. This chapter deals with the tire heating cycle. necessary requirements for the appropriate management of the important control points of the sterilization process for T0－Tb T0－Tb L＝log－1 ＝10 Z the parametric release of products, without performing steril- Z ity tests on products which have been subjected to terminal T0＝Temperature inside the chamber or inside the product sterilization (in the case of radiation sterilization, called dosi- to be sterilized metric release). Parametric release is a method that can be ap- Tb＝Reference temperature (1219C) plied in cases where the sterilization system is clearly deˆned, t important control points are clearly speciˆed, and the sterili- 1 F0＝fLdt zation system process can be validated by microbiological t0 methods using appropriate biological indicators. t1－t0＝Processing time (minutes) 1. Deˆnitions The deˆnitions of the terminology used in this chapter are 1.10 Control device provided below. A general term for the devices and measurement equip- 1.1 Terminal sterilization ment, including the equipment for controlling, measuring A process whereby a product is sterilized in its ˆnal con- and recording the physical parameters that can be measured tainer or packaging, and which permits the measurement and (temperature, humidity, pressure, time, radiation dose, etc.). evaluation of quantiˆable microbial lethality. 1.11 Parametric release A release procedure based on an evaluation of the produc- JP XV General Information / Sterility Assurance 1749 tion records and critical parameters of the sterilization i. Other required matters for the implementation of the process (temperature, humidity, pressure, time, radiation applicable sterilization validation dose, etc.) based on the results of validation, in lieu of release 2.3.2 The following sterilization validation is implement- based on testing results of the ˆnal product. ed according to the plan deˆning the items above. a. When the manufacturing license and additional 2. Sterilization Validation (modiˆcation) licenses for product production are obtained, 2.1 Subject of the Implementation implementation items for the sterilization validation to be ex- A manufacturer of sterile pharmaceuticals (hereafter, ecuted ``manufacturer'') must establish a quality system, implement 1 Product qualiˆcation product sterilization validation for the categories below as a 2 FacilityWequipment qualiˆcation general rule, and continuously control the sterilization 1) Installation qualiˆcation process based on the results of the sterilization validation. 2) Operation qualiˆcation a. Sterilization process 3 Performance qualiˆcation b. Sterilization process support system 1) Physical performance qualiˆcation 2.2 Documenting Sterilization Validation Procedure 2) Microbiological performance qualiˆcation 2.2.1 The manufacturer must prepare a ``Sterilization b. Sterilization validation to be executed until it is time to Validation Procedure'' deˆning the items listed below regard- renew the manufacturing license ing the procedures for managing the sterilization process. 1 Re-validation when there are changes a. Details related to the range of duties of the persons 2 Periodic re-validation (The items implemented, etc. responsible for the validation, as well as the extent of their must be determined based on a consideration of authority relevant factors such as the sterilization method.) b. Details related to the implementation period for the 2.3.3 Evaluate the results of the sterilization validation sterilization validation and verify that sterility is assured. c. Details related to the creation, modiˆcation, and ap- 2.3.4 Makeawrittenreportoftheresultsofthesteriliza- proval of the sterilization validation plan documents tion validation to the manufacturer's authorized person. d. Details related to the reporting, evaluation, and ap- 2.3.5 Perform the day-to-day management of the sterili- proval of the sterilization validation implementation results zation process. e. Details related to the storage of documentation con- cerning the sterilization validation 3. Microorganism Control Program f. Other required matters When parametric release is adopted, it is important to con- 2.2.2 The sterilization validation procedure must list the trol the bioburden in the raw materials of the product, the names of the enactors, the date of enactment, and when there containers and stoppers, and in the product before steriliza- are revisions, must also list the revisers, date of revisions, tion. The bioburden is measured with a previously speciˆed revised sections and reasons for the revisions. method and frequency, and when required, surveys of the 2.2.3 The manufacturer must properly store and main- characteristics of the isolated microorganisms are made to in- tain the sterilization validation procedure after clarifying the vestigate their resistance to the applicable sterilization procedures related to alterations and deletions of the contents method. Refer to the ``Microbiological Evaluation of Process of the sterilization validation procedure. Areas for Sterile Pharmaceutical Products'' regarding the 2.3 Persons Responsible for the Validation method for evaluating the environmental microorganisms in The manufacturer must assign persons to be responsible the processing areas of pharmaceutical products. for the sterilization validation. The responsible parties must 4. Sterilization Indicators perform each of the duties listed below according to the Biological indicators (BI), chemical indicators (CI), and sterilization validation procedure. dosimeters are among the means used to monitor a steriliza- 2.3.1 For products that are to be produced according to tion process and as indices of sterility (refer to Terminal the sterilization validation procedure, a written sterilization Sterilization and Sterilization Indicators). When using sterili- validation implementation plan must be prepared. The im- zation indicators it is important to consider environmental plementation plan will specify the following points based on and human safety, and to take all necessary precautions. The a consideration of the implementation details of the steriliza- BI used for sterilization validation and daily process control tion validation. must be deˆned in the speciˆcation, and recorded in writing. a. Subject pharmaceutical name (product name) When BI are used for daily process control it must be veriˆed b. Purpose of the applicable sterilization validation that the loading pattern on the form, product, or simulated c. Expected results product has a resistance equal to or greater than that used for d. Veriˆcation methods (including inspection results and the microbiological performance qualiˆcation. evaluation methods) e. Period of veriˆcation implementation 5. Establishment of a Change Control System f. Names of persons performing the sterilization valida- Changes which have a large eŠect on the sterile quality, tion (persons-in-charge) such as changes in sterilization equipment, loading pattern, g. Names of the persons who created the plan, creation and sterilization conditions, correspond to changes of the date, and in the event of revisions, the names of the revisers, parametric release conditions for the relevant pharmaceutical date of the revisions, revised sections, and reasons for revi- product. A change control system must be deˆned in the sion. sterilization validation procedure; and when there are h. Technical requirements for the applicable sterilization changes in the causes of variation that have been previously validation speciˆed, there must be an investigation of the causes of vari- 1750 Sterility Assurance / General Information JP XV ation and of acceptable conditions to verify that the phar- g) Other maceutical product is guaranteed always to conform to the 7.2 Ethylene oxide gas sterilization quality standards. Furthermore, before modiˆcations are Ethylene oxide gas allows sterilization at low temperatures, made to a sterilization process that has been validated, it is so there is typically little injury to the substance being steri- mandatory to obtain approval for the implementation of the lized; however, since the gas is toxic it must be handled with modiˆcations in question from the appropriate authorized extreme caution. The sterilization process consists of precon- person. ditioning, a sterilization cycle, and aeration. The precon- ditioning is performed before the sterilization cycle to process 6. Release Procedure the product so that temperature and relative humidity in the A release procedure must be created to clarify the condi- room or container are within the range in the speciˆcations. tions required for shipment based on parametric release of The sterilization cycle indicates the stage at which the actual terminally sterilized products. The following points must be sterilization is performed, and consists of removal of the air, evaluated and recorded when a product is released. conditioning (when used), injection of the sterilization gas, Depending on the sterilization method, some of these items maintenance of the sterilization conditions, removal of the may be omitted or modiˆed. sterilization gas, and replacement of the air. The aeration is a) Batch record the process of eliminating the residual ethylene oxide gas b) Microorganism evaluation data of production en- from the product, either inside the sterilization chamber or in vironment a separate location. c) Bioburden data for the raw materials and product be- 7.2.1 Important control points fore sterilization The important control points for the ethylene oxide gas d) Data related to the sterilization indicators sterilization are indicated below. e) Data on the maintenance management of the steriliza- 7.2.1.1 Preconditioning (when performed) tion process and sterilization process support systems a) Time, temperature, humidity f) Data on the management of sterilization parameters b) Product loading patternWloading density g) Data on the calibrations of measurement equipment c) Sterilization loading temperature andWor humidity h) Re-validation data d) Time from the end of preconditioning until the start of i) Other the sterilization 7. Critical Control Points e) Other necessary matters The important control points for each sterilization method 7.2.1.2 Conditioning are presented. a) If pressure reduction is performed, the pressure 7.1 Moist heat sterilization achieved and required time Moist heat sterilization is a method for killing microorgan- b) Reduced pressure maintenance period isms in which saturated water vapor is generated or in- c) Time, temperature, pressure, humidity troduced into a sterilization chamber at the appropriate tem- d) Sterilization loading temperature and humidity perature and pressure, and the chamber is then heated for a e) Other necessary matters certain period of time. It is roughly classiˆed into saturated 7.2.1.3 Sterilization cycle vapor sterilization, in which the target microorganisms are a) Pressure increase, injection time, and ˆnal pressure directly exposed to the saturated vapor, and unsaturated for the injection of the sterilization gas vapor sterilization, in which the ‰uid inside a container, such b) Concentration of the ethylene oxide gas (it is desirable as an ampule, is subjected to moist heat energy or highfre- to analyze directly the gas concentration inside the steriliza- quency energy from the outside. tion chamber, but the following alternatives are acceptable if 7.1.1 Important control points direct analysis is di‹cult) A process control procedure must be created, specifying i) Mass of gas used the process parameters that aŠect the sterile quality of the ii) Volume of gas used pharmaceutical product, and the permissible range of varia- iii) Conversion calculation using the initial low pressure tion for each parameter. The important control points for the level and the gas injection pressure moist heat sterilization are indicated below. c) Temperature within the sterilization chamber

a) Heating history (usually indicated by F0 value) d) Temperature of the loaded products to be sterilized b) Temperature e) EŠect time (exposure time) c) Pressure f) Product loading patternWloading density d) Time g) BI placement points and cultivation results e) Product loading formatWloading density h) Other necessary matters f) Other necessary matters 7.2.1.4 Aeration 7.1.2 Utilities a) Time, temperature The utilities and control devices required for moist heat b) Loaded sterilized substance temperature sterilization determine the quality and precision. c) Pressure variation in the sterilization chamber andWor a) Quality of the vapor used the aeration room b) Quality of the air introduced into the sterilization d) Rate of change of the air or other gases in the aeration chamber to restore pressure, etc. room c) Quality of the water used for cooling e) Other necessary matters d) Precision of the temperature control devices 7.2.2 Utilities e) Precision of the pressure control devices The utilities and control devices required for ethylene oxide f) Precision of the time control devices sterilization determine the quality and precision. JP XV General Information / Tablet Friability Test 1751

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 Notiˆcation No.158, sterilization Ministry of Health and Welfare 1995 d) Quality of the BI 2) Sterilization Validation Standards, PMSBWIGD Notiˆ- 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, PAB g) Precision of the humidity control devices Notiˆcation No.1128, Ministry of Health and Welfare h) Precision of the time control devices 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 electron 8) ISO 11138 Biological indicators beams and bremsstrahling (X-ray) generated from an elec- 9) ISO 11140 Chemical indicators tron accelerator. In the case of g-rays, the cells are killed by 10) ISO 11737-1 Microbiological Methods Part 1: Estima- secondarily generated electrons, while in the case of the elec- tion of population of microorganisms on products tron beam, the cells are killed by the electrons generated 11) USP 〈1222〉 Terminally Sterilized Pharmaceutical directly from the electron accelerator. For this reason, the Products - Parametric Release processing time for electron beam sterilization is generally shorter than that for g-ray sterilization; but, since the penetration of the g-rays is better than that of the electron beam, there must be appropriate consideration of the density 26. Tablet Friability Test and thickness of the substance being sterilized when choosing This test is harmonized with the European Pharmacopoeia between these methods. For an irradiation sterilization and the U.S. Pharmacopeia. process, the control procedures primarily make use of The Tablet Friability Test is a method to determine the dosimeters and measure the absorbed dose in the substance friability of compressed uncoated tablets. The test procedure being sterilized. This is called dosimetric release. presented in this chapter is generally applicable to most com- 7.3.1 Important control points pressed tablets. Measurement of tablets friability supple- The important control points for the irradiation steriliza- ments other physical strength measurement, such as tablet tion are indicated below. crushing strength. 7.3.1.1 g-ray radiation Use a drum, with an internal diameter between 283 and 291 a) Irradiation time (timer setting or conveyor speed) mm and a depth between 36 and 40 mm, of transparent syn- b) Absorbed dose thetic polymer with polished internal surface, and not subject c) Product loading pattern to minimum static build-up (see ˆgure for a typical appara- d) Other necessary matters tus). One side of the drum is removable. The tablets are tum- 7.3.1.2 Electron beam and X-ray radiation bled at each turn of the drum by a curved projection with an a) Electron beam characteristics (average electron beam inside radius between 75.5 and 85.5 mm that extends from current, electron energy, scan width) the middle of the drum to the outer wall. The outside di- b) Conveyor speed ameter of the central shaft ring is between 24.5 and 25.5 mm. c) Absorbed dose The drum is attached to the horizontal axis of a device that d) Product loading pattern rotates at 25±1 rpm. Thus, at each turn the tablets roll or e) Other necessary matters slide and fall onto the drum wall or onto each other. 7.3.2 Utilities For tablets with a unit mass equal to or less than 650 mg, A traceable calibration, performed according to national take a sample of whole tablets n corresponding as near as standards, must be performed for the radiation devices and possible to 6.5 g. For tablets with a unit mass of more than dose measurement systems. This calibration must be per- 650 mg, take a sample of 10 whole tablets. The tablets should formed as speciˆed in a written plan in order to verify that be carefully dedusted prior to testing. Accurately weigh the the equipment is kept within the required range of accuracy. tablet sample, and place the tablets in the drum. Rotate the 7.3.2.1 Required calibration items for gamma-radiation drum 100 times, and remove the tablets. Remove any loose equipment dust from the tablets as before, and accurately weigh. a) Cycle time or conveyor speed Generally, the test is run once. If obviously cracked, b) Weighing device cleaved, or broken tablets are present in the tablet sample c) Dose measurement system after tumbling, the sample fails the test. If the results are d) Other di‹cult to interpret or if the weight loss is greater than the 7.3.2.2 Required calibration items for electron-beam and targeted value, the test should be repeated twice and the mean X-ray radiation equipment of the three tests determined. A maximum mean weight loss a) Electron beam characteristics from the three samples of not more than 1z is considered ac- b) Conveyor speed ceptable for most products. c) Weighing device If tablet size or shape causes irregular tumbling, adjust the d) Dose measurement system drum base so that the base forms an angle of abut 109with e) Other 1752 Terminal Sterilization / General Information JP XV

of pharmaceutical products, and performing microbiological tests speciˆed in the monographs, see Disinfection and Sterilization Methods. 1. Deˆnitions Thedeˆnitionsofthetermsusedinthistextareasfollows. Terminal sterilization: A process whereby a product is sterilized in its ˆnal container or packaging, and which per- mits the measurement and evaluation of quantiˆable microbial lethality. Product: A generic term used to describe raw materials, in- termediate products, and ˆnished products, to be sterilized. Bioburden: Numbers and types of viable microorganisms in a product to be sterilized. Sterility assurance level (SAL): Probability of a viable microorganism being present in a product unit after exposure to the proper sterilization process, expressed as 10－n. the bench top and the tablets no longer bind together when Integrity test: A non-destructive test which is used to lying next to each other, which prevents them from falling predict the functional performance of a ˆlter instead of the freely. microorganism challenge test. EŠervescent tablets and chewable tablets may have diŠer- D value: The value which shows the exposure time (decimal ent speciˆcations as far as friability is concerned. In the case reduction time) or absorbed dose (decimal reduction dose) re- of hygroscopic tablets, an appropriate humidity-controlled quired to cause a 1-logarithm or 90z reduction in the popu- environment is required for testing. lation of test microorganisms under stated exposure condi- Drums with dual scooping projections, or apparatus with tions. more than one drum, for the running of multiple samples at Sterilization indicator: Indicators used to monitor the one time, are also permitted. sterilization process, or as an index of sterility, including bio- logical indicators (BI), chemical indicators (CI), dosimeters and the like. 27. Terminal Sterilization and 2. Sterilization Sterilization Indicators 2-1. Heat Method In the heat method, microorganisms are killed by heating. Sterilization is a process whereby the killing or removal of (i) Moist heat method all forms of viable microorganisms in substances is accom- Microorganisms are killed in saturated steam under pres- plished. It is achieved by terminal sterilization or a ˆltration sure. In this method, factors which may aŠect the steriliza- method. For substances to which terminal sterilization can be tion include temperature, steam pressure and exposure time. applied, an appropriate sterilization method should be select- Therefore, in routine sterilization process control, it is re- ed in accordance with the properties of the product, including quired to monitor continuously the temperature, steam pres- the packaging, after full consideration of the advantages and sure and exposure time, and they should be included in the disadvantages of each sterilization method, from among the speciˆcations of the sterilizer. heat method, irradiation method and gas method. After in- (ii) Dry-heat method stallation of the sterilizer (including design and development Microorganisms are killed in dry heated air. This method is of the sterilization process), validation is required to conˆrm usually conducted in a batch-type dry heat sterilizer or a tun- that the sterilization process is properly performing its nel-type dry heat sterilizer. In this method, factors which may designed function, under conditions of loading and unload- aŠect the sterilization include temperature and exposure ing of the product, on the basis of su‹cient scientiˆc evi- time. Therefore, in routine sterilization process control, it is dence. After the process has been validated and the steriliza- required to monitor continuously the temperature and ex- tion of the product commenced, the process must be con- posure time, and they should be included in the speciˆcations trolled correctly, and qualiˆcation tests of the equipment and of the sterilizer. procedures must be performed regularly. The bioburden per 2-2. Irradiation method product, prior to terminal sterilization, must be evaluated Microorganisms are directly killed by ionizing radiation, or periodically or on the basis of batches. Refer to the ISO stan- by the heat generated by microwave radiation. dard (ISO 11737-1) relevant to bioburden estimation. For a (i) Radiation method substance to which terminal sterilization can be applied, Ionizing radiations which may be used are gamma (g)rays generally use sterilization conditions such that a sterility as- emitted from a radioisotope such as 60, an electron surance level of less than 10－6 can be obtained. The propriety beam and bremsstrahlung (X rays) generated from an elec- of the sterilization should be judged by employing an ap- tron accelerator. Although any procedure can be applied to propriate sterilization process control, with the use of a suita- thermally unstable products with no radioactivity residue, it ble sterilization indicator, and if necessary, based on the is necessary to consider the possibility of material degrada- result of the sterility test. The ˆltration procedure is used for tion. Although a 25 kGy dose is traditionally used as a sterili- the sterilization of a liquid product, to which terminal sterili- zationdose,therearesomewaystocalculatethedoseasfol- zation can not be applied. Concerning the disinfection andW lows: the bioburden of the substance to be sterilized is meas- or sterilization necessary for processing equipment and areas ured and the sterilization dose is calculated based on the JP XV General Information / Terminal Sterilization 1753 mean bioburden and the standard resistance distribution ‰ow rate, ˆlter unit characteristics and the like. In routine (Method 1 in ISO 11137), the dose is calculated from the frac- ˆltration process control, it is required to perform integrity tion positive information from a sterility test in which tests of the sterilizing ˆlter after each ˆltration process (also representative product samples are exposed to a substerilizing prior to the ˆltration process, if necessary). dose (Method 2 in ISO 11137), or the dose is calculated based 4. Sterilization Indicators on the bioburden and D value of the most resistant microor- 4-1. Biological indicator (BI) ganisms (Log method) (see 5-3). In the case of the radiation A BI is prepared from speciˆc microorganisms resistant to sterilization procedure, factors which may aŠect the steriliza- the speciˆed sterilization process and is used to develop andW tion include dose (absorbed dose) and exposure time. There- or validate a sterilization process. The dry type BI is classiˆed fore, in g ray sterilization process control, it is required to de- into two kinds. In one, bacterial spores are added to a carrier termine the dose (the absorbed dose) at appropriate intervals such as ˆlter paper, glass or plastic and then the carriers are and to monitor continuously the exposure time in terms of dried and packaged. In the other, bacterial spores are added the operating parameters (the conveyer speed, the cycle time). to representative units of the product to be sterilized or to The dose control mechanism should be included in the simulated products. Packaging materials of the BI should speciˆcations of the sterilizer. In the case of electron beam or show good heat penetration in dry heat sterilization and good bremsstrahlung irradiation, it is required to monitor the ac- gas or steam penetration in ethylene oxide and moist heat celeration voltage, the beam current and beam scanning sterilizations. It should be conˆrmed that any carrier does not width besides the above-mentioned items. aŠect the D value of the spores. In the case of a liquid (ii) Microwave method product, the spores may be suspended in the same solution as Microorganisms are killed by the heat generated by micro- the product or in a solution showing an equivalent eŠect in wave radiation, usually at the frequency of 2450±50 MHz. the sterilization of biological indicator. However, when the This method is applied to liquids or water-rich products in spores are suspended in liquid, it is necessary to ensure that sealed containers. Since a glass or plastic container may be the resistance characteristics of the spores are not aŠected destroyed or deformed due to the rise of the inner pressure, due to germination. the containers must be certiˆed 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 su‹ciently low level to cause no harm to humans and no in- Representative Sterilization microorganisms* Strain name terference with radio communications and the like. In this method, factors which may aŠect the sterilization include Moist heat Geobacillus ATCC 7953, NBRC temperature, processing time and microwave output power. method stearo- 13737, JCM 9488 Therefore, in routine sterilization process control, it is re- thermophilus ATCC 12980, NBRC quired to monitor continuously the temperature, time and the 12550, JCM 2501 microwave output power, and they should be included in the Dry heat Bacillus speciˆcations of the sterilizer. method atrophaeus ATCC 9372, NBRC 13721 2-3. Gas method Gas method Bacillus Ethylene oxide (EO) is widely used as a sterilization gas. atrophaeus ATCC 9372, NBRC 13721 Since EO gas has an explosive nature, a 10 – 30z mixture with carbon dioxide is commonly used. Also, as EO gas is a * In addition to these microorganisms, other microorgan- strong alkylating agent, it can not be applied to the products isms with the greatest resistance to the sterilization proce- which are likely to react with or absorb it. Furthermore, be- dure concerned, found in the bioburden, can be used as the cause EO gas is toxic, the residual concentration of EO gas biological indicator. and other secondarily generated toxic gases in products steri- 4-1-1. D value of BI lized with EO gas must be reduced to less than the safe levels Methods for determination of the D value include the sur- thereof by means of aeration and the like before the product vival curve method and the fraction negative method is shipped. In this method, factors which may aŠect the (Stumbo, Murphy & Cochran procedure, Limited Spearman- sterilization include temperature, gas concentration (pres- Karber procedure and the like). In using marketed BIs, it is sure), humidity and exposure time. Therefore, in routine usually unnecessary to determine the D value before use if the sterilization process control, it is required to monitor con- D value indicated on the label has been determined by a stan- tinuously the temperature, gas concentration (pressure), hu- dardized biological indicator evaluation resistometer (BIER) midity and exposure time, and they should be included in the under strictly prescribed conditions in accordance with ISO speciˆcations of the sterilizer. 11138-1. It is acceptable that the D value indicated on the 3. Filtration method label shows a scattering of not more than ±30 seconds. Microorganisms are removed by using a sterilizing ˆlter 4-1-2. Setting up procedure of BI made of an appropriate material. However, this method is (i) In the case of dry materials not intended for microorganisms smaller than bacteria. A Dry type BI is placed at predetermined cold spots in the Generally, a sterilizing ˆlter challenged with more than 107 product to be sterilized or a suitable product showing an microorganisms of a strain of Brevundimonas diminuta equivalent eŠect in the sterilization. The BIs are usually pri- (ATCC 19146, NBRC 14213, JCM 2428), cultured under the mary packaged in the same way as the product, including a appropriate conditions, per square centimeter of eŠective secondary packaging, if applicable. ˆlter area should provide a sterile eŒuent. In this method, (ii) Inthecaseofwetmaterials factors which may aŠect the sterilization include pressure, Spores are suspended as the BI in the same solution as the 1754 Test for Trace Amounts / General Information JP XV product or in an appropriate similar solution, and should be logarithmic reduction (12D) of a known count of BI of more placed at cold spots in the sterilizer. than 1.0 D value is used. 4-1-3. Culture conditions of BI 5-3. Combination of BI and bioburden Soybean casein digest medium is generally used. General Generally, a count of mean bioburden added three times of culture conditions are at 55 – 609C for 7 days in the case of its standard deviation obtained by an extensive bioburden es- G. stearothermophilus andat30–359C for 7 days in the case timation is considered as the maximum bioburden count, and of B. atrophaeus. the sterilization time (or radiation dose) is calculated with the 4-2. Chemical indicator (CI) bioburden count based on an objective sterility assurance lev- CI is an indicator which shows a color change of a sub- el. When this procedure is used, it is required to determine stance applied to a paper slip, etc. as a result of physical andW the resistance of the bioburden to the sterilization as well as or chemical change due to exposure to heat, gas or radiation. the bioburden count in the product being sterilized. If a more The CI can be classiˆed into three types. The ˆrst is employed resistant microorganism than the BI spore is found in the bio- to identify whether or not sterilization has already been im- burden estimation, it should be used as the BI. plemented, the second is employed to control the sterilization N process (for example, its color changes after sterilization for Sterilization time (or radiation dose) ＝D×log 0 N a su‹cient time), and the third is the Bowie & Dick type used to evaluate the eŠectiveness of air removal during the pre- D: D value of the BI vacuum phase of the pre-vacuum sterilization cycle. N: Sterility assurance level

4-3. Dosimeter N0: Maximum bioburden count in the product In the radiation (g-ray) method, the sterilization eŠect de- 5-4. Absolute bioburden method pends on the absorbed radiation dose, so the sterilization The sterilization conditions are determined by employing process control is mainly performed by measuring the dose. the D value of the most resistant microorganism found in the A dosimeter is installed at a position corresponding to the product or environment by the resistant estimation and being minimum dose region of an exposed container or a position based on the bioburden count in the product. Generally, a where the dose is in a known relation to that in the above count of mean bioburden added three times of its standard region. Measurement should be done for each radiation deviation obtained by an extensive bioburden estimation is batch. If there are many containers in the same batch, employed as the bioburden count. When this procedure is dosimeters should be employed so that more than one used, it is required to make frequent counting and resistance dosimeter is always installed at the eŠective radiation section determination of microorganisms in daily bioburden estima- of the irradiation chamber. It should be noted that tion. dosimeters may be aŠected by environmental conditions (temperature, humidity, ultraviolet light, time until reading, References etc.) before and during irradiation. Practical dosimeters for 1) ISO 11134 Industrial moist heat sterilization g-ray and bremsstrahlung sterilization include the 2) ISO 11135 Ethylene oxide sterilization dyed polymethylmethacrylate dosimeter, clear poly- 3) ISO 11137 Radiation sterilization methylmethacrylate dosimeter, ceric-cerous sulfate 4) ISO 11138 Biological indicators dosimeter, alanine-EPR dosimeter and the like. A dosimeter 5) ISO 11140 Chemical indicators for gamma radiation can not generally be used for steriliza- 6) ISO 11737 Microbiological methods tion process control with an electron beam of less than 3 MeV Part 1: Estimation of population of micro- energy. Dosimeters for electron beam sterilization include the organisms on products cellulose acetate dosimeter, radiochromic ˆlm dosimeter and the like. A practical dosimeter must be calibrated against an appropriate national or international standard dosimetry sys- tem. 28. Test for Trace Amounts of 5. Determination of sterilization conditions using microor- Aluminum in Trans Parenteral ganism as an indicator (TPN) Solutions Taking account of the characteristics upon the sterilization concerned, bioburden, etc. of a product to be sterilized, Trans parenteral nutrition solutions (TPNs) are nutrient chose a suitable method from the followings and determine preparations for intravenous injection packed in a large the conditions. volume. Since toxic eŠects to the central , 5-1. Half-cycle method bone, etc. due to trace amounts of aluminum have recently In this method, a sterilization time of twice as long as that been reported in several countries, testing methods for trace required to inactive all of 106 counts of BI placed in the amounts of aluminum contaminating TPNs are required for product is used, regardless of bioburden count in the product the o‹cial standard. The following three analytical methods being sterilized or the resistance of the objective microorgan- are available: (1) High-Performance Liquid Chromatography isms to the sterilization. using a ‰uorescence photometric detector (HPLC with 5-2. Overkill method ‰uorescence detection), (2) Inductivity Coupled Plasma- In this method, a sterilization condition giving a sterility Atomic Emission Spectrometry (ICP-AES method), (3) In- assurance level of not more than 10－6 counts is used, regard- ductivity Coupled Plasma- (ICP-MS less of bioburden count in the product being sterilized or the method). Detection sensitivity by HPLC with ‰uorescence resistance of the objective microorganisms to the steriliza- detection is about 1 mg/L (ppb), while ICP-AES ˆtted with tion. Generally, a sterilization condition providing 12 special apparatus and ICP-MS have higher sensitivity. JP XV General Information / Test for Trace Amounts 1755

Since TPNs are nutrient preparations, they contain many minum. Other than the above- mentioned diŠerences, the size such as sugars, amino acids, electrolytes, etc., in of column, column temperature, and the mobile phase are various compositions. Thus, care is needed in the selection of also diŠerent from those used in the standard method, so a suitable analytical method, because these coexisting compo- suitable analytical conditions should be established for per- nents may aŠect the measurement of trace amounts of alumi- forming precise and reproducible examinations of trace num. amounts of aluminum in the sample specimen. In view of the general availability of HPLC apparatus, the (2) Lumogallion complexing method present general information describes procedures for the de- After forming a complex of aluminum ion in the sample termination of trace levels of aluminum in TPNs by means of specimen with the ‰uorescent reagent of lumogallion, the so- HPLC with a ‰uorescence photometric detector, using two lution is examined by HPLC ˆtted with a ‰uorescence pho- kinds of ‰uorescent chelating agents, i.e., (1) Quinolinol tometer. complexing method, (2) Lumogallion complexing method. Preparation of sample solution (1) Quinolinol complexing method Pipet 70 mL of the sample specimen (TPN) exactly, add After forming a complex of aluminum ion in the sample 0.15 mL of lumogallion hydrochloric acid TS and 0.6 mL of solution with quinolinol, the assay for aluminum by HPLC buŠer solution for aluminum test, pH 7.2 exactly, then mix ˆtted with a ‰uorescence photometer is performed. the solution. After this solution has been allowed to stand for 4 hours at 409C, it can be used for the measurement as a sam- Preparation of sample solution ple solution. Pipet 1 mL of the sample (TPNs) exactly, and after adding 10 mL of water for aluminum test, make up the sample solu- Preparation of a series of standard solutions for calibration tion to 10 mL exactly by adding the mobile phase. curve Preparation of a series of standard solutions for calibra- Pipet 1 mL each of standard aluminum solutions (1) – (5) tion curve exactly, and add diluted nitric acid for aluminum test (1 in Pipet 1 mL of water for aluminum test exactly, and after 100) to make exactly 100 mL. Pipet 70 mL each of these solu- adding 10 mL each of standard solutions of aluminum (1)?? tions exactly, and add exactly 0.15 mL of lumogallion (5), make up the standard solutions for calibration curve to hydrochloric acid TS and exactly 0.6 mL of buŠer solution 10 mL (Aluminum concentration: 0, 1.25, 2.5, 5.0, and 10.0 for aluminum test, pH 7.2 then allow to stand for 4 hours at ppb). 409C to make a series of standard solutions for obtaining the calibration curve (Aluminum: 0, 1.07, 2.13, 4.27, and 8.54 Standard testing method ppb). Pipet 0.1 mL each of the sample solution and standard so- lutions, and perform the test by HPLC under the following Standard examination method conditions. Calculate the aluminum cotent in the sample so- Take 0.1 mL each of the sample solution and standard alu- lution using a calibration curve method. minum solutions for the calibration curve, and perform Operating conditions— HPLC analysis under the following conditions. Calculate the Detector: Fluorescence photometer(excitation wavelength: aluminum content in the sample solution by using a calibra- 380 nm, emission wavelength: 520 nm) tion curve method. Column: A stainless steel column 4.6 mm in inside di- Operating conditions— ameter and 15 cm in length, packed with phenylsilanized sili- Detector: Fluorescence photometer(excitation wavelength ca gel for liquid chromatography (5 mm in particle diameter). 505 nm, emission wavelength 574 nm) Column temperature: A constant temperature of about Column: A stainless steel column 6.0 mm in inside di- 409C. ameter and 10 cm in length, packed with octylsilanized silica Mobile phase: A mixture of 8-quinolinol in acetonitrile (3 gel for liquid chromatography (5 mm in particle diameter). in 100) and diluted 0.5 mol/L ammonium acetate TS (2 in 5) Column temperature: A constant temperature of about (1:1). 409C. Flow rate: Adjust the ‰ow rate so that the retention time of Mobile phase: Take 100 mL of 2-propanol, and add a aluminum/8-quinolinol complex is about 9 minutes. diluted 1 mol/L acetic acid-sodium acetate buŠer solution of System suitability— pH 5.0 (1 in 10) to make 1000 mL. The correlation coe‹cient of the calibration curve, which Flow rate: Adjust the ‰ow rate so that the retention time of is prepared using a series of standard solutions, is not less aluminum/lumogallion complex is about 5 minutes. than 0.99. System suitability— Furthermore there is an alternative method, in which the The correlation coe‹cient of the calibration curve, which chelating agent 8-quinolinol is not included in the mobile is prepared using a series of standard solutions, is not less phase. In this method also, aluminum is detected as a com- than 0.99. plex with 8-quinolinol in the sample solution by using HPLC The following points should be noted in connection with ˆtted with ‰uorescence photometer. But it is necessary to measuring trace amounts of aluminum in TPN preparations. form a more stable aluminum/8-quinolinol complex in the 1) Regarding water, solvents, reagents, vessels and other sample solution, because the chelating agent is not included tools used for the examination, select those not contami- in the mobile phase. Further, since the analytical wavelength nated with aluminum. Further, keep the testing environ- for the ‰uorescence detection is diŠerent from that in the ment clean and free from dust in the testing room. standard method, excitation WL: 370 nm, emission WL: 504 2) Before the measurement, it is necessary to conˆrm that the nm, the detection sensitivity is diŠerent. Thus, it is appropri- characteristic properties of the sample do not aŠect the for- ate to obtain the calibration curve between 0 – 25 ppb of alu- mation of the complex. 1756 Total Protein Assay / General Information JP XV

3) Reference substances of river water for analysis of trace Method 1 elements, distributed by the Japan Society for Analytical Protein in solution absorbs UV light at a wavelength of 280 Chemistry, contain certiˆed amounts of aluminum: JSAC nm, due to the presence of aromatic amino acids, mainly 0301-1 and JSAC 0302 (a known amount of aluminum is tyrosine and tryptophan. This property is the basis of this artiˆcially added to JSAC 0301-1). method. Protein determination at 280 nm is mainly a func- Standard Solutions, Reagents and Test Solutions tion of the tyrosine and tryptophan content of the protein. If Other than the standard solutions, reagents and test solu- the buŠer used to dissolve the protein has a high absorbance tions speciˆed in the Japanese Pharmacopoeia, those de- relative to that of water, there is an interfering substance in scribed below can be used in this test. the buŠer. This interference can be compensated for when the spectrophotometer is adjusted to zero buŠer absorbance. N,N-Bis(2-hydroxyethyl)-2-aminoethane sulfonic acid C 6 If the interference results in a large absorbance that H NO S White crystals or powder. 15 5 challenges the limit of sensitivity of the spectrophotometer, Hydrochloric acid for aluminum test Same as the reagent the results may be compromised. Furthermore, at low con- Hydrochloric acid.Further,itcontainsnotmorethan1ppb centrations protein can be absorbed onto the cuvette, thereby of aluminum. reducing the content in solution. This can be prevented by preparing samples at higher concentrations or by using a non- Lumogallion [5-Chloro-2-hydroxy-3(2,4-dihydrox- ionic detergent in the preparation. yphenylazo)benzenesulfonic acid] C H ClN O SRed-brown 12 9 2 6 Note: Keep the Test Solution, the Standard Solution, and to dark brown powder. Further, it contains not more than 1 the buŠer at the same temperature during testing. ppm of aluminum. Standard Solution Unless otherwise speciˆed in the Lumogallion hydrochloric acid TS Dissolve 0.86 g of lu- individual monograph, prepare a solution of the reference mogallion in 300 mL of 2-propanol, and add 350 mL of dilut- standard or reference material for the protein under test in ed Hydrochloric acid for aluminum test (9 in 50) and Water the same buŠer and at the same concentration as the Test for aluminum test to make 1000 mL exactly. Solution. Test Solution Dissolve a suitable quantity of the protein un- Nitric acid for aluminum test Same as the reagent Nitric der test in the appropriate buŠer to obtain a solution having a acid. Further, it contains not more than 1 ppb of aluminum. concentration of 0.2 to 2 mg per mL. pH buŠer solution for aluminum test, pH 7.2 Dissolve Procedure Concomitantly determine the absorbances of the 106.6 g of N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic Standard Solution and the Test Solution in quartz cells at a acid in 800 mL of Water for aluminum test, adjust the pH 7.2 wavelength of 280 nm, with a suitable spectrophotometer, by using Tetramethylammonium hydroxide aqueous solu- using the buŠer as the blank. To obtain accurate results, the tion,andaddWater for aluminum test to make 1000 mL. response should be linear in the range of protein concentra- tions to be assayed. Standard aluminum solution Pipet a constant volume Light-Scattering The accuracy of the UV spectroscopic each of Water for aluminum test or the Standard aluminum determination of protein can be decreased by the scattering stock solution, dilute and adjust the aluminum concentration of light by the test specimen. If the proteins in solution exist to 0, 1.25, 2.5, 5.0, and 10 ppm by using diluted Nitric acid as particles comparable in size to the wavelength of the meas- for aluminum test (1 in 100), to make Standard aluminum so- uring light (250 to 300 nm), scattering of the light beam lutions (1) – (5). results in an apparent increase in absorbance of the test speci-

Tetramethylammonium hydroxide TS (CH3)4NOH men. To calculate the absorbance at 280 nm due to light-scat- It is a 25z aqueous solution, prepared for aluminum test. tering, determine the absorbances of the Test Solution at Further, it contains not more than 1 ppb of aluminum. wavelengths of 320, 325, 330, 335, 340, 345, and 350 nm. Us- ing the linear regression method, plot the log of the observed Water for aluminum test Same as the monograph Puri- absorbance versus the log of the wavelength, and determine ˆed Water. Further, it contains not more than 1 ppb of alu- the standard curve best ˆtting the plotted points. From the minum. graph so obtained, extrapolate the absorbance value due to light-scattering at 280 nm. Subtract the absorbance from light-scattering from the total absorbance at 280 nm to ob- 29. Total Protein Assay tain the absorbance value of the protein in solution. Filtra- tion with a ˆlter having a 0.2-mm porosity or clariˆcation by This test is harminized with the European Pharmacopoeia centrifugation may be performed to reduce the eŠect of light- and the U.S. Pharmacopeia. The parts of the text that are not scattering, especially if the solution is noticeably turbid.  harmonized are marked with symbols ( ). Calculations Calculate the concentration, CU, of protein in The following procedures are provided as illustrations of the test specimen by the formula: the determination of total protein content in pharmacopoeial C ＝C (A /A ), preparations. Other techniques, such as HPLC, are also ac- U S U S ceptable if total protein recovery is demonstrated. Many of in which CS is the concentration of the Standard Solution; the total protein assay methods described below can be per- and AU and AS are the corrected absorbances of the Test formed successfully using kits from commercial sources. Solution and the Standard Solution, respectively. Note: Where water is required, use distilled water. Method 2 This method, commonly referred to as the Lowry assay, is based on the reduction by protein of the phosphomolybdic- JP XV General Information / Total Protein Assay 1757 tungstic mixed acid chromogen in the Folin-Ciocalteu's the absorbances of the solutions from the Standard Solutions phenol reagent, resulting in an absorbance maximum at versus the protein concentrations, and determine the stan- 750 nm. The Folin-Ciocalteu's phenol reagent (Folin's TS) dard curve best ˆtting the plotted points. From the standard reacts primarily with tyrosine residues in the protein, which curve so obtained and the absorbance of the Test Solution, can lead to variation in the response of the assay to diŠerent determine the concentration of protein in the Test Solution. proteins. Because the method is sensitive to interfering Interfering Substances In the following procedure, deoxy- substances, a procedure for precipitation of the protein from cholate-trichloroacetic acid is added to a test specimen to the test specimen may be used. Where separation of interfer- remove interfering substances by precipitation of proteins be- ing substances from the protein in the test specimen is fore testing. This technique also can be used to concentrate necessary, proceed as directed below for Interfering proteins from a dilute solution. Substances prior to preparation of the Test Solution. The Sodium Deoxycholate Reagent Prepare a solution of eŠect of interfering substances can be minimized by dilution sodium deoxycholate in water having a concentration of provided the concentration of the protein under test remains 150 mg in 100 mL. su‹cient for accurate measurement. Variations of the Lowry Trichloroacetic Acid Reagent Prepare a solution of test that are indicated in national regulatory documents1) can trichloroacetic acid in water having a concentration of 72 g in be substituted for the method described below. 100 mL. Standard Solutions Unless otherwise speciˆed in the Procedure Add 0.1 mL of Sodium Deoxycholate Reagent individual monograph, dissolve the reference standard or to 1 mL of a solution of the protein under test. Mix on a reference material for the protein under test in the buŠer used vortex mixer, and allow to stand at room temperature for 10 to prepare the Test Solution. Dilute portions of this solution minutes. Add 0.1 mL of Trichloroacetic Acid Reagent, and with the same buŠer to obtain not fewer than ˆve Standard mix on a vortex mixer. Centrifuge at 3000×gfor30minutes, Solutions having concentrations between 5 and 100 mgof decant the liquid, and remove any residual liquid with a protein per mL, the concentrations being evenly spaced. pipet. Redissolve the protein pellet in 1 mL of Alkaline Cop- Test Solution Dissolve a suitable quantity of the protein un- per Reagent. Proceed as directed for Test Solution. [Note: der test in the appropriate buŠer to obtain a solution having a Color development reaches a maximum in 20 to 30 minutes concentration within the range of the concentrations of the during incubation at room temperature, after which there is a Standard Solutions. An appropriate buŠer will produce a pH gradual loss of color. Most interfering substances cause a in the range of 10 to 10.5. lower color yield; however, some detergents cause a slight in- Blank Use the buŠer used for the Test Solution and the crease in color. A high salt concentration may cause a Standard Solutions. precipitate to form. Because diŠerent protein species may Reagents and Solutions— give diŠerent color response intensities, the standard protein Copper Sulfate Reagent Dissolve 100 mg of copper (II) and test protein should be the same.] sulfate pentahydrate and 200 mg of sodium dihy- Method 3 drate in water, dilute with water to 50 mL, and mix. Dissolve This method, commonly referred to as the Bradford assay, 10 g of anhydrous sodium carbonate in water to a ˆnal is based on the absorption shift from 470 nm to 595 nm ob- volume of 50 mL, and mix. Slowly pour the sodium car- served when the brilliant blue G-250 dye binds to protein. bonate solution into the copper sulfate solution with mixing. The G-250 dye binds most readily to Prepare this solution fresh daily. arginyl and lysyl residues in the protein, which can lead to 5z SDS TS Dissolve 5 g of sodium dodecyl sulfate in variation in the response of the assay to diŠerent proteins. water, and dilute with water to 100 mL. Standard Solutions Unless otherwise speciˆed in the Alkaline Copper Reagent Prepare a mixture of 5z SDS individual monograph, dissolve the reference standard or the TS, Copper Sulfate Reagent, and Sodium Hydroxide reference material for the protein under test in the buŠer used Solution (4 in 125) (2:1:1). This reagent may be stored at to prepare the Test Solution. Dilute portions of this solution room temperature for up to 2 weeks. with the same buŠer to obtain not fewer than ˆve Standard Diluted Folin's TS Mix 10 mL of Folin's TS with 50 mL Solutions having concentrations between 100 mgand1mgof of water. Store in an amber bottle, at room temperature. protein per mL, the concentrations being evenly spaced. Procedure To 1 mL of each Standard Solution, the Test So- Test Solution Dissolve a suitable quantity of the protein un- lution, and the Blank, add 1 mL of Alkaline Copper Reagent, der test in the appropriate buŠer to obtain a solution having a and mix. Allow to stand at room temperature for 10 minutes. concentration within the range of the concentrations of the Add0.5mLoftheDilutedFolin'sTStoeachsolution,and Standard Solutions. mix each tube immediately after the addition, and allow to Blank Use the buŠer used to prepare the Test Solution and stand at room temperature for 30 minutes. Determine the ab- the Standard Solutions. sorbances of the solutions from the Standard Solutions and Coomassie Reagent Dissolve 100 mg of brilliant blue G-250 the Test Solution at the wavelength of maximum absorbance 2) in 50 mL of ethanol (95). [Note: Not all dyes have the same at 750 nm, with a suitable spectrophotometer, using the solu- brilliant blue G content, and diŠerent products may give tion from the Blank to set the instrument to zero. diŠerent results.] Add 100 mL of phosphoric acid, dilute with Calculations [Note: The relationship of absorbance to water to 1000 mL, and mix. Filter the solution through ˆlter protein concentration is nonlinear; however, if the standard paper (Whatman No.1 or equivalent), and store the ˆltered curve concentration range is su‹ciently small, it will reagent in an amber bottle at room temperature. [Note: Slow approach linearity.] Using the linear regression method, plot precipitation of the dye will occur during storage of the rea-

1) Example: the Minimum Requirements for Biological Products and in- 2) Dye purity is important in the reagent preparation. dividual monograph of JP. 1758 Total Protein Assay / General Information JP XV gent.Filterthereagentbeforeuse.] bances of the solutions from the Standard Solutions and the Procedure Add 5 mL of the Coomassie Reagent to 100 mL Test Solution in quartz cells at 562 nm, with a suitable of each Standard Solution, the Test Solution, and the Blank, spectrophotometer, using the Blank to set the instrument to and mix by inversion. Avoid foaming, which will lead to poor zero. After the solutions are cooled to room temperature, the reproducibility. Determine the absorbances of the solutions color intensity continues to increase gradually. If substances from the Standard Solutions and the Test Solution at 595 that will cause interference in the test are present, proceed as nm, with a suitable spectrophotometer, using the Blank to set directed for Interfering Substances under Method 2. Because the instrument to zero. diŠerent protein species may give diŠerent color response in- [Note: Do not use quartz (silica) spectrophotometer cells: tensities, the standard protein and test protein should be the the dye binds to this material. Because diŠerent protein same. species may give diŠerent color response intensities, the Calculations [Note: The relationship of absorbance to pro- standard protein and test protein should be the same.] There tein concentration is nonlinear; however, if the standard are relatively few interfering substances, but detergents and curve concentration range is su‹ciently small, it will ampholytes in the test specimen should be avoided. Highly approach linearity.] Using the linear regression method, plot alkaline specimens may interfere with the acidic reagent. the absorbances of the solutions from the Standard Solutions Calculations [Note: The relationship of absorbance to versus the protein concentrations, and determine the stan- protein concentration is nonlinear; however, if the standard dard curve best ˆtting the plotted points. From the standard curve concentration range is su‹ciently small, it will curve so obtained and the absorbance of the Test Solution, approach linearity.] Using the linear regression method, plot determine the concentration of protein in the Test Solution. the absorbances of the solutions from the Standard Solutions Method 5 versus the protein concentrations, and determine the stan- This method, commonly referred to as the Biuret assay, is dard curve best ˆtting the plotted points. From the standard based on the interaction of cupric (Cu2＋) ion with protein in curve so obtained and the absorbance of the Test Solution, an alkaline solution and the resultant development of absor- determine the concentration of protein in the Test Solution. bance at 545 nm. Method 4 Standard Solutions Unless otherwise speciˆed in the in- This method, commonly referred to as the bicinchoninic dividual monograph, prepare a solution of Albumin Human acid or BCA assay, is based on reduction of the cupric (Cu2＋) for which the protein content has been previously determined ion to cuprous (Cu＋) ion by protein. The bicinchoninic acid by nitrogen analysis (using the nitrogen-to-protein conver- reagent is used to detect the cuprous ion. The method has few sion factor of 6.25) or of the reference standard or reference interfering substances. When interfering substances are material for the protein under test in sodium chloride solu- present, their eŠect may be minimized by dilution, provided tion (9 in 1000). Dilute portions of this solution with sodium that the concentration of the protein under test remains chloride solution (9 in 1000) to obtain not fewer than three su‹cient for accurate measurement. Standard Solutions having concentrations between 0.5 and 10 Standard Solutions Unless otherwise speciˆed in the mg per mL, the concentrations being evenly spaced. [Note: individual monograph, dissolve the reference standard or the Low responses may be observed if the sample under test has reference material for the protein under test in the buŠer used signiˆcantly diŠerent level of proline than that of Albumin to prepare the Test Solution. Dilute portions of this solution Human. A diŠerent standard protein may be employed in with the same buŠer to obtain not fewer than ˆve Standard such cases.] Solutions having concentrations between 10 and 1200 mgof Test Solution Prepare a solution of the test protein in protein per mL, the concentrations being evenly spaced. sodium chloride solution (9 in 1000) having a concentration Test Solution Dissolve a suitable quantity of the protein un- within the range of the concentrations of the Standard der test in the appropriate buŠer to obtain a solution having a Solutions. concentration within the range of the concentrations of the Blank Use sodium chloride solution (9 in 1000). Standard Solutions. Biuret Reagent Dissolve about 3.46 g of copper (II) sulfate Blank Use the buŠer used to prepare the Test Solution and pentahydrate in 10 mL of water, with heating if necessary, the Standard Solutions. and allow to cool (Solution A). Dissolve about 34.6 g of Reagents and Solutions— sodium citrate dihydrate and 20.0 g of anhydrous sodium BCA Reagent Dissolve about 10 g of bicinchoninic acid, carbonate in 80 mL of water, with heating if necessary, and 20 g of sodium carbonate monohydrate, 1.6 g of sodium allow to cool (Solution B). Mix Solutions A and B, and dilute tartrate dihydrate, 4 g of sodium hydroxide, and 9.5 g of with water to 200 mL. This Biuret Reagent is stable at room sodium hydrogen carbonate in water. Adjust, if necessary, temperature for 6 months. Do not use the reagent if it de- with sodium hydroxide or sodium hydrogen carbonate to a velops turbidity or contains any precipitate. pH of 11.25. Dilute with water to 1000 mL, and mix. Procedure To one volume of the Standard Solutions and a Copper Sulfate Reagent Dissolve about 2 g of copper (II) solution of the Test Solution add an equal volume of sodium sulfate pentahydrate in water to a ˆnal volume of 50 mL. hydroxide solution (6 in 100), and mix. Immediately add a Copper-BCA Reagent Mix 1 mL of Copper Sulfate volume of Biuret Reagent equivalent to 0.4 volume of the Reagent and 50 mL of BCA Reagent. Test Solution, and mix. Allow to stand at a temperature Procedure Mix 0.1 mL of each Standard Solution, the Test between 159Cand259C for not less than 15 minutes. Within Solution, and the Blank with 2 mL of the Copper-BCA 90 minutes after the addition of the Biuret Reagent, deter- Reagent. Incubate the solutions at 379C for 30 minutes, note mine the absorbances of the Standard Solutions and the thetime,andallowtocometoroomtemperature.Within60 solution from the Test Solution at the wavelength of maxi- minutes following the incubation time, determine the absor- mum absorbance at 545 nm, with a suitable spectrophotome- JP XV General Information / Total Protein Assay 1759 ter, using the Blank to set the instrument to zero. [Note: Any Reagents and Solutions— solution that develops turbidity or a precipitate is not accept- Borate BuŠer Dissolve about 61.83 g of boric acid in able for calculation of protein concentration.] water, and adjust with potassium hydroxide to a pH of 10.4. Calculations Using the least-squares linear regression Dilute with water to 1000 mL, and mix. method, plot the absorbances of the Standard Solutions Stock OPA Reagent Dissolve about 120 mg of o- versus the protein concentrations, and determine the stan- phthalaldehyde in 1.5 mL of methanol, add 100 mL of dard curve best ˆtting the plotted points, and calculate the Borate BuŠer, and mix. Add 0.6 mL of polyoxyethylene (23) correlation coe‹cient for the line. [Note: Within the given lauryl ether, and mix. This solution is stable at room temper- range of the standards, the relationship of absorbance to pro- ature for at least 3 weeks. tein concentration is approximately linear.] A suitable system OPA Reagent To 5 mL of Stock OPA Reagent add 15 mL is one that yields a line having a correlation coe‹cient of not of 2-mercaptoethanol. Prepare at least 30 minutes prior to less than 0.99. From the standard curve and the absorbance use. This reagent is stable for one day. of the Test Solution, determine the concentration of protein Procedure Adjust each of the Standard Solutions and the in the test specimen, making any necessary correction. Test Solution to a pH between 8.0 and 10.5. Mix 10 mLofthe Interfering Substances To minimize the eŠect of interfering Test Solution and each of the Standard Solutions with 100 mL substances, the protein can be precipitated from the initial of OPA Reagent, and allow to stand at room temperature for test specimen as follows. Add 0.1 volume of 50z trichloroa- 15 minutes. Add 3 mL of 0.5 mol/L sodium hydroxide TS, cetic acid to 1 volume of a solution of the test specimen, and mix. Using a suitable ‰uorometer, determine the ‰uores- withdraw the supernatant layer, and dissolve the precipitate cent intensities of solutions from the Standard Solutions and in a small volume of 0.5 mol/L sodium hydroxide TS. Use the Test Solution at an excitation wavelength of 340 nm and the solution so obtained to prepare the Test Solution. an emission wavelength between 440 and 455 nm. [Note: The Comments This test shows minimal diŠerence between ‰uorescence of an individual specimen is read only once be- equivalent IgG and albumin samples. Addition of the sodium cause irradiation decreases the ‰uorescent intensity.] hydroxide and the Biuret Reagent as a combined reagent, in- Calculations The relationship of ‰uorescence to protein su‹cient mixing after the addition of the sodium hydroxide, concentration is linear. Using the linear regression method, or an extended time between the addition of the sodium plot the ‰uorescent intensities of the solutions from the Stan- hydroxide solution and the addition of the Biuret Reagent dard Solutions versus the protein concentrations, and deter- will give IgG samples a higher response than albumin sam- mine the standard curve best ˆtting the plotted points. From ples. The trichloroacetic acid method used to minimize the the standard curve so obtained and the ‰uorescent intensity eŠects of interfering substances also can be used to determine of the Test Solution, determine the concentration of protein the protein content in test specimens at concentrations below in the test specimen. 500 mgpermL. Method 7 Method 6 This method is based on nitrogen analysis as a means of This ‰uorometric method is based on the derivatization of protein determination. Interference caused by the presence of the protein with o-phthalaldehyde (OPA), which reacts with other nitrogen-containing substances in the test protein can the primary amines of the protein (i.e., NH2-terminal amino aŠect the determination of protein by this method. Nitrogen acid and the e-amino group of the lysine residues). The analysis techniques destroy the protein under test but are not sensitivity of the test can be increased by hydrolyzing the pro- limited to protein presentation in an aqueous environment. tein before testing. Hydrolysis makes the a-amino group of Procedure A Determine the nitrogen content of the protein the constituent amino acids of the protein available for reac- under test as directed elsewhere in the Pharmacopoeia. Com- tion with the OPA reagent. The method requires very small mercial instrumentation is available for the Kjeldahl nitrogen quantities of the protein. assay. Primary amines, such as tris(hydroxymethyl)amino- Procedure B Commercial instrumentation is available for methaneandaminoacidbuŠers,reactwithOPAandmustbe nitrogen analysis. Most nitrogen analysis instruments use avoided or removed. Ammonia at high concentrations will pyrolysis (i.e., combustion of the sample in oxygen at react with OPA as well. The ‰uorescence obtained when a- temperatures approaching 10009C), which produces nitric mine reacts with OPA can be unstable. The use of automated oxide (NO) and other oxides of nitrogen (NOx) from the procedures to standardize this procedure may improve the ac- nitrogen present in the test protein. Some instruments curacy and precision of the test. convert the nitric oxides to nitrogen gas, which is quantiˆed Standard Solutions Unless otherwise speciˆed in the with a thermal conductivity detector. Other instruments mix individual monograph, dissolve the reference standard or the (NO) with ozone (O3) to produce excited nitrogen . reference material for the protein under test in the buŠer used dioxide (NO2 ), which emits light when it decays and can be to prepare the Test Solution. Dilute portions of this solution quantiˆed with a chemiluminescence detector. A protein with the same buŠer to obtain not fewer than ˆve Standard reference standard or reference material that is relatively pure Solutions having concentrations between 10 and 200 mgof and is similar in composition to the test proteins is used to op- protein per mL, the concentrations being evenly spaced. timize the injection and pyrolysis parameters and to evaluate Test Solution Dissolve a suitable quantity of the protein un- consistency in the analysis. der test in the appropriate buŠer to obtain a solution having a Calculations The protein concentration is calculated by concentration within the range of the concentrations of the dividing the nitrogen content of the sample by the known Standard Solutions. nitrogen content of the protein. The known nitrogen content Blank Use the buŠer used to prepare the Test Solution and of the protein can be determined from the chemical composi- the Standard Solutions. tion of the protein or by comparison with the nitrogen 1760 Validation of Analytical / General Information JP XV content of the appropriate reference standard or reference The terminology and deˆnitions of the validation charac- material. teristics may possibly vary depending upon the ˆelds to which analytical procedures are applied. The terminology and deˆ- nitions shown in this document are established for the pur- 30. Validation of Analytical pose of the Japanese Pharmacopoeia. Typical methods for assessing the validation characteristics are shown in the item Procedures of assessment. Various kinds of methods to determine the validation characteristics have been proposed and any The validation of an analytical procedure is the process of methods that are widely accepted will be accepted for the conˆrming that the analytical procedure employed for a test present purpose. However, since values of the validation of pharmaceutics is suitable for its intended use. In other characteristics may possibly depend upon methods of deter- word, the validation of an analytical procedure requires us to mination, it is required to present the methods of determining demonstrate scientiˆcally that risks in decision by testing the validation characteristics, the data and calculation caused by errors from analytical steps are acceptably small. methods in su‹cient detail. The performance of an analytical procedure is established by Although robustness is not listed as a validation charac- various kinds of validation characteristics. The validity of a teristic, it should be considered during the development of proposed analytical procedure can be shown by demonstrat- analytical procedures. Studying the robustness may help to ing experimentally that the validation characteristics of the improve analytical procedures and to establish appropriate analytical procedure satisfy the standards set up according to analytical conditions including precautions. the acceptable limits of testing. (1) AccuracyWTrueness When an analytical procedure is to be newly carried in the Deˆnition: The accuracy is a measure of the bias of ob- Japanese Pharmacopoeia, when a test carried in the Japanese served values obtained by an analytical procedure. The ac- Pharmacopoeia is to be revised, and when the test carried in curacy is expressed as the diŠerence between the average the Japanese Pharmacopoeia is to be replaced with a new test value obtained from a large series of observed values and the according to regulations in general notices, analytical proce- true value. dures employed for these tests should be validated according Assessment: The estimate of accuracy of an analytical to this document. method is expressed as the diŠerence between the total mean of observed values obtained during investigation of the Required data for analytical procedures to be carried in the reproducibility and the true value. The theoretical value is Japanese Pharmacopoeia used as the true value (e.g., in the case of titration methods, (1) Outline etc.). When there is no theoretical value or it is di‹cult to ob- This section should provide a brief explanation of the prin- tain a theoretical value even though it exists, a certiˆed value ciple of a proposed analytical procedure, identify the necessi- or a consensus value may be used as the true value. When an ty of the analytical procedure and its advantage compared analytical procedure for a drug product is considered, the ob- with other procedures, and summarize the validation. When served value of the standard solution of the drug substance an analytical procedure is revised, the limitation of the cur- may be used as the consensus value. rent analytical procedure and the advantage oŠered by the It may be inferred from speciˆcity data that an analytical new analytical procedure should be described. procedure is unbiased. (2) Analytical procedure The estimate of accuracy and a 95z conˆdence interval of This section should contain a complete description of the the accuracy should be calculated using the standard error analytical procedure to enable skilled persons to evaluate cor- based on the reproducibility (intermediate precision). It rectly the analytical procedure and replicate it if necessary. should be conˆrmed that the conˆdence interval includes zero Analytical procedures include all important operating proce- or that the upper or lower conˆdence limits are within the dures for performing analyses, the preparation of standard range of the accuracy required of the analytical procedure. samples, reagents and test solutions, precautions, procedures (2) Precision to verify system suitability (e.g. the veriˆcation of the Deˆnition: The precision is a measure of the closeness of separating performance of a chromatographic system), for- agreement between observed values obtained independently mulas to obtain results, the number of replications and so from multiple samplings of a homogenous sample and is ex- forth. Any instruments and apparatus that are not stated in pressed as the variance, standard deviation or relative stan- the Japanese Pharmacopoeia should be described in detail. dard deviation (coe‹cient of variation) of observed values. The physical, chemical or biological characteristics of any The precision should be considered at three levels with new reference standards should be clariˆed and their testing diŠerent repetition conditions; repeatability, intermediate methods should be established. precision and reproducibility. (3) Data showing the validity of analytical procedures (i) RepeatabilityWIntra-assay precision This section should provide complete data showing the The repeatability expresses the precision of observed values validity of the analytical procedures. This includes the ex- obtained from multiple samplings of a homogenous sample perimental designs to determine the validation characteris- over a short time interval within a laboratory, by the same tics, experimental data, calculation results and results of analyst, using the same apparatus and instruments, lots of hypothesis tests. reagents and so forth (repeatability conditions). Validation characteristics (ii) Intermediate precision The deˆnition of typical validation characteristics to be as- The intermediate precision expresses the precision of ob- sessed in validation of analytical procedures and examples of served values obtained from multiple samplings of a assessing procedures are given below. homogenous sample by changing a part of or all of the oper- JP XV General Information / Validation of Analytical 1761 ating conditions including analysts, experimental dates, ap- the standard deviation of responses of blank samples and the paratus and instruments and lots of reagents within a labora- slope of the calibration curve. tory (intermediate precision condition). DL＝3.3sWslope (iii) Reproducibility The reproducibility expresses the precision of observed DL: detection limit values obtained from multiple samplings of a homogenous s: the standard deviation of responses of blank sample in diŠerent laboratories (reproducibility condition). samples Assessment: A su‹cient volume of a homogenous sample slope: slope of the calibration curve should be prepared before studying the precision. The solu- The noise level may be used as the standard deviation of tion is assumed to be homogenous. When it is di‹cult to ob- responses of blank samples in chromatographic methods. It tain a homogenous sample, the following samples may be should be ensured that the detection limit of the analytical used as homogenous samples; e.g., a large amount of drug procedure is lower than the speciˆed limit for testing. products or mixture of drug substance and vehicles that are (5) Quantitation limit crushed and mixed well until they can be assumed to be Deˆnition: The quantitation limit is the lowest amount or homogenous. concentration of the analyte in a sample that can be deter- Suitable experimental designs such as one-way layout may mined. The precision expressed as the relative standard devia- be employed when more than one level of precision is to be tion of samples containing an analyte at the quantitation investigated simultaneously. A su‹cient number of repeti- limit is usually 10z. tions, levels of operating conditions and laboratories should Assessment: The quantitation limit may be calculated us- be employed. Sources of variations aŠecting analytical results ing the standard deviation of responses of blank samples or should be evaluated as thoroughly as possible through the samples containing an analyte close to the quantitation limit validation. and the slope of the calibration curve close to the quantita- It is required to show the variance, standard deviation and tion limit. The following equation is an example to determine relative standard deviation (coe‹cient of variation) of each the quantitation limit using the standard deviation of level of precision. The 90z conˆdence interval of the vari- responses of blank samples and the slope of the calibration ance and corresponding intervals of the standard deviation curve. and relative standard deviation should also be established. The validity of the proposed analytical procedure for its in- QL＝10sWslope tended use may be conˆrmed by comparing obtained values QL: quantitation limit with the required values of the analytical procedure. Whether s: the standard deviation of responses of blank the proposed analytical procedure is acceptable may normal- samples ly be decided based on the reproducibility. slope: slope of the calibration curve (3) Speciˆcity Deˆnition: The speciˆcity is the ability of an analytical The noise level may be used as the standard deviation of procedure to measure accurately an analyte in the presence of responses of blank samples in chromatographic methods. It components that may be expected to be present in the sample should be ensured that the quantitation limit of the analytical matrix. The speciˆcity is a measure of discriminating ability. procedure is lower than the speciˆed limit for testing. Lack of speciˆcity of an analytical procedure may be com- (6) Linearity pensated by other supporting analytical procedures. Deˆnition: The linearity is the ability of an analytical Assessment: It should be conˆrmed that the proposed ana- procedure to elicit responses linearly related to the amount or lytical procedure can identify an analyte or that it can ac- concentration of an analyte in samples. A well-deˆned curately measure the amount or concentration of an analyte mathematical transformation may sometimes be necessary to in a sample. The method to conˆrm the speciˆcity depends obtain a linear relationship. very much upon the purpose of the analytical procedure. For Assessment: Responses are obtained after analyzing sam- example, the speciˆcity may be assessed by comparing analyt- ples with various amounts or concentrations of an analyte ac- ical results obtained from a sample containing the analyte cording to described operating procedures. The linearity may only with results obtained from samples containing ex- be evaluated in terms of the correlation coe‹cient, and the cipients, related substances or degradation products, and in- slope and y-intercept of the regression line. It may be also cluding or excluding the analyte. If reference standards of helpful for evaluating the linearity to plot residual errors impurities are unavailable, samples that are expected to con- from the regression line against the amount or concentration tain impurities or degradation products may be used (e.g. and to conˆrm that there is no particular tendency in the samples after accelerated or stress tests). graph. Samples with ˆve diŠerent amounts or concentrations (4) Detection limit of an analyte should be usually investigated. Deˆnition: The detection limit is the lowest amount or con- (7) Range centration of the analyte in a sample that is detectable, but Deˆnition: The range for the validation of analytical not necessarily quantiˆable. procedures is the interval between the lower and upper limits Assessment: The detection limit should be normally deter- of the amount or concentration of an analyte providing mined so that producer's and consumer's risks are less than su‹cient accuracy and precision. The range for the valida- 5z. The detection limit may be calculated using the standard tion of analytical procedures for an analytical procedure with deviation of responses of blank samples or samples contain- linearity is the interval between the lower and upper limits ing an analyte close to the detection limit and the slope of the providing su‹cient accuracy, precision and linearity. calibration curve close to the detection limit. The following Assessment: When the range for the validation of analyti- equation is an example to determine the detection limit using cal procedures is investigated, 80 to 120z of speciˆed limits 1762 Validation of Analytical / General Information JP XV of testing should be usually considered. The accuracy, preci- procedures including the number of replications should be sion and linearity should be evaluated using samples contain- validated. This is diŠerent from repetition in the validation of ing the lower and upper limits and in the middle of the range. analytical procedures to obtain accuracy or precision. Observed value: The value of a characteristic obtained as Categories of tests employing analytical procedures the result of performing an analytical procedure. Tests covered with this document are roughly classiˆed Consumer's risk: This is the probability that products out into three categories shown below according to their pur- of the speciˆcation of tests are decided to be accepted after poses. The table lists the normally required validation charac- testing. It is usually expressed as b, and is called the probabil- teristics to be evaluated in the validation of analytical proce- ityoftypeIIerrorortheprobabilityoffalsenegativeinim- dures used in these tests. This list should be considered to purity tests. represent typical validation characteristics. A diŠerent ap- Producer's risk: This is the probability that products satis- proach to validating analytical procedures should be consi- fying the speciˆcation of tests are decided to be rejected after dered depending upon the characteristics of analytical proce- testing. It is usually expressed as a, and is called the probabil- dures and their intended use. ityoftypeIerrorortheprobabilityoffalsepositiveinim- Type I Identiˆcation. Tests for identifying major compo- purity tests. nents in pharmaceuticals according to their characteristics. Robustness: The robustness is a measure of the capacity to Type II Impurity tests. Tests for determination of impur- remain unaŠected by small but deliberate variations in ana- ities in pharmaceuticals. lytical conditions. The stability of observed values may be Type III Tests for assaying drug substances, active in- studied by changing various analytical conditions within suit- gredients, and major components in pharmaceuticals. able ranges including pH values of solutions, reaction tem- (Additives such as stabilizing agents and preservatives are in- perature, reaction time or amount of reagents added. When cluded in major components.) Tests for determining perfor- observed values are unstable, the analytical procedure should mance of pharmaceuticals, such as dissolution testing. be improved. Results of studying robustness may be re‰ected Table Lists of validation characteristics required to be in the developed analytical procedure as precautions or sig- evaluated in tests of each type niˆcant digits describing analytical conditions. Test: Tests mean various tests described in general tests Type III ＼ Type of test Type I Type II and o‹cial monographs in the Japanese Pharmacopoeia such ＼Validation Quantitation Limit as impurity tests and assay. They includes sampling methods, ＼characteristics test test speciˆcation limits and analytical procedures. AccuracyWTrueness －＋－＋ Precision Repeatability －＋－＋ Intermediate precision －－* －－* Reproducibility －＋* －＋* Specificity** ＋＋＋＋ Detection limit －－＋－ Quantitation limit －＋－－ 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 Terminology used in the validation of analytical procedures Analytical procedure: This document covers analytical procedures applied to identiˆcation, and ones that provides responses depending upon the amount or concentration of analytes in samples. Laboratory: The laboratory means an experimental room or facility where tests are performed. In this document diŠer- ent laboratories are expected to perform an analytical proce- dure using diŠerent analysts, diŠerent experimental appara- tus and instruments, diŠerent lots of reagents and so forth. Number of replications: The number of replications is one that is described in analytical procedures. An observed value is often obtained by more than one measurement in order to achieve good precision of analytical procedures. Analytical Atomic Weight Table (2004)

In 1961 it was decided that the atomic weights of the The atomic weight of a single nuclide element is the elements would be based on values relative to the mass most accurate and the precision is also high. This is number of 12 (no fractions) for carbon (12C). Ever because it is not necessary to consider the isotope ratio since, there has been a marked improvement in the since single nuclide elements do not possess a multiple quality and quantity of data on the nuclide masses and number of stable isotopes. The atomic weights of such isotope ratios of the elements using physical methods elements are determined based on the mass3) of each such as mass spectrometry. The Commission on nuclide determined by physical techniques, taking into Atomic Weights and Isotope Abundances (CAWIA) of consideration the uncertainty with constant criteria. the International Union of Pure and Applied Chemis- Among the elements, the majority of samples try (IUPAC) collected and examined newly measured gathered on Earth exhibit a constant isotope composi- data and then published an atomic weight table. Based tion, however, some specific samples have isotope on this table, in April of each year the Atomic Weight compositions that are different to these. These kinds of Subcommittee of the Chemical Society of Japan also elements are indicated by a ``g'', which means the value publishes an atomic weight table. A simple explanation in the atomic weight table cannot be used as is, depend- is provided below so that the table can be used correctly ing on the sample, as the atomic weight of these and effectively. For a more detailed explanation, the elements. In relation to this, oxygen for example exists user is referred to a report1) and a review2) published by in a number of forms on Earth, such as in air, salt the CAWIA. water, , and in rocks, and because the The atomic weight values of each of the elements isotope compositions fluctuate among these sub- shown in the atomic weight tables are, as stated in the stances, oxygen is not an element for which only one preface to the table, for elements that originate on value can be used. Thus, an ``r'' is attached to an Earth and are present in substances that exist naturally. element for which a precise atomic weight cannot be Atomic weights are, with the exception of single given, no matter how much progress is made in nuclide elements (elements consisting of one stable techniques for measuring the isotope composition. On nuclide), not natural constants like the speed of light, the other hand, it is also possible, depending on the but rather change depending on a variety of factors, element, to use an isotope that has undergone artificial such as the method of treatment or the origin of the fractionation as a reagent. Typical elements that are substance containing that element. This is because the representative include hydrogen, , boron, and atomic weight is dependent on the relative frequency uranium. This type of element is identified by an ``m'', (isotope ratio) of the stable nuclides comprising each of and particularly in cases where the atomic weight is a the respective elements. Due to advancements in problem, it is necessary to be careful by referring to the measurement techniques, the isotopic frequencies of label of the reagent. each of the elements are not necessarily constant, and fluctuate due to a variety of processes that occur on the 1) IUPAC Inorganic Chemistry Division, CAWIA: Atomic Earth. We have come to learn that this is reflected in Weights of the Elements 2001. Pure Appl. Chem., 75,1107 the atomic weights. The result of this is that differences (2003). have arisen in the accuracy of the atomic weights 2) J. R. De Laeter et al.: Atomic Weights of the Elements: between elements. The figures in parentheses that Review 2000. Pure Appl. Chem., 75, 683 (2003). 3) G. Audi and A. H. Wapstra: The 1993 Atomic Mass follow the atomic weight values in the atomic weight Evaluation (I). Atomic Mass Table. Nucl. Phys., A565.1 tables represent the uncertainty with respect to the last (1993). digit in the atomic weight. For example, in the case of hydrogen, 1.00794(7) means 1.00794±0.00007.

1763 1764 Appendix JP XV

Standard Atomic Weights 2004

(scaled to Ar(12C)＝12, where 12C is a neutral atom in its nuclear and electronic ground state)

The atomic weights of many elements are not invariant but depend on the origin and treatment of the material. The standard values of Ar(E) and the uncertainties (in parentheses, following the last significant figure to which they are attributed) apply to elements of natural terrestrial origin. The footnotes to this Table elaborate the types of variation which may occur for individual elements and which may be larger than the listed uncertainties of values of Ar(E). Names of elements with atomic number 112 to 116 are provisional.

*: Element has no stable isotopes. †: Commercially available Li materials have atomic weights that range between 6.939 and 6.996; if a more accurate value is required, it must be determined for the specific material. g: geological specimens are known in which the element has an isotopic composition outside the limits for normal material. The difference between the atomic weight of the element in such specimens and that given in the Table may exceed the stated uncertainty. m: modified isotopic compositions may be found in commercially available material because it has been subjected to an undisclosed or inadvertent isotopic fraction- ation. Substantial deviations in atomic weight of the element from that given in the Table can occur. r: range in isotopic composition of normal terrestrial material prevents a more precise Ar(E) being given: the tabulated Ar(E) value should be applicable to any normal material. ,Atomic Weights Subcommittee of the Chemical Society of Japan INDEX

Solution, 293 Sulfate Injection, 312 A Aluminum Areca, 1258 Acetylsalicylate, 319 Arginine Hydrochloride, 313 Absorptive Ointment, 265 Monostearate, 291 Injection, 314 Acacia, 1251 Potassium Sulfate Hydrate, 292 L-Arginine, 313 Hydrochloride, 265 Sucrose Sulfate Ester, 1118 Hydrochloride, 313 Aluminum, 266 Hydrochloride, 293 Hydrochloride Injection, 314 Acetaminophen, 267 Ambenonium Chloride, 294 Aromatic Castor Oil, 415 , 268 Amidotrizoic Acid, 295 Hydrochloride, 314 Acetic Acid, 268 Sulfate, 296 Arsenical Paste, 315 , 269 Aminoacetic Acid, 704 Trioxide, 316 Chloride for Injection, Aminobenzylpenicillin, 307 Arsenous Acid, 316 271 Sodium, 308 Artemisia Capillaris Flower, 1258 Acetylsalicylic Acid, 318 Ascorbic Acid, 316 Tablets, 319 Hydrate, 297 Injection, 317 Achyranthes Root, 1252 Injection, 297 Powder, 317 Aclarubicin Hydrochloride, 272 Hydrochloride, 298 Asiasarum Root, 1259 Acrinol Tablets, 299 Asparagus Tuber, 1259 and Oxide Oil, 273 Ammonia Water, 299 L-Aspartic Acid, 318 and Zinc Oxide Ointment, 274 , 300 , 318 Hydrate, 274 Sodium for Injection, 300 Aluminum, 319 Actinomycin D, 275 Amomum Seed, 1256 Tablets, 319 , 276 Amorphous Insulin Zinc Injection Aspoxicillin Hydrate, 320 Injection, 276 (Aqueous Suspension), 764 Astragalus Root, 1260 Solution, 277 , 301 Astromicin Sulfate, 322 Adsorbed Hydrate, 302 , 323 Diphtheria Toxoid for Adult Use, , 303 Atractylodes 596 for Injection, 304 Lancea Rhizome, 1261 Diphtheria-Puriˆed Pertussis-Teta- Syrup, 305 Rhizome, 1260 nus Combined Vaccine, 596 Tablets, 305 Diphtheria-Tetanus Combined Sulfate Hydrate, 324 Toxoid, 596 Ethoxycarbonyloxyethyl Hydro- Sulfate Injection, 324 Habu-venom Toxoid, 716 chloride, 329 , 325 , 716 Hydrate, 307 Tablets, 326 Puriˆed Pertussis Vaccine, 980 Sodium, 308 Hydrate, 327 Tetanus Toxoid, 1156 Ampicillinphthalidyl Hydrochloride, , 328 Agar, 1252 1138 A‰oqualone, 277 , 278 Amyl , 309 Tablets, 279 Anemarrhena Rhizome, 1256 B Akebia Stem, 1253 Anesthamine, 644 Alacepril, 279 Ether, 641 Hydrochloride, 329 Tablets, 280 Angelica Dahurica Root, 1257 , 330 Albumin Tannate, 282 Anhydrous , 331 Alcohol, 638 Aminobenzylpenicillin, 306 Tablets, 332 for Disinfection, 640 Ampicillin, 306 Sulfate, 333 Aldioxa, 282 CaŠeine, 386 , 333 Tartrate, 283 Citric Acid, 514 , 334 Alisma Rhizome, 1253 Dibasic Calcium Phosphate, 396 Bearberry Leaf, 1262 , 284 Ethanol, 639 Bear Bile, 1262 Aloe, 1254 Lactose, 802 Dipropionate, 335 Alpinia O‹cinarum Rhizome, 1256 Antipyrine, 310 Tallow, 336 , 284 Apricot Bekanamycin Sulfate, 337 Hydrochloride, 285 Kernel, 1257 Belladonna Alprostadil, 286 Kernel Water, 1257 Extract, 1263 Alfadex, 287 Arbekacin Root, 1263 Alum, 292 Sulfate, 311 Hydrochloride, 338

1765 1766 Index JP XV

Tablets, 339 Hydrochloride, 383 Hydrate, 387 Benincasa Seed, 1264 Bupleurum Root, 1266 , 415 Benoxinate Hydrochloride, 951 Hydrochloride, 383 Capsules, 416 Benserazide Hydrochloride, 340 Burdock Fruit, 1267 Compound Granules, 419 Bentonite, 341 Burnt Alum, 292 Fine Granules, 417 Benzalkonium Chloride, 342 , 384 , 421 Concentrated Solution 50, 343 Butropium Bromide, 385 , 422 Solution, 342 Butyl Parahydroxybenzoate, 386 Sodium, 423 , 343 Sodium, 424 Benzethonium Chloride, 344 C Propylene Glycolate, 425 Solution, 345 Sodium, 426 , 644 Cacao Butter, 386 Hydrate, 428 , 345 Calciferol, 624 Sodium, 429 Benzoin, 1265 Calcium Pivoxil Hydrochloride Benzyl Chloride Hydrate, 390 Fine Granules, 432 Alcohol, 346 Chloride Injection, 390 Hydrate, 430 Benzoate, 347 Folinate, 391 Tablets, 433 Gluconate Hydrate, 391 , 434 Benzathine Hydrate, 348 Hydroxide, 392 Capsules, 435 Potassium, 349 Lactate Hydrate, 393 Fine Granules, 436 Leucovorin, 391 Pivoxil, 437 Chloride Hydrate, 351 Oxide, 393 Fine Granules, 438 Tannate, 352 Pantothenate, 394 Tablets, 438 Mesilate, 353 Paraaminosalicylate Granules, 394 Dihydrochloride Tablets, 354 Paraaminosalicylate Hydrate, 395 for Injection, 441 , 355 Polystyrene Sulfonate, 398 Hydrate, 439 Dipropionate, 358 Stearate, 400 Hydrochloride, 443 Sodium Phosphate, 359 Calumba, 1267 Sodium, 445 Tablets, 356 Camellia Oil, 400 Sodium Hydrate, 446 Valerate, 360 Mesilate, 400 Sodium, 447 Valerate and Sulfate d-, 401 Sodium, 448 Cream, 361 dl-Camphor, 402 Sodium, 449 Valerate and Gentamicin Sulfate Capsicum, 1268 , 451 Ointment, 362 and Spirit, 1270 Chloride, 363 Tincture, 1269 Hexetil Hydrochloride, 453 Bezaˆbrate, 364 Capsules, 403 Hydrochloride, 455 Sustained Release Tablets, 365 , 403 Hydrochloride for Injection, 456 , 366 , 404 Hydrochloride, 457 Hydrochloride, 366 Sodium Sulfonate for Injection, 458 Bisacodyl, 367 Hydrate, 405 Sodium, 458 Suppositories, 368 Carbetapentane Citrate, 974 Sulfate, 460 Carbetapentene Citrate, 974 Proxetil, 461 Subgallate, 368 Hydrate, 406 Hydrate, 462 Subnitrate, 369 Carbolic Acid, 985 Sodium, 464 Bitter for Disinfection, 986 Hydrate, 466 Cardamon, 1265 L-, 407 Pivoxil, 468 Orange Peel, 1265 Carbon Dioxide, 407 Fine Granules, 469 Tincture, 1266 Carboxymethylcellulose, 408 Hydrate, 470 Calcium, 409 Sodium, 471 Hydrochloride, 370 Sodium, 410 Sodium Hydrate, 472 Sulfate, 372 Cardamon, 1271 Boric Acid, 374 Carmellose, 408 Axetil, 474 , 374 Calcium, 409 Sodium, 476 Hydrochloride, 375 Sodium, 410 Cellacefate, 480 Mesilate, 376 Carmofur, 411 Celmoleukin (Genetical Recombina- Bromovalerylurea, 377 Carnauba Wax, 411 tion), 481 Bucillamine, 377 Hydrochloride, 412 Cetanol, 484 Hydrochloride, 378 Sodium, 412 Hydrochloride, 485 Hydrochloride, 379 Cassia Seed, 1271 Ceˆxime, 442 , 380 Castor Oil, 414 , 486 Cream, 380 Catalpa Fruit, 1271 Hydrate, 487 Ointment, 381 CaŠeine , 487 , 382 and , 388 Palmitate, 488 JP XV Index 1767

Sodium Succinate, 489 Clove, 1274 Crystalline Insulin Zinc Injection , 490 Oil, 1275 (Aqueous Suspension), 765 Powder, 491 Sodium Hydrate, 533 , 881 Tablets, 492 , 534 Cyanamide, 548 Cloˆbrate, 525 , 548 Gluconate Solution, 493 Capsules, 526 Injection, 549 Hydrochloride, 493 CMC, 408 Hydrochloride, 549 Chlorinated Lime, 494 Calcium, 409 Hydrate, 550 , 494 Sodium, 410 , 551 , 495 Cnidium Cyperus Rhizome, 1280 , 496 Monnieri Fruit, 1275 Hydrochloride Hy- Chlorpheniramine Rhizome, 1275 drate, 551 and Calcium Powder, 497 Hydrochloride, 535 , 552 Maleate, 498 Oil, 535 Maleate Injection, 499 Phosphate D Maleate Powder, 499 Hydrate, 536 Maleate Tablets, 500 Tablets, 537 Daiokanzoto Extract, 1280 d-Chlorpheniramine Maleate, 501 1 Codeine Phosphate Powder, 536 Sodium Hydrate, 553 Hydrochloride, 502 10 Codeine Phosphate Powder, 537 Hydrochloride, 553 Injection, 503 Cod Oil, 538 Mesilate, 554 Tablets, 503 Coix Seed, 1276 Dehydrated Alcohol, 639 , 504 , 538 Dehydrocholate Sodium Injection, Tablets, 505 557 , 506 Sodium Methanesulfonate, 540 Dehydrocholic Acid, 556 , 506 Sulfate, 541 Injection, 557 , 507 Colophonium, 1347 Demethylchlortetracycline Hydro- Chorionic Gonadotrophin, 707 Compound chloride, 558 for Injection, 708 Acrinol and Zinc Oxide Oil, 273 Dental Chrysanthemum Flower, 1271 and Antiformin, 310 , 507 Powder, 567 Glycerin, 771 , 509 Hycodenone Injection, 952 Paraformaldehyde Paste, 969 A, 509 Iodine Glycerin, 770 Phenol with Camphor, 987 Cilastatin Sodium, 510 Spirit, 879 Sodium Hypochlorite Solution, 310 , 511 and Atropine Injection, Triozinc Paste, 1210 Tablets, 512 953 Dermatol, 368 , 513 Oxycodone Injection, 952 Deslanoside, 559 Cimicifuga Rhizome, 1272 Phellodendron Powder for Injection, 560 Hydrochloride, 569 Cataplasm, 1332 , 560 Cinnamon Rhubarb and Senna Powder, 1346 Dextran Bark, 1272 Salicylic Acid Spirit, 1080 40, 561 Oil, 1273 Scopolia Extract and Diastase Pow- 40 Injection, 562 , 513 der, 1355 70, 562 Citric Acid Hydrate, 515 Thianthol and Salicylic Acid Solu- Sulfate Sodium Sulfur 18, 565 Citrus Unshiu Peel, 1273 tion, 1165 Sulfate Sodium Sulfur 5, 564 , 516 B Powder, 1230 Dextrin, 565 Tablets, 517 Concentrated Hydrobromide Fumarate, 519 Glycerin, 703 Hydrate, 566 Clematis Root, 1274 Glycerol, 703 Diagnostic Sodium Citrate Solution, Hydrochloride, 519 Condurango, 1276 1100 Capsules, 520 Fluidextract, 1277 Diastase, 567 Clindamycin Phosphate, 521 Coptis Rhizome, 1277 and Sodium Bicarbonate Powder, Clinoˆbrate, 522 Corn 567 Hydrochloride Hydrate, Oil, 542 , 567 523 Starch, 542 Dibasic Hydrochloride, 524 Cornus Fruit, 1279 Calcium Phosphate Hydrate, 396 Citrate, 526 Cortisone Acetate, 543 Sodium Phosphate Hydrate, 568 Tablets, 527 Corydalis Tuber, 1279 Dibekacin Sulfate, 569 Hydrochloride, 528 , 544 Dibucaine Hydrochloride, 569 , 528 Cresol, 544 Dichlorphenamide, 571 Hydrochloride, 529 Solution, 545 Tablets, 572 Hydrochloride, 530 Hydrochloride, 546 Sodium, 570 , 531 Croscarmellose Sodium, 546 Diclofenamide, 571 , 532 Crude Glycyrrhiza Extract, 1297 Tablets, 572 1768 Index JP XV

Dicloxacillin Sodium Hydrate, 573 Hydrochloride, 605 Ethacridine Lactate, 274 Diethylcarbamazine Citrate, 574 Hydrochloride Hydrate, Hydrochloride, 637 Tablets, 574 606 Ethanol, 638 Diphosphate, 683 Dried for Disinfection, 640 Tablets, 684 Aluminum Hydroxide Gel, 288 , 640 Difenidol Hydrochloride, 575 Aluminum Hydroxide Gel Fine Ether, 641 Digenea, 1280 Granules, 289 , 642 , 576 Aluminum Potassium Sulfate, 292 Tablets, 642 Tablets, 576 Sodium Carbonate, 1096 , 643 , 577 Sodium Sulˆte, 1109 , 644 Injection, 578 Thyroid, 1171 Ethoxybenzamide, 640 Tablets, 580 Yeast, 1241 Ethyl Phosphate, 581 , 608 Aminobenzoate, 644 1 Dihydrocodeine Phosphate Pow- , 609 Cysteine Hydrochloride, 645 der, 582 Tablets, 610 L-Cysteine Hydrochloride, 645 10 Dihydrocodeine Phosphate Pow- Icosapentate, 646 der, 582 E Parahydroxybenzoate, 647 Mesilate, 583 Ethylenediamine, 648 Dihydroergotoxine Mesilate, 584 Ecarazine Hydrochloride, 1185 Hydrochloride Hy- Hydrochloride Hydrate, 586 Ecothiopate Iodide, 610 drate, 648 Hydrochloride, 586 Edrophonium Chloride, 611 Etidronate Disodium, 649 Dilute Injection, 612 Tablets, 650 Hydrochloric Acid, 726 EDTA Sodium Hydrate, 598 Hydrochloride, 650 Iodine Tincture, 769 Elcatonin, 612 Tablets, 651 Diluted Powder, 940 Eleutherococcus Senticosus Rhizome, , 652 Phosphate, 588 1283 , 653 , 588 Hydrate, 616 , 653 Tablets, 589 Enviomycin Sulfate, 616 Eucalyptus Oil, 654 , 590 En‰urane, 615 Eucommia Bark, 1284 Injection, 590 Hydrochloride, 618 Evodia Fruit, 1285 Dimorpholamine, 591 Ephedra Herb, 1283 Exsiccated Gypsum, 1298 Injection, 591 Hydrochloride, 619 Dinoprost, 592 Injection, 619 F Dionin, 648 Powder, 620 Dioscorea Rhizome, 1282 Tablets, 621 , 655 , 593 10 Ephedrine Hydrochloride Powder, for Injection, 656 and Bromovalerylurea Powder, 593 620 Powder, 657 Hydrochloride, 594 Epimedium Herb, 1284 Tablets, 657 Tannate, 595 , 276 Sodium Phenol and Zinc Oxide Liniment, Injection, 276 for Syrup, 659 595 Solution, 277 Hydrate, 658 Diphenylhydantoin, 991 , 621 Tablets, 660 Powder, 992 Hydrochloride, 622 , 660 Sodium for Injection, 992 , 624 Fennel, 1285 Tablets, 992 Maleate, 625 Oil, 1286 Diphtheria Injection, 625 Citrate, 661 Toxoid, 596 Tablets, 626 Ferrous Sulfate Hydrate, 661 Tetanus Combined Toxoid, 596 Tartrate, 627 Flavin Dinucleotide Sodium, , 597 , 627 662 Disodium Edetate Hydrate, 598 Ethylsuccinate, 629 Hydrochloride, 664 , 598 Lactobionate, 629 Sodium, 664 Distigmine Bromide, 599 Stearate, 630 for Injection, 666 Tablets, 600 , 631 , 667 Disulˆram, 600 Benzoate, 631 Capsules, 667 Hydrochloride, 601 Injection, 632 , 668 Dolichos Seed, 1282 Injection (Aqueous Suspension), , 669 Hydrochloride, 602 633 , 670 Injection, 602 , 633 , 670 Dover's Powder, 1324 Injection (Aqueous Suspension), , 672 Hydrochloride Hydrate, 634 Fluorescein Sodium, 673 603 Tablets, 635 , 673 Doxi‰, 604 Etacrynic Acid, 636 , 674 Capsules, 604 Tablets, 636 , 675 JP XV Index 1769

Fluphenazine Enanthate, 676 Geranium Herb, 1291 Hydrate, 734 , 677 Ginger, 1291 Hydrogenated Oil, 735 Capsules, 678 , 1292 Hydrophilic Hydrochloride, 678 Glacial Acetic Acid, 269 Ointment, 735 , 679 Glehnia Root, 1294 Petrolatum, 981 Foeniculated Ammonia Spirit, 1286 , 699 Hydrous Lanolin, 807 Folic Acid, 680 Glucose, 700 Acetate, 736 Injection, 681 Injection, 701 Hydroxypropylcellulose, 736 Tablets, 681 , 701 Hydroxypropylmethylcellulose, 741 Formalin, 681 Glycerin, 702 Water, 682 and Potash Solution, 704 Hydrochloride, 739 Fumarate Hydrate, 682 Glycerol, 702 Pamoate, 739 Forsythia Fruit, 1286 Glyceryl Monostearate, 704 , 740 , 683 Glycine, 704 Hypromellose, 741 Tablets, 684 Glycyrrhiza, 1295 Phthalate, 743 Extract, 1296 Calcium Hydrate, 685 Acetate, 705 I Sodium, 686 , 712 Sodium for Injection, 687 , 713 , 744 Fradiomycin Sulfate, 688 Guaiacol Glyceryl Ether, 714 Ichthammol, 744 Freeze-dried , 714 Idarubicin Hydrochloride, 745 BCG Vaccine (for Percutaneous Acetate, 715 for Injection, 746 Use), 335 Sulfate, 716 Idoxuridine, 747 Botulism Antitoxin, Equine, 374 Gypsum, 1297 Ophthalmic Solution, 747 Diphtheria Antitoxin, Equine, 596 Tartrate, 748 Habu Antivenom, Equine, 716 H Inactivated Tissue Culture Rabies and Cilastatin for Injection, 750 Vaccine, 1054 , 717 Hydrate, 749 Japanese Encephalitis Vaccine, 786 Tablets, 718 Hydrochloride, 751 Live Attenuated Measles Vaccine, , 719 Hydrochloride, 751 843 , 719 Tablets, 752 Live Attenuated Mumps Vaccine, Fruit, 1298 Immature Orange, 1302 902 Sodium, 721 Imperata Rhizome, 1302 Live Attenuated Rubella Vaccine, Injection, 722 Hydrochloride, 753 1074 Hochuekkito Extract, 1298 Indigocarmine, 754 Mamushi Antivenom, Equine, 841 Hydrobromide, 722 Injection, 755 Smallpox Vaccine, 1091 Hydrochloride, Indium (111In) Chloride Injection, 755 Smallpox Vaccine Prepared in Cell 723 , 755 Culture, 1091 Honey, 1301 Capsules, 756 Tetanus Antitoxin, Equine, 1156 Houttuynia Herb, 1302 Suppositories, 757 Fritillaria Bulb, 1287 Human Insulin, 758 Fructose, 689 Chorionic Gonadotrophin, 707 Human (Genetical Recombination), Injection, 689 Chorionic Gonadotrophin for 760 , 690 Injection, 708 Injection, 762 Tablets, 691 Menopausal Gonadotrophin, 708 Zinc Injection (Aqueous Suspen- Hydrochloride, 692 Normal Immunoglobulin, 724 sion), 763 Hycoato Injection, 953 Zinc Injection (Aqueous G Hydrochloride, 724 Suspension), 766 for Injection, 724 In‰uenza HA Vaccine, 758 Gabexate Mesilate, 693 Powder, 725 , 767 b-Galactosidase Tablets, 725 Iodinated (131I) (Aspergillus), 694 Hydrochloric Acid, 725 Injection, 768 (Penicillium), 695 Lemonade, 727 Iodine, 768 Gallium (67Ga) Citrate Injection, 696 , 727 Salicylic Acid and Phenol Spirit, Gambir, 1287 , 728 772 Gardenia Fruit, 1288 Acetate, 729 Tincture, 769 Gas Gangrene Antitoxin, Equine, 696 and Diphenhydramine Ointment, Iodoform, 773 Gastrodia Tuber, 1289 730 , 773 Gelatin, 696 Butyrate, 730 , 774 Gentamicin Sulfate, 698 Sodium Phosphate, 731 Iotroxic Acid, 775 Gentian, 1290 Sodium Succinate, 732 Ipecac, 1303 and Sodium Bicarbonate Powder, Succinate, 733 Syrup, 1304 1290 Hydrocotarnine Hydrochloride Hydrate, 776 1770 Index JP XV

Iproveratril Hydrochloride, 1225 Lenampicillin Hydrochloride, 810 Sulfate Injection, 839 Isepamicin Sulfate, 777 L-Leucine, 812 Sulfate Mixture, 839 L-Isoleucine, 780 Leucomycin, 798 Magnolia , 780 Acetate, 799 Bark, 1315 Injection, 781 Tartrate, 800 Flower, 1317 Tablets, 781 Tartrate, 813 Mallotus Bark, 1317 Isophane Insulin Injection (Aqueous Injection, 814 Maltose Hydrate, 840 Suspension), 762 Levodopa, 814 D-Mannite Injection, 842 l- Hydrochloride, 782 Maleate, 815 D-Mannitol, 841 Isopropanol, 783 Sodium Injection, 842 , 783 Hydrate, 816 Hydrochloride, 842 Isopropylantipyrine, 783 Tablets, 817 Hydrochloride, 843 Isosorbide, 784 , 818 Mecobalamin, 844 Dinitrate, 785 Hydrochloride Injection, 818 , 845 Dinitrate Tablets, 786 Injection, 818 Medicinal Isotonic Light Carbon, 845 Salt Solution, 1098 Anhydrous Silicic Acid, 1087 Soap, 846 Sodium Chloride Injection, 1098 Liquid Para‹n, 967 , 847 Sodium Chloride Solution, 1098 Limaprost Alfadex, 819 , 849 Iso‰urane, 778 Hydrochloride Hydrate, Tablets, 849 820 , 850 J Lindera Root, 1313 Amidotrizoate Injection, 851 Liothyronine Sodium, 821 Iotalamate Injection, 852 Japanese Tablets, 822 Sodium Amidotrizoate Injection, Angelica Root, 1305 Liqueˆed 853 Encephalitis Vaccine, 786 Carbolic Acid, 986 Sodium Iodamide Injection, 854 Gentian, 1306 Phenol, 986 , 855 , 1306 Liquid Para‹n, 966 , 855 Josamycin, 786 Propionate, 788 Hydrate, 823 Herb, 1317 Jujube, 1307 Tablets, 824 Oil, 1318 Seed, 1307 , 826 Water, 1318 Lithospermum Root, 1313 dl-, 856 K Live Oral Poliomyelitis Vaccine, 1005 l-Menthol, 857 Longgu, 1313 Bromide, 857 Lonicera Leaf and Stem, 1314 Mepirizole, 621 and Santonin Powder, 790 Loquat Leaf, 1314 , 858 Hydrate, 789 , 827 Hydrochloride, 859 Kakkonto Extract, 1308 Low Substituted Hydroxypropylcellu- Injection, 860 Kallidinogenase, 790 lose, 738 , 861 Kamishoyosan Extract, 1309 Sodium Hydrate, 828 Merbromin, 862 Kanamycin Lycium Solution, 863 Monosulfate, 793 Bark, 1315 Hydrate, 861 Sulfate, 794 Fruit, 1315 Mercurochrome, 862 Kaolin, 795 Lysine Hydrochloride, 829 Solution, 863 Hydrochloride, 795 L-Lysine Hydrochloride, 829 Hydrate, 863 , 796 Hydrochloride, 830 , 864 Fumarate, 797 Kitasamycin, 798 M Acetate, 865 Acetate, 799 Enanthate, 866 Tartrate, 800 Enanthate Injection, 866 400, 830 Hydrochloride, 867 L 1500, 831 Tablets, 867 4000, 832 Hydrochloride, Lactic Acid, 801 6000, 832 868 Lactose, 803 20000, 833 L-Methionine, 869 Hydrate, 803 Ointment, 833 , 869 Lactulose, 804 Magnesium , 870 Lanatoside C, 805 Carbonate, 834 Methylbenactyzium Bromide, 871 Tablets, 806 Oxide, 835 Methylcellulose, 871 Lard, 808 Silicate, 836 Sodium, 809 Stearate, 837 Hydrate, 873 Lauromacrogol, 810 Sulfate Hydrate, 838 Tablets, 874 JP XV Index 1771 dl-Methylephedrine Hydrochloride, Natural Aluminum Silicate, 289 Oil, 946 875 Nelumbo Seed, 1320 Peel Syrup, 1325 Powder, 876 Sulfate, 688 Peel Tincture, 1325 10 dl-Methylephedrine Hydrochlo- Methylsulfate, 909 Sulfate, 946 ride Powder, 876 Injection, 910 Oriental Bezoar, 1325 Maleate, 876 Netilmicin Sulfate, 910 , 947 Tablets, 877 Hydrochloride, 911 , 948 Methyl Injection, 912 , 948 Parahydroxybenzoate, 878 , 913 , 949 Salicylate, 881 Powder, 914 Oxethazaine, 949 , 879 Tablets, 915 Hydrochloride, 950 Succinate, 880 Niceritrol, 916 Hydrochloride, 951 Methylrosanilinium Chloride, 881 Nicomol, 917 Oxycodone Hydrochloride Hydrate, , 882 Tablets, 918 951 Tablets, 883 , 919 Oxydol, 954 Meticrane, 884 , 919 Oxygen, 955 Metildigoxin, 885 Nicotinic Acid, 920 , 956 , 886 Injection, 921 Oxytetracycline Hydrochloride, 956 Tablets, 886 , 922 , 958 Tartrate, 887 , 923 Injection, 960 Tablets, 888 Tablets, 924 Oyster Shell, 1326 , 889 , 925 O‰oxacin, 938 Tablets, 889 , 926 , 890 Tablets, 927 P Hydrochloride, 891 Nitrogen, 928 Me‰oquine Hydrochloride, 848 Nitroglycerin Tablets, 928 Panax Japonicus Rhizome, 1326 , 892 , 929 Pancreatin, 961 Nitrate, 893 Noradrenaline, 930 , 962 Microcrystalline Cellulose, 477 Hydrochloride Injection, 931 , 962 Micronomicin Sulfate, 893 Noradrenaline Injection, 931 , 964 Midecamycin, 894 , 930 Hydrochloride, 965 Acetate, 895 Hydrochloride Injection, 931 Injection, 965 Migrenin, 896 Injection, 931 , 267 Hydrochloride, 897 , 932 Paraformaldehyde, 968 , 898 , 933 Para‹n, 966 Monobasic Calcium Phosphate and Ethinylestradiol Tablets, 934 Parnaparin Sodium, 969 Hydrate, 397 Hydrochloride, 935 Pas-calcium Monosodium Trichloroethyl Phos- Nor‰oxacin, 932 Granules, 394 phate, 1202 , 936 Hydrate, 395 Syrup, 1203 Hydrochloride Hydrate, 937 Peach Kernel, 1327 Notopterygium Rhizome, 1321 Oil, 971 and Atropine Injection, 899 Nuphar Rhizome, 1321 Sulfate, 972 Hydrochloride Hydrate, 900 Nux Vomica, 1321 G Potassium, 349 Hydrochloride Injection, 901 Extract, 1322 , 972 Hydrochloride Tablets, 902 Extract Powder, 1323 Calcium, 973 Moutan Bark, 1318 Tincture, 1323 Citrate, 974 Mulberry Bark, 1320 , 937 Peony Root, 1328 Calcium Hydrate, 902 Peplomycin Sulfate, 975 O Perilla Herb, 1329 N , 977 Olive Oil, 939 Maleate, 978 , 904 Operidine, 980 Maleate Tablets, 979 , 905 Injection, 981 Tablets, 977 Hydrochloride, 906 Ophiopogon Tuber, 1324 Hydrochloride, 980 Opium Injection, 981 and Chlorpheniramine Solution, Ipecac Powder, 1324 Petroleum Benzin, 982 906 Tincture, 940 Pharbitis Seed, 1330 Hydrochloride, 907 Opium Phellodendron, Nitrate, 908 and Atropine Injection, 942 Albumin Tannate and Bismuth Sub- , 908 and Injection, 943 nitrate Powder, 1332 Narcotine, 936 Hydrochlorides, 941 Bark, 1330 Hydrochloride, 937 Hydrochlorides Injection, 942 , 310 , 994 Orange Phenethicillin Potassium, 983 1772 Index JP XV

Phenobarbital, 984 Potassium Senna Leaf, 1362 Powder, 985 Bromide, 1008 Smilax Rhizome, 1364 10 Powder, 985 Canrenoate, 1008 Sophora Root, 1364 Phenol, 985 Carbonate, 1009 Sweet Hydrangea Leaf, 1365 and Zinc Oxide Liniment, 987 Chloride, 1009 Swertia Herb, 1366 for Disinfection, 987 Clavulanate, 1010 Tragacanth, 1369 Phenolated Water, 987 , 1011 Zanthoxylum Fruit, 1371 for Disinfection, 986 Hydroxide, 1012 , 1018 Phenolsulfonphthalein, 988 Iodide, 1013 Sodium, 1018 Injection, 988 Permanganate, 1013 , 1020 L-Phenylalanine, 989 Sulfate, 1014 Tablets, 1021 , 990 Potato Starch, 1014 Precipitated , 389 Hydrochloride, 990 Povidone, 1015 , 1021 , 991 Iodine, 1017 Acetate, 1023 Powder, 992 Powdered Sodium Succinate for Injection, Sodium for Injection, 992 Acacia, 1251 1025 Tablets, 992 Agar, 1253 Succinate, 1024 , 993 Alisma Rhizome, 1253 Tablets, 1022 Phytonadione, 993 Aloe, 1255 , 1026 Picrasma Wood, 1333 Amomum Seed, 1256 , 1027 Hydrochloride, 994 Atractylodes Lancea Rhizome, Tablets, 1027 Pimaricin, 994 1261 Hydrochloride, 1028 , 995 Atractylodes Rhizome, 1260 Injection, 1029 Pinellia Tuber, 1333 Calumba, 1268 Tablets, 1029 Hydrate, 996 Capsicum, 1269 Hydrochloride, 1030 Sodium, 997 Cellulose, 480 Injection, 1030 for Injection, 998 Cinnamon Bark, 1273 Hydrochloride, 1031 Clove, 1274 Hydrochloride Hydrate, Adipate, 999 Cnidium Rhizome, 1276 1032 Phosphate Hydrate, 999 Coix Seed, 1276 Maleate, 1033 Phosphate Tablets, 1000 Coptis Rhizome, 1278 Tablets, 1033 Pirarubicin, 1000 Cyperus Rhizome, 1280 Processed Pirenoxine, 1001 Dioscorea Rhizome, 1282 Aconite Root, 1338 Hydrochloride Hydrate, Fennel, 1285 Ginger, 1341 1002 Gambir, 1287 , 1034 , 1003 Gardenia Fruit, 1288 Injection, 1034 Hydrochloride, 1004 Gentian, 1290 , 1035 Plantago Geranium Herb, 1291 Hydrochloride, 1036 Herb, 1334 Ginger, 1292 , 1036 Seed, 1334 Ginseng, 1293 Hydrochloride, 1037 Platycodon Glycyrrhiza, 1296 Tablets, 1038 Fluidextract, 1334 Ipecac, 1303 Propylene Glycol, 1039 Root, 1334 Japanese Angelica Root, 1305 Propyl Parahydroxybenzoate, 1039 Polyethylene Glycol Japanese Gentian, 1306 Propylthiouracil, 1040 400, 830 Japanese Valerian, 1307 Tablets, 1040 1500, 831 Magnolia Bark, 1316 , 783 4000, 832 Moutan Bark, 1319

6000, 832 Opium, 939 E1,286 20000, 833 Oyster Shell, 1326 E1 a-Cyclodextrin Clathrate Com- Ointment, 833 Panax Japonicus Rhizome, 1327 pound, 287

Polygala Root, 1335 Peach Kernel, 1327 F2a,592 Polygonatum Rhizome, 1336 Peony Root, 1329 , 1041 Polygonum Root, 1336 Phellodendron Bark, 1331 Injection, 1042 Polymixin B Sulfate, 1006 Picrasma Wood, 1333 Prothionamide, 1042 Polyoxyethylene Lauryl Alcohol Ether, Platycodon Root, 1335 Protirelin, 1043 810 Polygala Root, 1335 Tartrate Hydrate, 1044 Polyoxyl 40 Stearate, 1007 Polyporus Sclerotium, 1337 Prunella Spike, 1341 Polyporus Sclerotium, 1336 Poria Sclerotium, 1337 Pueraria Root, 1341 Polysorbate 80, 1007 Processed Aconite Root, 1339 Pullulan, 1045 Polyvidone, 1015 Rhubarb, 1345 Puriˆed Polyvinylpyrrolidone, 1015 Rose Fruit, 1346 Dehydrocholic Acid, 556 Poria Sclerotium, 1337 Root, 1359 Gelatin, 697 Potash Soap, 1007 Senega, 1361 Lanolin, 808 JP XV Index 1773

Shellac, 1085 Saireito Extract, 1349 Chromate (51Cr) Injection, 1099 Water, 1236 Salazosulfapyridine, 1077 Citrate Hydrate, 1099 Pamoate, 1045 Sulfate, 1078 Citrate Injection for Transfusion, , 1046 Salicylated Alum Powder, 1078 1099 Pyridostigmine Bromide, 1047 Salicylic Acid, 1079 Cromoglicate, 1100 Hydrochloride, 1047 Adhesive Plaster, 1080 Fusidate, 1101 Injection, 1048 Spirit, 1080 Hydrogen Sulfite, 1094 Pyroxylin, 1049 Santonin, 1081 Hydroxide, 1101 Pyrrolnitrin, 1049 Saponated Cresol Solution, 545 Iodide, 1102 Saposhnikovia Root, 1352 Iodide (123I) Capsules, 1103 Q Sappan Wood, 1352 Iodide (131I) Capsules, 1103 Saussurea Root, 1352 Iodide (131I) Solution, 1103 Quick Lime, 393 SaŠron, 1349 Iodohippurate (131I) Injection, 1103 Sulfate Hydrate, 1050 SaŒower, 1348 Iotalamate Injection, 1103 Schisandra Fruit, 1352 Lauryl Sulfate, 1104 Ethyl Carbonate, 1051 Schizonepeta Spike, 1353 Metabisulˆte, 1108 Hydrochloride Hydrate, 1052 Scopolamine Pertechnetate (99mTc) Injection, Sulfate Hydrate, 1053 Butylbromide, 1082 1105 Hydrobromide Hydrate, 1083 Picosulfate Hydrate, 1105 R Scopolia Extract, 1353 Polystyrene Sulfonate, 1106 Papaverine and Ethyl Aminobenzo- Sulfate Hydrate, 1107 Hydrochloride, 1054 ate Powder, 1356 Pyrosulˆte, 1108 Rape Seed Oil, 1055 and Carbon Powder, 1355 Salicylate, 1108 Red Ginseng, 1342 and Ethyl Aminobenzoate Powder, Thiosulfate Hydrate, 1109 Rehmannia Root, 1343 1355 Thiosulfate Injection, 1110 , 1055 and Tannic Acid Suppositories, , 1110 Injection, 1056 1357 0.9 Sodium Chloride Injection, Powder, 1057 Powder, 1353 1098 Tablets, 1057 Rhizome, 1357 10 Sodium Chloride Injection, 1098 0.1 Reserpine Powder, 1057 Scutellaria Root, 1358 Sophora Root, 1364 Senega, 1360 Sorbitan Sesquioleate, 1111 Acetate, 1058 Syrup, 1361 D-Sorbitol, 1112 Palmitate, 1059 Senna Leaf, 1361 Solution, 1113 Rhubarb, 1344 Serrapeptase, 1083 Soybean Oil, 1113 Sulfate, 1063 Serum Gonadotrophin, 710 Hydrochloride Hydrate, Ribo‰avin, 1059 for Injection, 711 1114 Butyrate, 1060 Sesame Oil, 1084 Spiramycin Acetate, 1114 Phosphate, 1061 Siccanin, 1086 , 1116 Phosphate Injection, 1062 Silver Stearic Acid, 1116 Powder, 1060 Nitrate, 1088 Stearyl Alcohol, 1117 Sodium Phosphate, 1061 Nitrate Ophthalmic Solution, 1088 Sterile Puriˆed Water, 1236 Sodium Phosphate Injection, 1062 Protein, 1088 Streptomycin Sulfate, 1117 Rice Starch, 1346 Protein Solution, 1089 Hydrate, 1118 , 1064 Simple Sucrose, 1120 Capsules, 1065 Ointment, 1089 Sodium, 1122 Ringer's Solution, 1066 Syrup, 1089 Sodium, 1123 Hydrochloride, 1067 Sinomenium Stem, 1363 Silver, 1124 Tablets, 1068 Sisomicin Sulfate, 1090 , 1128 Rokitamycin, 1069 Slaked Lime, 392 , 1125 Rose Fruit, 1346 Smilax Rhizome, 1364 , 1126 Rosin, 1347 Sodium Sulfamonomethoxine Hydrate, 1126 Hydrochloride, Acetate Hydrate, 1091 , 1077 1070 Aurothiomalate, 1092 Sulfobromophthalein Sodium, 1129 Extended-release Capsules, 1071 Benzoate, 1093 Injection, 1130 , 1073 Bicarbonate, 1093 Sulfur, 1130 Ryokeijutsukanto Extract, 1347 Bicarbonate and Bitter Tincture Mix- Salicylic Acid and Thianthol Oint- ture, 1364 ment, 1131 S Bicarbonate Injection, 1094 and Camphor Lotion, 1130 Bisulfite, 1094 , 1131 Saccharated Pepsin, 1074 Borate, 1095 Capsules, 1132 Saccharin, 1075 Bromide, 1095 Tablets, 1132 Sodium, 1076 Carbonate Hydrate, 1096 Sulpyrine Sodium Hydrate, 1076 Chloride, 1097 Hydrate, 1133 1774 Index JP XV

Injection, 1134 , 1170 Injection, 1213 Tosilate Hydrate, 1134 , 1170 Hydrochloride, 1213 Sultiame, 1136 Tiaramide Hydrochloride, 1171 Turpentine Oil, 1214 Sulˆnpyrazone, 1127 Tablets, 1172 Tablets, 1128 Hydrochloride, 1173 U Sulˆsomezole, 1126 Hydrate, 1174 Sulˆsoxazole, 1128 Maleate, 1174 Ubidecarenone, 1214 , 1175 , 1215 for Injection, 1137 Hibenzate, 1176 Uncaria Hook, 1370 Hydrate, 1137 Tablets, 1177 , 1217 Injection, 1138 Titanium Oxide, 1178 Urea, 1218 Sweet Hydrangea Leaf, 1365 Hydrochloride, 1179 Urokinase, 1219 Swertia Toad Venom, 1368 , 1220 and Sodium Bicarbonate Powder, Tobramycin, 1180 Uva Ursi Fluidextract, 1371 1367 , 1181 Herb, 1365 Acetate, 1182 V Synthetic Calcium Succinate, 1183 Aluminum Silicate, 290 Nicotinate, 1184 L-Valine, 1221 Camphor, 402 dl-a-Tocopherol, 1181 Hydrochloride, 1221 Acetate, 1182 for Injection, 1223 T Nicotinate, 1184 Injection, 1223 Todralazine Hydrochloride Hydrate, Hydrochloride, 1225 Hydrochloride, 1138 1185 Tablets, 1226 , 1139 , 1186 Sulfate, 1226 Hydrochloride, 1140 , 1187 for Injection, 1227 Tannic Acid, 1141 , 1188 Sulfate, 1228 Tartaric Acid, 1141 Tablets, 1188 , 1142 , 1189 Acetate, 1058 Teceleukin Solution, 1189 Capsules, 1230 (Genetical Recombination), 1143 Hydrochloride, 1190 Oil, 1229 for Injection (Genetical Recombina- Tragacanth, 1369 Oil Capsules, 1230 tion), 1148 , 1191 Palmitate, 1059

Tegafur, 1149 Capsules, 1192 Vitamin B1 , 1150 Injection, 1193 Hydrochloride, 1160 Sulfate, 1153 Tablets, 1193 Hydrochloride Injection, 1161 Termeric, 1367 , 1194 Hydrochloride Powder, 1162 Trepibutone, 1195 Nitrate, 1162

Enanthate, 1154 Tretoquinol Hydrochloride, 1209 Vitamin B2, 1059 Enanthate Injection, 1154 , 1195 Phosphate Ester, 1061 Propionate, 1155 Acetonide, 1196 Phosphate Ester Injection, 1062 Propionate Injection, 1155 , 1197 Powder, 1060

Tetracaine Hydrochloride, 1156 Tribulus Fruit, 1369 , 1047 Hydrochloride, 1157 , 1198 Injection, 1048 201 ( Tl) Chloride Injection, Tablets, 1199 , 548 1158 Trichomycin, 1201 Injection, 549 , 1158 Trichosanthes Root, 1369 , 316 Thiamazole, 1159 Sodium, 1202 Injection, 317 Tablets, 1160 Syrup, 1203 Powder, 317

Thiamine Hydrochloride, 1204 Vitamin D2, 624 Chloride Hydrochloride, 1160 Tablets, 1204 Vitamin D3, 506 Chloride Hydrochloride Injection, Maleate, 1206 , 1181 1161 Trimetazidine Hydrochloride, 1207 Acetate, 1182 Chloride Hydrochloride Powder, Tablets, 1207 Calcium Succinate, 1183 1162 , 1208 Nicotinate, 1184

Nitrate, 1162 Tablets, 1209 Vitamin K1,993 Sodium, 1163 Trimetoquinol Hydrochloride Hydrate, Voglibose, 1231 for Injection, 1164 1209 Tablets, 1232 Thianthol, 1165 , 1211 Thiopental Sodium, 1166 L-Tryptophan, 1211 W for Injection, 1167 Hydrochloride Hydrochloride, 1168 Hydrate, 1212 Potassium, 1233 , 1168 Injection, 1213 Tablets, 1234 L-Threonine, 1169 Tubocurarine Hydrochloride, 1212 Water, 1235 JP XV Index 1775

for Injection, 1235 Weak Opium Alkaloids and Scopola- X Z mine Injection, 945 Weil's Disease and Akiyami Combined Xylitol, 1240 , 1242 Vaccine, 1237 Injection, 1241 Tablets, 1243 Wheat Starch, 1237 Zanthoxylum Fruit, 1371 White Y Zedoary, 1372 Beeswax, 336 Zinc Ointment, 1238 Yellow Chloride, 1244 Petrolatum, 981 Beeswax, 336 Oxide, 1245 Shellac, 1085 Petrolatum, 982 Oxide Oil, 1245 Soft Sugar, 1121 Oxide Ointment, 1246 Whole Human Blood, 1238 Oxide Starch Powder, 1246 Wine, 1238 Sulfate Hydrate, 1246 Sulfate Ophthalmic Solution, 1247 Zinostatin Stimalamer, 1247 INDEX IN NAME

A C E

Achyranthis Radix, 1252 Calumbae Eleutherococci senticosi Rhizoma, Adeps Radix, 1267 1283 Lanae Ruriˆcatus,808 Radix Pulverata, 1268 Ephdrae Herba,1283 Suillus,808 Fructus, 1298 Epimedii Herba, 1284 Agar, 1252 Capsici Eriobotryae Folium, 1314 Pulveratum,1253 Fructus,1268 Eucommiae Cortex, 1284 Akebiae Caulis, 1253 Fructus Pulveratus, 1269 Evodiae Fructus,1285 Alismatis Cardamomi Fructus, 1271 Extractum Belladonnae, 1263 Rhizoma,1253 Carthami Flos, 1348 Rhizoma Pulveratum,1253 Caryophylli F Aloe, 1254 Flos Pulveratus, 1274 Pulverata, 1255 Flos, 1274 Fel Ursi,1262 Alpiniae Cassiae Semen, 1271 Foeniculi Fructus, 1265 Catalpae Fructus, 1271 Fructus, 1285 o‹cinari Rhizoma, 1256 Cellulose Acetate Phthalate,480 Fructus Pulveratus, 1285 Amomi Cera Forsythiae Fructus,1286 Semen,1256 Alba, 336 Fossilia Ossis Mastodi,1313 Semen Pulveratum, 1256 Carnauba,411 Fritillariae Bulbus, 1287 Amylum Flava,336 Maydis,542 Chrysanthemi Flos,1271 G Oryzae, 1346 Cimicifugae Rhizoma,1272 Solani,1014 Cinnamomi Gambir, 1287 Tritici, 1237 Cortex, 1272 Pulveratum, 1287 Anemarrhenae Rhizoma,1256 Cortex Pulveratus,1273 Gardeniae Angelicae Clematidis Radix, 1274 Fructus, 1288 Radix, 1305 Cnidii Fructus Pulveratus, 1288 Radix Pulverata,1305 Monnieris Fructus,1275 Gastrodiae Tuber, 1289 Arctii Fructus, 1267 Rhizoma,1275 Gentianae Arecae Semen,1258 Rhizoma Pulveratum, 1276 Radix,1290 Armeniacae Semen, 1257 Coicis Radix Pulverata, 1290 Artemisiae Capillaris Flos, 1258 Semen, 1276 Scabrae Radix, 1306 Asiasari Radix, 1259 Semen Pulveratum, 1276 Scabrae Radix Pulverata, 1306 Asparagi Tuber,1259 Condurango Cortex,1276 Geranii Astragali Radix,1260 Coptidis Herba, 1291 Atractylodis Rhizoma,1277 Herba Pulverata,1291 Lanceae Rhizoma,1261 Rhizoma Pulveratum, 1278 Ginseng Lanceae Rhizoma Pulveratum, Corni Fructus,1279 Radix,1292 1261 Corydalis Tuber,1279 Radix Pulverata, 1293 Rhizoma,1260 Crocus, 1349 Radix Rubra, 1342 Rhizoma Pulveratum,1260 Curcumae Rhizoma,1367 Glehniae Radix cum Rhizoma, 1294 Aurantii Cyperi Glycyrrhizae Bobilis Pericarpium, 1273 Rhizoma,1280 Radix,1295 Fructus Immaturus, 1302 Rhizoma Pulveratum, 1280 Radix Pulverata, 1296 Pericarpium,1265 Gummi D Arabicum, 1251 B Arabicum Pulveratum,1251 Digenea, 1280 Gypsum Belladonnae Radix, 1263 Dioscoreae Exsiccatum, 1298 Benincasae Semen, 1264 Rhizoma,1282 Fibrosum, 1297 Benzoinum,1265 Rhizoma Pulveratum, 1282 Bezoar Bovis,1325 Dolichi Semen, 1282 H Bufonis Venenum, 1368 Bupleuri Radix, 1266 Houttuyniae Herba, 1302

1777 1778 Index in Latin name JP XV

Hydrangeae Ostreae Saussureae Radix, 1352 Dulcis Folium,1365 Testa,1326 Schisandrae Fructus,1352 Dulcis Folium Pulveratum,1365 Testa Pulverata, 1326 Schizonepetae Spica, 1353 Scopoliae Rhizoma, 1357 I P Scutellariae Radix,1358 Imperatae Rhizoma,1302 Paeoniae Radix Pulverata, 1359 Ipecacuanhae Radix, 1328 Senegae Radix, 1303 Radix Pulverata, 1329 Radix,1360 Radix Pulverata,1303 Panacis Japonici Radix Pulverata, 1361 Rhizoma,1326 Sennae L Rhizoma Pulveratum, 1327 Folium,1361 Perillae Herba, 1329 Folium Pulveratum, 1362 Linderae Radix, 1313 Persicae Sevum Bovinum,336 Lithospermi Radix, 1313 Semen, 1327 Sinomeni Caulis et Rhizoma, 1363 Lonicerae Folium Cum Caulis, 1314 Semen Pulveratum, 1327 Smilacis Lycii Pharbitidis Semen,1330 Rhizoma, 1364 Cortex, 1315 Phellodendri Rhizoma Pulveratum, 1364 Fructus, 1315 Cortex, 1330 Sophorae Cortex Pulveratus,1331 Radix,1364 M Picrasmae Radix Pulverata, 1364 Lignum,1333 Strychni Semen,1321 Magnoliae Lignum Pulveratum,1333 Swertiae Cortex Pulveratus, 1316 Pinelliae Tuber, 1333 Herba, 1365 Flos, 1317 Plantaginis Herba Pulverata,1366 Malloti Cortex, 1317 Herba,1334 Syrupus Mel, 1301 Semen, 1334 Ipecacuanha, 1304 Menthae Herba, 1317 Platycodi Senegae,1361 Mori Cortex,1320 Radix, 1334 Moutan Radix Pulverata, 1335 T Cortex, 1318 Polygalae Cortex Pulveratus, 1319 Radix, 1335 Tinctura Amara,1266 Radix Pulverata, 1335 Tragacantha,1369 N Polygonati Rhizoma, 1336 Pulverata,1369 Polygoni Multi‰ori Radix,1336 Tribuli Fructus, 1369 Nelumbis Semen,1320 Polyporus,1336 Trichosanthis Radix, 1369 Notopterygii Rhizoma,1321 Pulveratus,1337 Nupharis Rhizoma, 1321 Poria, 1337 U Pulveratum, 1337 O Processi Uncariae Uncis Cum Ramulus,1370 Aconiti Radix Pulverata, 1339 Uvae Ursi Folium,1262 Oleum Aconiti Radix,1338 Arachidis, 971 Prunellae Spica,1341 V Aurantii,946 Puerariae Radix, 1341 Cacao, 386 Valerianae Camelliae,400 R Radix,1306 Caryophylli, 1275 Radix Pulverata, 1307 Cinnamomi, 1273 Rehmanniae Radix,1343 Cocois,535 Resina Pini, 1347 Z Eucalypti, 654 Rhei Foeniculi, 1286 Rhizoma,1344 Zanthoxyli Maydis,542 Rhizoma Pulveratum, 1345 Fructus, 1371 Menthae Japonicae, 1318 Rosae Fructus Pulveratus, 1371 Olivae,939 Fructus,1346 Zedoariae Rhizoma,1372 Rapae, 1055 Fructus Pulveratus, 1346 Zingiberis Ricini,414 Processum Rhizoma,1341 Sesami, 1084 S Rhizoma, 1291 Sojae,1113 Rhizoma Pulveratum, 1292 Terebinthinae, 1214 Saposhnikoviae Radix,1352 Zizyphi Ophiopogonis Tuber,1324 Sappan Lignum, 1352 Fructus, 1307 Opium Pulveratum,939 Semen, 1307 INDEX IN JAPANESE

アマンタジン塩酸塩 293 1131 ア アミカシン硫酸塩 296 イオタラム酸 774 アミトリプチリン塩酸塩 298 イオタラム酸ナトリウム注射液 亜鉛華デンプン 1246 アミトリプチリン塩酸塩錠 299 1103 亜鉛華軟膏 1246 アミドトリゾ酸 295 イオタラム酸メグルミン注射液 852 亜酸化窒素 929 アミドトリゾ酸ナトリウムメグルミン イオトロクス酸 775 アカメガシワ 1317 注射液 853 イオパミドール 773 アクチノマイシン D 275 アミドトリゾ酸メグルミン注射液 イクタモール 744 アクラルビシン塩酸塩 272 851 イコサペント酸エチルエステル 646 アクリノール・チンク油 273 アミノ安息香酸エチル 644 イセパマイシン硫酸塩 777 アクリノール・亜鉛華軟膏 274 アミノフィリン水和物 297 イソソルビド 784 アクリノール水和物 274 アミノフィリン注射液 297 イソニアジド 780 アザチオプリン 325 アムホテリシン B シロップ 305 イソニアジド錠 781 アザチオプリン錠 326 アムホテリシン B 錠 305 イソニアジド注射液 781 亜硝酸アミル 309 アムホテリシン B303 イソフェンインスリン水性懸濁注射液 アジスロマイシン水和物 327 アモキサピン 301 762 アジピン酸塩ピペラジン 999 アモキシシリン水和物 302 イソフルラン 778 アジマリン 278 アモバルビタール 300 l-イソプレナリン塩酸塩 782 アジマリン錠 279 アラセプリル 279 イソプロパノール 783 アスコルビン酸 316 アラセプリル錠 280 イソプロピルアンチピリン 783 アスコルビン酸散 317 アラビアゴム 1251 L-イソロイシン 780 アスコルビン酸注射液 317 アラビアゴム末 1251 イダルビシン塩酸塩 745 アストロマイシン硫酸塩 322 アリメマジン酒石酸塩 283 イドクスウリジン 747 L-アスパラギン酸 318 亜硫酸水素ナトリウム 1094 イドクスウリジン点眼液 747 アスピリン 318 L-アルギニン 313 イフェンプロジル酒石酸塩 748 アスピリンアルミニウム 319 L-アルギニン塩酸塩 313 イブプロフェン 744 アスピリン錠 319 L-アルギニン塩酸塩注射液 314 イプラトロピウム臭化物水和物 776 アスポキシシリン水和物 320 アルジオキサ 282 イミプラミン塩酸塩 751 アズトレオナム 328 アルプラゾラム 284 イミプラミン塩酸塩錠 752 アセグルタミドアルミニウム 266 アルプレノロール塩酸塩 285 イミペネム水和物 749 アセタゾラミド 268 アルプロスタジル 286 イレイセン 1274 アセトアミノフェン 267 アルプロスタジルアルファデクス インジゴカルミン 754 アセトヘキサミド 269 287 インジゴカルミン注射液 755 アセブトロール塩酸塩 265 アルベカシン硫酸塩 311 インスリン 758 アセンヤク 1287 アルベカシン硫酸塩注射液 312 インスリン亜鉛水性懸濁注射液 763 アセンヤク末 1287 アロエ 1254 インスリン注射液 762 アテノロール 323 アロエ末 1255 インチンコウ 1258 アトロピン硫酸塩水和物 324 アロチノロール塩酸塩 314 インデノロール塩酸塩 753 アトロピン硫酸塩注射液 324 アロプリノール 284 インドメタシン 755 アドレナリン 276 安息香酸 345 インドメタシンカプセル 756 アドレナリン液 277 安息香酸ナトリウム 1093 インドメタシン坐剤 757 アドレナリン注射液 276 安息香酸ナトリウムカフェイン 388 インフルエンザ HA ワクチン 758 亜ヒ酸パスタ 315 安息香酸ベンジル 347 インヨウカク 1284 アフロクアロン 277 アンソッコウ 1265 アヘン・トコン散 1324 アンチピリン 310 ウ アヘンアルカロイド・アトロピン注射 アンピシリンナトリウム 308 液 942 アンピシリン水和物 307 ウイキョウ 1285 アヘンアルカロイド・スコポラミン注 アンベノニウム塩化物 294 ウイキョウ末 1285 射液 943 アンモニア・ウイキョウ精 1286 ウイキョウ油 1286 アヘンアルカロイド塩酸 941 アンモニア水 299 ウコン 1367 アヘンアルカロイド塩酸注射液 942 ウヤク 1313 アヘン散 940 イ ウラピジル 1217 アヘンチンキ 940 ウリナスタチン 1215 アヘン末 939 イオウ 1130 ウルソデオキシコール酸 1220 アマチャ 1365 イオウ・カンフルローション 1130 ウロキナーゼ 1219 アマチャ末 1365 イオウ・サリチル酸・チアントール軟膏 ウワウルシ 1262

1779 1780 Index in Japanese JP XV

ウワウルシ流エキス 1371 塩化亜鉛 1244 注射液 1105 酸化亜鉛 1245 果糖 689 エ 塩化インジウム (111In) 注射液 755 果糖注射液 689 塩化カリウム 1009 カナマイシン一硫酸塩 793 エーテル 641 塩化カルシウム水和物 390 カナマイシン硫酸塩 794 エイジツ 1346 塩化カルシウム注射液 390 カノコソウ 1306 エイジツ末 1346 塩化タリウム (201Tl) 注射液 1158 カノコソウ末 1307 液状フェノール 986 塩化ナトリウム 1097 カフェイン水和物 387 エコチオパートヨウ化物 610 10 塩化ナトリウム注射液 1098 カプセル 403 エスタゾラム 631 エンゴサク 1279 カプトプリル 403 エストラジオール安息香酸エステル 塩酸 725 過マンガン酸カリウム 1013 631 塩酸キニーネ 1052 加味逍遙散エキス 1309 エストラジオール安息香酸エステル水 塩酸ドパミン注射液 602 カモスタットメシル酸塩 400 性懸濁注射液 633 塩酸モルヒネ注射液 901 カリジノゲナーゼ 790 エストラジオール安息香酸エステル注 塩酸リモナーデ 727 カリ石ケン 1007 射液 632 エンビオマイシン硫酸塩 616 カルテオロール塩酸塩 412 エストリオール 633 エンフルラン 615 カルナウバロウ 411 エストリオール錠 635 カルバゾクロムスルホン酸ナトリウム エストリオール水性懸濁注射液 634 オ 水和物 405 エタクリン酸 636 カルバマゼピン 404 エタクリン酸錠 636 オウギ 1260 カルビドパ水和物 406 エタノール 638 オウゴン 1358 L-カルボシステイン 407 エタンブトール塩酸塩 637 オウゴン末 1359 カルメロース 408 エチオナミド 643 黄色ワセリン 982 カルメロースカルシウム 409 エチゾラム 652 オウセイ 1336 カルメロースナトリウム 410 エチドロン酸二ナトリウム 649 オウバク 1330 カルモナムナトリウム 412 エチドロン酸二ナトリウム錠 650 オウバク・タンナルビン・ビスマス散 カルモフール 411 エチニルエストラジオール 642 1332 カロコン 1369 エチニルエストラジオール錠 642 オウバク末 1331 カンキョウ 1341 L-エチルシステイン塩酸塩 645 オウレン 1277 乾燥はぶウマ抗毒素 716 エチルモルヒネ塩酸塩水和物 648 オウレン末 1278 乾燥まむしウマ抗毒素 841 エチレフリン塩酸塩 650 オキサゾラム 948 乾燥ジフテリアウマ抗毒素 596 エチレフリン塩酸塩錠 651 オキサピウムヨウ化物 947 乾燥ボツリヌスウマ抗毒素 374 エチレンジアミン 648 オキサプロジン 948 乾燥亜硫酸ナトリウム 1109 エテンザミド 640 オキシコドン塩酸塩水和物 951 乾燥甲状腺 1171 エデト酸ナトリウム水和物 598 オキシテトラサイクリン塩酸塩 956 乾燥酵母 1241 エトスクシミド 644 オキシトシン 958 乾燥細胞培養痘そうワクチン 1091 エトドラク 653 オキシトシン注射液 960 乾燥弱毒生おたふくかぜワクチン エトポシド 653 オキシドール 954 902 エドロホニウム塩化物 611 オキシブプロカイン塩酸塩 951 乾燥弱毒生風しんワクチン 1074 エドロホニウム塩化物注射液 612 オキシメトロン 956 乾燥弱毒生麻しんワクチン 843 エノキサシン水和物 616 オキセサゼイン 949 乾燥水酸化アルミニウムゲル 288 エピリゾール 621 オクスプレノロール塩酸塩 950 乾燥水酸化アルミニウムゲル細粒 エピルビシン塩酸塩 622 オフロキサシン 938 289 エフェドリン塩酸塩 619 オリブ油 939 乾燥組織培養不活化狂犬病ワクチン エフェドリン塩酸塩錠 621 オルシプレナリン硫酸塩 946 1054 エフェドリン塩酸塩注射液 619 オレンジ油 946 乾燥炭酸ナトリウム 1096 エフェドリン塩酸塩 10 620 オンジ 1335 乾燥痘そうワクチン 1091 エペリゾン塩酸塩 618 オンジ末 1335 乾燥日本脳炎ワクチン 786 エリスロマイシン 627 乾燥破傷風ウマ抗毒素 1156 エリスロマイシンエチルコハク酸エス カ 乾燥硫酸アルミニウムカリウム 292 テル 629 乾燥 BCG ワクチン 335 エリスロマイシンステアリン酸塩 カイニン酸・サントニン散 790 カンゾウ 1295 630 カイニン酸水和物 789 カンゾウエキス 1296 エリスロマイシンラクトビオン酸塩 カオリン 795 カンゾウ粗エキス 1297 629 カカオ脂 386 カンゾウ末 1296 エルカトニン 612 加香ヒマシ油 415 カンテン 1252 エルゴカルシフェロール 624 カゴソウ 1341 カンテン末 1253 エルゴタミン酒石酸塩 627 カシュウ 1336 dl-カンフル 402 エルゴメトリンマレイン酸塩 625 加水ラノリン 807 d-カンフル 401 エルゴメトリンマレイン酸塩錠 626 カッコン 1341 肝油 538 エルゴメトリンマレイン酸塩注射液 葛根湯エキス 1308 カンレノ酸カリウム 1008 625 過テクネチウム酸ナトリウム (99mTc) ガジュツ 1372 JP XV Index in Japanese 1781

ガスえそウマ抗毒素 696 クロトリマゾール 532 ケイ酸マグネシウム 836 b-ガラクトシダーゼ（アスペルギルス） クロナゼパム 528 軽質無水ケイ酸 1087 694 クロニジン塩酸塩 529 軽質流動パラフィン 967 b-ガラクトシダーゼ（ペニシリウム） クロフィブラート 525 ケイヒ 1272 695 クロフィブラートカプセル 526 ケイヒ末 1273 含糖ペプシン 1074 クロフェダノール塩酸塩 524 ケイヒ油 1273 クロペラスチン塩酸塩 530 ケタミン塩酸塩 795 キ クロミフェンクエン酸塩 526 結晶セルロース 477 クロミフェンクエン酸塩錠 527 結晶性インスリン亜鉛水性懸濁注射液 希塩酸 726 クロミプラミン塩酸塩 528 765 キキョウ 1334 クロム酸ナトリウム (51Cr) 注射液 血清性性腺刺激ホルモン 710 キキョウ末 1335 1099 ケツメイシ 1271 キキョウ流エキス 1334 クロモグリク酸ナトリウム 1100 ケトチフェンフマル酸塩 797 キクカ 1271 クロラムフェニコール 487 ケトプロフェン 796 キササゲ 1271 クロラムフェニコールコハク酸エステ ケノデオキシコール酸 486 キシリトール 1240 ルナトリウム 489 ケンゴシ 1330 キシリトール注射液 1241 クロラムフェニコールパルミチン酸エ 滅菌精製水 1236 キジツ 1302 ステル 488 ゲンタマイシン硫酸塩 698 キタサマイシン 798 クロルジアゼポキシド 490 ゲンチアナ 1290 キタサマイシン酒石酸塩 800 クロルジアゼポキシド散 491 ゲンチアナ・重曹散 1290 キタサマイシン酢酸エステル 799 クロルジアゼポキシド錠 492 ゲンチアナ末 1290 キニーネエチル炭酸 1051 クロルフェニラミン・カルシウム散 ゲンノショウコ 1291 キニーネ硫酸塩水和物 1053 497 ゲンノショウコ末 1291 キニジン硫酸塩水和物 1050 クロルフェニラミンマレイン酸塩 吸水軟膏 265 498 コ キョウカツ 1321 クロルフェニラミンマレイン酸塩散 キョウニン 1257 499 コウカ 1348 キョウニン水 1257 クロルフェニラミンマレイン酸塩錠 硬化油 735 希ヨードチンキ 769 500 コウジン 1342 金チオリンゴ酸ナトリウム 1092 クロルフェニラミンマレイン酸塩注射 コウブシ 1280 牛脂 336 液 499 コウブシ末 1280 クロルフェネシンカルバミン酸エステ コウボク 1315 ク ル 496 コウボク末 1316 d-クロルフェニラミンマレイン酸塩 コカイン塩酸塩 535 クエン酸ガリウム (67Ga) 注射液 696 501 コムギデンプン 1237 クエン酸ナトリウム 1099 クロルプロパミド 504 コメデンプン 1346 クエン酸水和物 515 クロルプロパミド錠 505 コリスチンメタンスルホン酸ナトリウ クコシ 1315 クロルプロマジン塩酸塩 502 ム 540 クジン 1364 クロルプロマジン塩酸塩錠 503 コリスチン硫酸塩 541 クジン末 1364 クロルプロマジン塩酸塩注射液 503 コルチゾン酢酸エステル 543 苦味重曹水 1364 クロルヘキシジングルコン酸塩液 コルヒチン 538 苦味チンキ 1266 493 コレカルシフェロール 506 クラブラン酸カリウム 1010 クロルヘキシジン塩酸塩 493 コレステロール 507 クラリスロマイシン 516 クロルマジノン酢酸エステル 494 コレラワクチン 506 クラリスロマイシン錠 517 クロロブタノール 495 コロンボ 1267 クリノフィブラート 522 グアイフェネシン 714 コロンボ末 1268 クリンダマイシンリン酸エステル グアナベンズ酢酸塩 715 コンズランゴ 1276 521 グアネチジン硫酸塩 716 コンズランゴ流エキス 1277 クリンダマイシン塩酸塩 519 グアヤコールスルホン酸カリウム 合成ケイ酸アルミニウム 290 クリンダマイシン塩酸塩カプセル 1011 ゴオウ 1325 520 グラミシジン 712 ゴシツ 1252 クレオソート 544 グリシン 704 ゴシュユ 1285 クレゾール 544 グリセオフルビン 713 705 クレゾール水 545 グリセリン 702 ゴボウシ 1267 クレゾール石ケン液 545 グリセリンカリ液 704 ゴマ油 1084 クレマスチンフマル酸塩 519 グリベンクラミド 699 ゴミシ 1352 クロカプラミン塩酸塩水和物 523 グルコン酸カルシウム水和物 391 クロキサシリンナトリウム水和物 グルタチオン 701 サ 533 クロキサゾラム 534 ケ サイクロセリン 551 クロコナゾール塩酸塩 546 サイコ 1266 クロスカルメロースナトリウム 546 ケイガイ 1353 サイシン 1259 クロチアゼパム 531 経口生ポリオワクチン 1005 柴苓湯エキス 1349 1782 Index in Japanese JP XV

酢酸 268 シュクシャ 1256 ジフェンヒドラミン・バレリル尿素散 酢酸ナトリウム水和物 1091 シュクシャ末 1256 593 サッカリン 1075 酒石酸 1141 ジフェンヒドラミン・フェノール・亜鉛 サッカリンナトリウム水和物 1076 ショウキョウ 1291 華リニメント 595 サフラン 1349 ショウキョウ末 1292 ジフェンヒドラミン塩酸塩 594 サラシ粉 494 硝酸イソソルビド 785 ジフテリアトキソイド 596 サラシミツロウ 336 硝酸イソソルビド錠 786 ジフテリア破傷風混合トキソイド サラゾスルファピリジン 1077 硝酸銀 1088 596 サリチル・ミョウバン散 1078 硝酸銀点眼液 1088 ジブカイン塩酸塩 569 サリチル酸 1079 ショウズク 1271 ジベカシン硫酸塩 569 サリチル酸ナトリウム 1108 消毒用エタノール 640 次没食子酸ビスマス 368 サリチル酸メチル 881 消毒用フェノール 986 ジメモルファンリン酸塩 588 サリチル酸精 1080 消毒用フェノール水 987 ジメルカプロール 590 サリチル酸絆創膏 1080 ショウマ 1272 ジメルカプロール注射液 590 サルブタモール硫酸塩 1078 シラスタチンナトリウム 510 ジメンヒドリナート 588 酸化カルシウム 393 シロスタゾール 511 ジメンヒドリナート錠 589 酸化チタン 1178 シロスタゾール錠 512 ジモルホラミン 591 酸化マグネシウム 835 シロップ用ファロペネムナトリウム ジモルホラミン注射液 591 サンキライ 1364 659 弱アヘンアルカロイド・スコポラミン サンキライ末 1364 シンイ 1317 注射液 945 三酸化ヒ素 316 親水ワセリン 981 ジャショウシ 1275 サンシシ 1288 親水軟膏 735 ジュウヤク 1302 サンシシ末 1288 診断用クエン酸ナトリウム液 1100 常水 1235 サンシュユ 1279 ジアスターゼ 567 ジョサマイシン 786 サンショウ 1371 ジアスターゼ・重曹散 567 ジョサマイシンプロピオン酸エステル サンショウ末 1371 ジアゼパム 567 788 酸素 955 ジエチルカルバマジンクエン酸塩 ジラゼプ塩酸塩水和物 586 サンソウニン 1307 574 ジルチアゼム塩酸塩 586 サントニン 1081 ジエチルカルバマジンクエン酸塩錠 人全血液 1238 サンヤク 1282 574 サンヤク末 1282 ジオウ 1343 ス ザルトプロフェン 1242 ジギトキシン 576 ザルトプロフェン錠 1243 ジギトキシン錠 576 水酸化カリウム 1012 ジクロキサシリンナトリウム水和物 水酸化カルシウム 392 シ 573 水酸化ナトリウム 1101 ジクロフェナクナトリウム 570 スキサメトニウム塩化物水和物 1137 シアナミド 548 ジクロフェナミド 571 スキサメトニウム塩化物注射液 1138 シアノコバラミン 548 ジクロフェナミド錠 572 スクラルファート水和物 1118 シアノコバラミン注射液 549 ジコッピ 1315 スコポラミン臭化水素酸塩水和物 歯科用アンチホルミン 310 ジゴキシン 577 1083 歯科用トリオジンクパスタ 1210 ジゴキシン錠 580 ステアリルアルコール 1117 歯科用パラホルムパスタ 969 ジゴキシン注射液 578 ステアリン酸 1116 歯科用フェノール・カンフル 987 次硝酸ビスマス 369 ステアリン酸カルシウム 400 歯科用ヨード・グリセリン 771 ジスチグミン臭化物 599 ステアリン酸ポリオキシル 40 1007 シクラシリン 507 ジスチグミン臭化物錠 600 ステアリン酸マグネシウム 837 シクロスポリン 509 ジスルフィラム 600 ストレプトマイシン硫酸塩 1117 シクロペントラート塩酸塩 549 ジソピラミド 598 スピラマイシン酢酸エステル 1114 シクロホスファミド水和物 550 ジドロゲステロン 609 スピロノラクトン 1116 シコン 1313 ジドロゲステロン錠 610 スペクチノマイシン塩酸塩水和物 シゴカ 1283 ジノスタチン スチマラマー 1247 1114 シスプラチン 513 ジノプロスト 592 スルタミシリントシル酸塩水和物 シソマイシン硫酸塩 1090 ジヒドロエルゴタミンメシル酸塩 1134 シタラビン 552 583 スルチアム 1136 シッカニン 1086 ジヒドロエルゴトキシンメシル酸塩 スルバクタムナトリウム 1122 シツリシ 1369 584 スルピリド 1131 シプロヘプタジン塩酸塩水和物 551 ジヒドロコデインリン酸塩 581 スルピリドカプセル 1132 シメチジン 513 ジヒドロコデインリン酸塩散 10 スルピリド錠 1132 シャクヤク 1328 582 スルピリン水和物 1133 シャクヤク末 1329 ジヒドロコデインリン酸塩散 1 スルピリン注射液 1134 シャゼンシ 1334 582 スルファジアジン銀 1124 シャゼンソウ 1334 ジピリダモール 597 スルファメチゾール 1125 臭化カリウム 1008 ジフェニドール塩酸塩 575 スルファメトキサゾール 1126 臭化ナトリウム 1095 ジフェンヒドラミン 593 スルファモノメトキシン水和物 1126 JP XV Index in Japanese 1783

スルフィンピラゾン 1127 セフテラムピボキシル 468 大黄甘草湯エキス 1280 スルフィンピラゾン錠 1128 セフテラム ピボキシル細粒 469 ダイズ油 1113 スルフイソキサゾール 1128 セフトリアキソンナトリウム水和物 ダウノルビシン塩酸塩 553 スルベニシリンナトリウム 1123 472 ダントロレンナトリウム水和物 553 スルホブロモフタレインナトリウム セフピラミドナトリウム 458 1129 セフピロム硫酸塩 460 チ スルホブロモフタレインナトリウム注 セフブペラゾンナトリウム 429 射液 1130 セフポドキシムプロキセチル 461 チアマゾール 1159 セフミノクスナトリウム水和物 446 チアマゾール錠 1160 セ セフメタゾールナトリウム 445 チアミラールナトリウム 1163 セフメノキシム塩酸塩 443 チアミン塩化物塩酸塩 1160 成人用沈降ジフテリアトキソイド セフロキサジン水和物 462 チアミン塩化物塩酸塩散 1162 596 セフロキシムアキセチル 474 チアミン塩化物塩酸塩注射液 1161 精製セラック 1085 セフロキシムナトリウム 476 チアミン硝化物 1162 精製ゼラチン 697 セラセフェート 480 チアラミド塩酸塩 1171 精製デヒドロコール酸 556 セラペプターゼ 1083 チアラミド塩酸塩錠 1172 精製ラノリン 808 セルモロイキン（遺伝子組換え） 481 チアントール 1165 精製水 1236 センキュウ 1275 チオテパ 1168 精製白糖 1120 センキュウ末 1276 チオペンタールナトリウム 1166 生理食塩液 1098 センコツ 1321 チオリダジン塩酸塩 1168 石油ベンジン 982 センソ 1368 チオ硫酸ナトリウム水和物 1109 セタノール 484 センナ 1361 チオ硫酸ナトリウム注射液 1110 セッコウ 1297 センナ末 1362 チクセツニンジン 1326 セトラキサート塩酸塩 485 センブリ 1365 チクセツニンジン末 1327 セネガ 1360 センブリ・重曹散 1367 チクロピジン塩酸塩 1173 セネガシロップ 1361 センブリ末 1366 チザニジン塩酸塩 1179 セネガ末 1361 ゼラチン 696 窒素 928 セファクロル 415 チニダゾール 1175 セファクロルカプセル 416 ソ チペピジンヒベンズ酸塩 1176 セファクロル細粒 417 チペピジンヒベンズ酸塩錠 1177 セファクロル複合顆粒 419 ソウジュツ 1261 チメピジウム臭化物水和物 1174 セファゾリンナトリウム 426 ソウジュツ末 1261 チモ 1256 セファゾリンナトリウム水和物 428 ソウハクヒ 1320 チモール 1170 セファトリジンプロピレングリコール ソボク 1352 チモロールマレイン酸塩 1174 425 ソヨウ 1329 注射用アセチルコリン塩化物 271 セファドロキシル 421 ソルビタンセスキオレイン酸エステル 注射用アムホテリシン B304 セファピリンナトリウム 424 1111 注射用アモバルビタールナトリウム セファレキシン 422 D-ソルビトール 1112 300 セファロチンナトリウム 423 D-ソルビトール液 1113 注射用イダルビシン塩酸塩 746 セフィキシム 442 注射用イミペネム･シラスタチンナト セフェピム塩酸塩水和物 439 タ リウム 750 セフォジジムナトリウム 447 注射用スキサメトニウム塩化物 1137 セフォゾプラン塩酸塩 457 タイソウ 1307 注射用セフェピム塩酸塩 441 セフォタキシムナトリウム 449 タウリン 1142 注射用セフォゾプラン塩酸塩 458 セフォチアムヘキセチル塩酸塩 453 タクシャ 1253 注射用セフォチアム塩酸塩 456 セフォチアム塩酸塩 455 タクシャ末 1253 注射用チアミラールナトリウム 1164 セフォテタン 451 タムスロシン塩酸塩 1140 注射用チオペンタールナトリウム セフォペラゾンナトリウム 448 タランピシリン塩酸塩 1138 1167 セフカペン ピボキシル塩酸塩細粒 タルク 1139 注射用テセロイキン（遺伝子組換え） 432 炭酸カリウム 1009 1148 セフカペン ピボキシル塩酸塩錠 433 炭酸ナトリウム水和物 1096 注射用バンコマイシン塩酸塩 1223 セフカペン ピボキシル塩酸塩水和物 炭酸マグネシウム 834 注射用ヒト絨毛性性腺刺激ホルモン 430 炭酸リチウム 826 708 セフジトレン ピボキシル 437 炭酸水素ナトリウム 1093 注射用ヒドララジン塩酸塩 724 セフジトレン ピボキシル細粒 438 炭酸水素ナトリウム注射液 1094 注射用ピペラシリンナトリウム 998 セフジトレン ピボキシル錠 438 単シロップ 1089 注射用ファモチジン 656 セフジニル 434 単軟膏 1089 注射用フェニトインナトリウム 992 セフジニルカプセル 435 タンニン酸 1141 注射用フロモキセフナトリウム 666 セフジニル細粒 436 タンニン酸アルブミン 282 注射用プレドニゾロンナトリウムコハ セフスロジンナトリウム 464 タンニン酸ジフェンヒドラミン 595 ク酸エステル 1025 セフタジジム水和物 466 タンニン酸ベルベリン 352 注射用ホスホマイシンナトリウム セフチゾキシムナトリウム 471 ダイオウ 1344 687 セフチブテン水和物 470 ダイオウ末 1345 注射用血清性性腺刺激ホルモン 711 1784 Index in Japanese JP XV

注射用水 1235 デスラノシド 559 トルナフタート 1189 注射用硫酸ビンブラスチン 1227 デスラノシド注射液 560 トルナフタート液 1189 チョウジ 1274 デヒドロコール酸 556 トルブタミド 1188 チョウジ末 1274 デヒドロコール酸注射液 557 トルブタミド錠 1188 チョウジ油 1275 デフェロキサミンメシル酸塩 554 トルペリゾン塩酸塩 1190 チョウトウコウ 1370 デメチルクロルテトラサイクリン塩酸 L-トレオニン 1169 チョレイ 1336 塩 558 トレピブトン 1195 チョレイ末 1337 トロピカミド 1211 チンク油 1245 ト トロンビン 1170 沈降はぶトキソイド 716 豚脂 808 沈降ジフテリア破傷風混合トキソイド トウガシ 1264 ドキサプラム塩酸塩水和物 603 596 トウガラシ 1268 ドキシサイクリン塩酸塩水和物 606 沈降精製百日せきジフテリア破傷風混 トウガラシ・サリチル酸精 1270 ドキシフルリジン 604 合ワクチン 596 トウガラシチンキ 1269 ドキシフルリジンカプセル 604 沈降精製百日せきワクチン 980 トウガラシ末 1269 ドキソルビシン塩酸塩 605 沈降炭酸カルシウム 389 トウキ 1305 ドパミン塩酸塩 602 沈降破傷風トキソイド 1156 トウキ末 1305 ドブタミン塩酸塩 601 沈降 B 型肝炎ワクチン 716 トウニン 1327 ドロペリドール 608 チンピ 1273 トウニン末 1327 トウヒ 1265 ナ ツ トウヒシロップ 1325 トウヒチンキ 1325 ナイスタチン 937 ツバキ油 400 トウモロコシデンプン 542 ナタネ油 1055 ツボクラリン塩化物塩酸塩水和物 トウモロコシ油 542 ナドロール 904 1212 トコフェロール 1181 ナファゾリン・クロルフェニラミン液 ツボクラリン塩化物塩酸塩注射液 トコフェロールコハク酸エステルカル 906 1213 シウム 1183 ナファゾリン塩酸塩 907 ツロブテロール塩酸塩 1213 トコフェロールニコチン酸エステル ナファゾリン硝酸塩 908 1184 ナプロキセン 908 テ トコフェロール酢酸エステル 1182 ナリジクス酸 905 トコン 1303 ナロキソン塩酸塩 906 テイコプラニン 1150 トコンシロップ 1304 低置換度ヒドロキシプロピルセルロー トコン末 1303 ニ ス 738 トチュウ 1284 テオフィリン 1158 トドララジン塩酸塩水和物 1185 ニカルジピン塩酸塩 911 テガフール 1149 トフィソパム 1186 ニカルジピン塩酸塩注射液 912 テストステロンエナント酸エステル トブラマイシン 1180 ニガキ 1333 1154 トラガント 1369 ニガキ末 1333 テストステロンエナント酸エステル注 トラガント末 1369 ニコチン酸 920 射液 1154 トラザミド 1187 ニコチン酸アミド 919 テストステロンプロピオン酸エステル トラネキサム酸 1191 ニコチン酸注射液 921 1155 トラネキサム酸カプセル 1192 ニコモール 917 テストステロンプロピオン酸エステル トラネキサム酸錠 1193 ニコモール錠 918 注射液 1155 トラネキサム酸注射液 1193 ニコランジル 919 テセロイキン（遺伝子組換え） 1143 トラピジル 1194 二酸化炭素 407 テトラカイン塩酸塩 1156 トリアムシノロン 1195 ニセリトロール 916 テトラサイクリン塩酸塩 1157 トリアムシノロンアセトニド 1196 ニセルゴリン 913 テルブタリン硫酸塩 1153 トリアムテレン 1197 ニセルゴリン散 914 テレビン油 1214 トリクロホスナトリウム 1202 ニセルゴリン錠 915 天然ケイ酸アルミニウム 289 トリクロホスナトリウムシロップ ニトラゼパム 925 テンマ 1289 1203 ニトレンジピン 926 テンモンドウ 1259 トリクロルメチアジド 1198 ニトレンジピン錠 927 デキサメタゾン 560 トリクロルメチアジド錠 1199 ニトログリセリン錠 928 デキストラン硫酸エステルナトリウム トリコマイシン 1201 ニフェジピン 922 イオウ 5 564 L-トリプトファン 1211 日本脳炎ワクチン 786 デキストラン硫酸ナトリウム イオウ トリヘキシフェニジル塩酸塩 1204 乳酸 801 18 565 トリヘキシフェニジル塩酸塩錠 1204 乳酸カルシウム水和物 393 デキストラン 40 561 トリメタジオン 1208 乳糖水和物 803 デキストラン 40 注射液 562 トリメタジオン錠 1209 尿素 1218 デキストラン 70 562 トリメタジジン塩酸塩 1207 ニルバジピン 923 デキストリン 565 トリメタジジン塩酸塩錠 1207 ニルバジピン錠 924 デキストロメトルファン臭化水素酸塩 トリメトキノール塩酸塩水和物 1209 ニンジン 1292 水和物 566 トリメブチンマレイン酸塩 1206 ニンジン末 1293 JP XV Index in Japanese 1785

ニンドウ 1314 パラオキシ安息香酸プロピル 1039 ピラジナミド 1046 パラオキシ安息香酸メチル 878 ピラルビシン 1000 ネ パラフィン 966 ピランテルパモ酸塩 1045 パラホルムアルデヒド 968 ピリドキシン塩酸塩 1047 ネオスチグミンメチル硫酸塩 909 パルナパリンナトリウム 969 ピリドキシン塩酸塩注射液 1048 ネオスチグミンメチル硫酸塩注射液 パンクレアチン 961 ピリドスチグミン臭化物 1047 910 パンクロニウム臭化物 962 ピレノキシン 1001 ネチルマイシン硫酸塩 910 パンテチン 964 ピレンゼピン塩酸塩水和物 1002 パントテン酸カルシウム 394 ピロールニトリン 1049 ノ ピロ亜硫酸ナトリウム 1108 ヒ ピロカルピン塩酸塩 994 濃グリセリン 703 ピロキシカム 1003 濃ベンザルコニウム塩化物液 50 343 ヒトインスリン（遺伝子組換え） 760 ピロキシリン 1049 ノスカピン 936 ヒト下垂体性性腺刺激ホルモン 708 ピンドロール 995 ノスカピン塩酸塩水和物 937 ヒト絨毛性性腺刺激ホルモン 707 ノルアドレナリン 930 人免疫グロブリン 724 フ ノルアドレナリン注射液 931 ヒドララジン塩酸塩 724 ノルエチステロン 932 ヒドララジン塩酸塩散 725 ファモチジン 655 ノルゲストレル 933 ヒドララジン塩酸塩錠 725 ファモチジン散 657 ノルゲストレル・エチニルエストラジ ヒドロキシジンパモ酸塩 739 ファモチジン錠 657 オール錠 934 ヒドロキシジン塩酸塩 739 ファロペネムナトリウム錠 660 ノルトリプチリン塩酸塩 935 ヒドロキシプロピルセルロース 736 ファロペネムナトリウム水和物 658 ノルフロキサシン 932 ヒドロキソコバラミン酢酸塩 736 フィトナジオン 993 ヒドロクロロチアジド 727 フェニトイン 991 ハ ヒドロコタルニン塩酸塩水和物 734 フェニトイン散 992 ヒドロコルチゾン 728 フェニトイン錠 992 白色セラック 1085 ヒドロコルチゾン・ジフェンヒドラミ フェニルブタゾン 990 白色ワセリン 981 ン軟膏 730 L-フェニルアラニン 989 白色軟膏 1238 ヒドロコルチゾンコハク酸エステル フェニレフリン塩酸塩 990 白糖 1121 733 フェネチシリンカリウム 983 ハチミツ 1301 ヒドロコルチゾンコハク酸エステルナ フェノール 985 ハッカ 1317 トリウム 732 フェノール・亜鉛華リニメント 987 ハッカ水 1318 ヒドロコルチゾンリン酸エステルナト フェノールスルホンフタレイン 988 ハッカ油 1318 リウム 731 フェノールスルホンフタレイン注射液 ハマボウフウ 1294 ヒドロコルチゾン酢酸エステル 729 988 ハロキサゾラム 719 ヒドロコルチゾン酪酸エステル 730 フェノール水 987 ハロタン 719 ヒプロメロース 741 フェノバルビタール 984 ハロペリドール 717 ヒプロメロースフタル酸エステル フェノバルビタール散 10 985 ハロペリドール錠 718 743 フェンタニルクエン酸塩 661 ハンゲ 1333 ヒマシ油 414 フェンブフェン 660 バイモ 1287 ヒメクロモン 740 複方アクリノール・チンク油 273 バカンピシリン塩酸塩 329 氷酢酸 269 複方オキシコドン・アトロピン注射液 バクモンドウ 1324 ビサコジル 367 953 バクロフェン 331 ビサコジル坐剤 368 複方オキシコドン注射液 952 バクロフェン錠 332 ビタミン A 油 1229 複方サリチル酸メチル精 879 バシトラシン 330 ビタミン A 油カプセル 1230 複方サリチル酸精 1080 バソプレシン注射液 1223 ビペリデン塩酸塩 366 複方ジアスターゼ・重曹散 567 バメタン硫酸塩 333 ビホナゾール 366 複方ダイオウ・センナ散 1346 L-バリン 1221 ビャクシ 1257 複方チアントール・サリチル酸液 バルビタール 333 ビャクジュツ 1260 1165 バルプロ酸ナトリウム 1110 ビャクジュツ末 1260 複方ビタミン B 散 1230 バレイショデンプン 1014 ビワヨウ 1314 複方ヨード・グリセリン 770 バンコマイシン塩酸塩 1221 ビンクリスチン硫酸塩 1228 複方ロートエキス・ジアスターゼ散 パップ用複方オウバク散 1332 ビンブラスチン硫酸塩 1226 1355 パニペネム 962 ビンロウジ 1258 フシジン酸ナトリウム 1101 パパベリン塩酸塩 965 ピコスルファートナトリウム水和物 フラジオマイシン硫酸塩 688 パパベリン塩酸塩注射液 965 1105 フラビンアデニンジヌクレオチドナト パラアミノサリチル酸カルシウム水和 ピブメシリナム塩酸塩 1004 リウム 662 物 395 ピペミド酸水和物 996 フラボキサート塩酸塩 664 パラアミノサリチル酸カルシウム顆粒 ピペラシリンナトリウム 997 フルオキシメステロン 675 394 ピペラジンリン酸塩錠 1000 フルオシノニド 672 パラオキシ安息香酸エチル 647 ピペラジンリン酸塩水和物 999 フルオシノロンアセトニド 670 パラオキシ安息香酸ブチル 386 ピマリシン 994 フルオレセインナトリウム 673 1786 Index in Japanese JP XV

フルオロウラシル 674 プロクロルペラジンマレイン酸塩 ベンセラジド塩酸塩 340 フルオロメトロン 673 1033 ベンゼトニウム塩化物 344 フルシトシン 668 プロクロルペラジンマレイン酸塩錠 ベンゼトニウム塩化物液 345 フルジアゼパム 669 1033 ベントナイト 341 フルスルチアミン塩酸塩 692 プログルミド 1035 ベルベリン塩化物水和物 351 フルニトラゼパム 670 プロゲステロン 1034 ペチジン塩酸塩 980 フルフェナジンエナント酸エステル プロゲステロン注射液 1034 ペチジン塩酸塩注射液 981 676 プロタミンインスリン亜鉛水性懸濁注 ペプロマイシン硫酸塩 975 フルラゼパム 677 射液 766 ペルフェナジン 977 フルラゼパムカプセル 678 プロタミン硫酸塩 1041 ペルフェナジンマレイン酸塩 978 フルラゼパム塩酸塩 678 プロタミン硫酸塩注射液 1042 ペルフェナジンマレイン酸塩錠 979 フルルビプロフェン 679 プロチオナミド 1042 ペルフェナジン錠 977 フロセミド 690 プロチレリン 1043 ペンタゾシン 972 フロセミド錠 691 プロチレリン酒石酸塩水和物 1044 ペントキシベリンクエン酸塩 974 フロプロピオン 667 プロテイン銀 1088 ペントバルビタールカルシウム 973 フロプロピオンカプセル 667 プロテイン銀液 1089 ペンブトロール硫酸塩 972 フロモキセフナトリウム 664 プロパンテリン臭化 1036 粉末セルロース 480 プロピルチオウラシル 1040 ホ ブクモロール塩酸塩 378 プロピルチオウラシル錠 1040 ブクリョウ 1337 プロピレングリコール 1039 ホウ酸 374 ブクリョウ末 1337 プロプラノロール塩酸塩 1037 ホウ砂 1095 ブシ 1338 プロプラノロール塩酸塩錠 1038 抱水クロラール 487 ブシ末 1339 プロベネシド 1027 ホスフェストロール 683 ブシラミン 377 プロベネシド錠 1027 ホスフェストロール錠 684 ブスルファン 384 プロメタジン塩酸塩 1036 ホスホマイシンカルシウム水和物 ブチルスコポラミン臭化物 1082 685 ブトロピウム臭化物 385 ヘ ホスホマイシンナトリウム 686 ブドウ酒 1238 補中益気湯エキス 1298 ブドウ糖 700 ヘパリンナトリウム 721 ホマトロピン臭化水素酸塩 722 ブドウ糖注射液 701 ヘパリンナトリウム注射液 722 ホミカ 1321 ブナゾシン塩酸塩 383 ヘンズ 1282 ホミカエキス 1322 ブフェキサマク 380 ベカナマイシン硫酸塩 337 ホミカエキス散 1323 ブフェキサマククリーム 380 ベクロメタゾンプロピオン酸エステル ホミカチンキ 1323 ブフェキサマク軟膏 381 335 ホモクロルシクリジン塩酸塩 723 ブフェトロール塩酸塩 379 ベザフィブラート 364 ホリナートカルシウム 391 ブプラノロール塩酸塩 383 ベザフィブラート徐放錠 365 ホルマリン 681 ブメタニド 382 ベタネコール塩化物 363 ホルマリン水 682 ブレオマイシン塩酸塩 370 ベタヒスチンメシル酸塩 353 ホルモテロールフマル酸塩水和物 ブレオマイシン硫酸塩 372 ベタヒスチンメシル酸塩錠 354 682 ブロマゼパム 374 ベタメタゾン 355 ボウイ 1363 ブロムヘキシン塩酸塩 375 ベタメタゾンジプロピオン酸エステル ボウコン 1302 ブロモクリプチンメシル酸塩 376 358 ボウフウ 1352 ブロモバレリル尿素 377 ベタメタゾンリン酸エステルナトリウ ボグリボース 1231 プラステロン硫酸エステルナトリウム ム 359 ボグリボース錠 1232 水和物 1107 ベタメタゾン吉草酸エステル 360 ボタンピ 1318 プラゼパム 1020 ベタメタゾン吉草酸エステル･ゲンタ ボタンピ末 1319 プラゼパム錠 1021 マイシン硫酸塩クリーム 361 ボレイ 1326 プラノプロフェン 1018 ベタメタゾン吉草酸エステル･ゲンタ ボレイ末 1326 プラバスタチンナトリウム 1018 マイシン硫酸塩軟膏 362 ポビドン 1015 プリミドン 1026 ベタメタゾン錠 356 ポビドンヨード 1017 プルラン 1045 ベニジピン塩酸塩 338 ポリスチレンスルホン酸カルシウム プレドニゾロン 1021 ベニジピン塩酸塩錠 339 398 プレドニゾロンコハク酸エステル ベラドンナエキス 1263 ポリスチレンスルホン酸ナトリウム 1024 ベラドンナコン 1263 1106 プレドニゾロン錠 1022 ベラパミル塩酸塩 1225 ポリソルベート 80 1007 プレドニゾロン酢酸エステル 1023 ベラパミル塩酸塩錠 1226 ポリミキシン B 硫酸塩 1006 プロカインアミド塩酸塩 1028 ベンザルコニウム塩化物 342 プロカインアミド塩酸塩錠 1029 ベンザルコニウム塩化物液 342 マ プロカインアミド塩酸注射液 1029 ベンジルアルコール 346 プロカイン塩酸塩 1030 ベンジルペニシリンカリウム 349 マーキュロクロム 862 プロカイン塩酸塩注射液 1030 ベンジルペニシリンベンザチン水和物 マーキュロクロム液 863 プロカテロール塩酸水和物 1032 348 マイトマイシン C898 プロカルバジン塩酸塩 1031 ベンズブロマロン 343 マオウ 1283 JP XV Index in Japanese 1787

マクリ 1280 メチルドパ錠 874 マクロゴール軟膏 833 メチルドパ水和物 873 ヨ マクロゴール 1500 831 メチルプレドニゾロン 879 マクロゴール 20000 833 メチルプレドニゾロンコハク酸エステ ヨーダミド 767 マクロゴール 400 830 ル 880 ヨーダミドナトリウムメグルミン注射 マクロゴール 4000 832 メチルベナクチジウム臭化物 871 液 854 マクロゴール 6000 832 メチルロザニリン塩化物 881 ヨード・サリチル酸・フェノール精 マシニン 1298 メテノロンエナント酸エステル 866 772 麻酔用エーテル 641 メテノロンエナント酸エステル注射液 ヨードチンキ 769 マプロチリン塩酸塩 842 866 ヨードホルム 773 マルトース水和物 840 メテノロン酢酸エステル 865 ヨウ化カリウム 1013 D-マンニトール 841 メトキサレン 870 ヨウ化人血清アルブミン (131I) 注射液 D-マンニトール注射液 842 メトクロプラミド 886 768 メトクロプラミド錠 886 ヨウ化ナトリウム 1102 ミ メトトレキサート 869 ヨウ化ナトリウム (123I) カプセル メトプロロール酒石酸塩 887 1103 ミクロノマイシン硫酸塩 893 メトプロロール酒石酸塩錠 888 ヨウ化ナトリウム (131I) 液 1103 ミグレニン 896 メトホルミン塩酸塩 867 ヨウ化ナトリウム (131I) カプセル ミコナゾール 892 メトホルミン塩酸塩錠 867 1103 ミコナゾール硝酸塩 893 メトロニダゾール 889 ヨウ化ヒプル酸ナトリウム (131I) 注射 ミツロウ 336 メトロニダゾール錠 889 液 1103 ミデカマイシン 894 メナテトレノン 855 葉酸 680 ミデカマイシン酢酸エステル 895 メピチオスタン 858 葉酸錠 681 ミノサイクリン塩酸塩 897 メピバカイン塩酸塩 859 葉酸注射液 681 ミョウバン水 293 メピバカイン塩酸塩注射液 860 ヨウ素 768 メフェナム酸 847 ヨクイニン 1276 ム メフルシド 849 ヨクイニン末 1276 メフルシド錠 849 無晶性インスリン亜鉛水性懸濁注射液 メフロキン塩酸塩 848 ラ 764 メペンゾラート臭化物 857 無水アンピシリン 306 メルカプトプリン水和物 861 ラウリル硫酸ナトリウム 1104 無水エタノール 639 メルファラン 855 ラウロマクロゴール 810 無水カフェイン 386 メロペネム 水和物 863 酪酸リボフラビン 1060 無水クエン酸 514 dl-メントール 856 ラクツロース 804 無水乳糖 802 l-メントール 857 ラタモキセフナトリウム 809 無水リン酸水素カルシウム 396 ラッカセイ油 971 ムピロシンカルシウム 水和物 902 モ ラナトシド C 805 ラナトシド C 錠 806 メ モクツウ 1253 ラニチジン塩酸塩 1054 モッコウ 1352 メキシレチン塩酸塩 891 モノステアリン酸アルミニウム 291 リ メキタジン 861 モノステアリン酸グリセリン 704 メクロフェノキサート塩酸塩 843 モルヒネ・アトロピン注射液 899 リオチロニンナトリウム 821 メグルミン 850 モルヒネ塩酸塩錠 902 リオチロニンナトリウム錠 822 メコバラミン 844 モルヒネ塩酸塩水和物 900 リシノプリル錠 824 メシル酸塩ガベキサート 693 リシノプリル水和物 823 メストラノール 864 ヤ L-リジン塩酸塩 829 メタンフェタミン塩酸塩 868 リゾチーム塩酸塩 830 メダゼパム 845 焼セッコウ 1298 リトドリン塩酸塩 1067 L-メチオニン 869 ヤクチ 1265 リトドリン塩酸塩錠 1068 メチクラン 884 薬用石ケン 846 リドカイン 818 メチラポン 890 薬用炭 845 リドカイン注射液 818 メチルエルゴメトリンマレイン酸塩 ヤシ油 535 リファンピシン 1064 876 ユーカリ油 654 リファンピシンカプセル 1065 メチルエルゴメトリンマレイン酸塩錠 リボスタマイシン硫酸塩 1063 877 ユ リボフラビン 1059 dl-メチルエフェドリン塩酸塩 875 リボフラビンリン酸エステルナトリウ dl-メチルエフェドリン塩酸塩散 10 ユウタン 1262 ム 1061 876 輸血用クエン酸ナトリウム注射液 リボフラビンリン酸エステルナトリウ メチルジゴキシン 885 1099 ム注射液 1062 メチルセルロース 871 ユビデカレノン 1214 リボフラビン散 1060 メチルテストステロン 882 リマプロスト アルファデクス 819 メチルテストステロン錠 883 リュウコツ 1313 1788 Index in Japanese JP XV

硫酸アルミニウムカリウム水和物 ロートエキス・アネスタミン散 1355 292 レ ロートエキス・カーボン散 1355 硫酸カリウム 1014 ロートエキス・タンニン坐剤 1357 硫酸バリウム 334 レセルピン 1055 ロートエキス・パパベリン・アネスタミ 硫酸マグネシウム水 839 レセルピン散 0.1 1057 ン散 1356 硫酸マグネシウム水和物 838 レセルピン錠 1057 ロートエキス散 1353 硫酸マグネシウム注射液 839 レセルピン注射液 1056 ロートコン 1357 硫酸亜鉛水和物 1246 レチノールパルミチン酸エステル L-ロイシン 812 硫酸亜鉛点眼液 1247 1059 ロキサチジン酢酸エステル塩酸塩 硫酸鉄水和物 661 レチノール酢酸エステル 1058 1070 リュウタン 1306 レナンピシリン塩酸塩 810 ロキサチジン酢酸エステル塩酸塩徐放 リュウタン末 1306 レバロルファン酒石酸塩 813 カプセル 1071 流動パラフィン 966 レバロルファン酒石酸塩注射液 814 ロキシスロマイシン 1073 リョウキョウ 1256 レボチロキシンナトリウム錠 817 ロキソプロフェンナトリウム水和物 苓桂朮甘湯エキス 1347 レボチロキシンナトリウム水和物 828 リンゲル液 1066 816 ロキタマイシン 1069 リンコマイシン塩酸塩水和物 820 レボドパ 814 ロジン 1347 リン酸コデイン散 10 537 レボメプロマジンマレイン酸塩 815 ロラゼパム 827 リン酸コデイン散 1 536 レンギョウ 1286 リン酸コデイン錠 537 レンニク 1320 ワ リン酸コデイン水和物 536 リン酸水素カルシウム水和物 396 ロ ワイル病秋やみ混合ワクチン 1237 リン酸水素ナトリウム水和物 568 ワルファリンカリウム 1233 リン酸二水素カルシウム水和物 397 ロートエキス 1353 ワルファリンカリウム錠 1234

Top View