Recombinant Human Parathyroid Hormone (Rhpth1-34)

Recombinant Human Parathyroid Hormone (rhPTH134)

General Notices

SVSEIQLMHN LGKHLNSMER VEWLRKKLQD VHNF

Molecular Formula: C181H291N55O51S2

Molecular Weight (Theoretical): 4117.8 Daltons

Action and use

Bone formation agent

DEFINITION

Recombinant human parathyroid hormone (rhPTH134) or teriparatide is a biologically active N- terminal fragment of endogenous (native) human parathyroid hormone produced by recombinant DNA technology in bacteria. The concentrated solution of rhPTH134 is present at a concentration not less than 1 mg / mL in buffer salts.

Content 95.0 per cent to 105.0 per cent ( dried substance )

PRODUCTION

Human Parathyroid Hormone concentrated solution is produced by a method based on recombinant DNA (r-DNA) technology using bacteria as host cells. It is produced under conditions designated to minimize microbial contamination of the product.

Each batch-derived Drug Substance material of rhPTH134 is released after testing by various analytical methods described below 

Version 4 dated 26th Sep 2012 1/14 Recombinant Human Parathyroid Hormone (rhPTH134)

Host cell-derived proteins

Not more than 100 ppm

Host cell-derived DNA

10ng per therapeutic dose

DESCRIPTION

White to practically white solid

IDENTIFICATION

A. It shows the expected Biological activity (see Assay)

B. Liquid Chormatography

Sample diluent: 69:31 Sulfate buffer, 0.27M,pH 2.3:CAN

Standard diluents: 75:25 SUlfate buffer, 0.20M, pH 2.3 : CAN

Adjust the pH of Sulfate buffer using 85% phosphoric acid or 1 N NaOH, if necessary

Test solution Prepare a fivefold dilution of drug product using the sample diluent

Reference solution Prepare reference standard solution with Standard diluent at approximately 0.1mg / mL ( prepare three separate solutions, in case of Potency)

Column

Stationary phase: octadecylsilyl silica gel for chromatography

– Size: l =15cm, Ø = 4.6 mm, 3.5µm

– Temperature: 40 °C

– Mobile phase

Mobile phase A: 90:10 Sulfate buffer,0.20M,pH 2.3 : ACN

Mobile phase B: 50:50 Sulfate buffer, 0.2M, pH 2.3 : CAN

Isocratic condition: 63% Mobile phase A and 37% Mobile phase B

Flow rate 0.8 mL / min.

Injection volume: 25µl

Refreidgerator Autosampler maintained at : 5 ºC

Detection Spectrophotometer at 214 nm.

Run time : 20min

Approximately rh PTH( 1-34) retention time : 8 min

System suitability

The relative standard deviation ( RSD) of the rh PTH ( 1-34) peaks area ratio must be ≤ 1.25%

Results

Integrate the peak attributed to rh-PTH (1-34) in the standard and sample chromatogram to determine the retention time.

Calculate the retention time ratio for identity determination

Retention time ratio = Retention time of the sample rhPTH(1- 34) peak

Retention time of the standard rhPTH(1- 34) peak

Retention time ratio must be 1.00±0.03.

Fig: Typical rh-PTH( 1-34) bulk drug substance potency chromatogram

C. Peptide mapping : Use method 1 or method 2

Method 1

Test solution Prepare 1 mg / mL solution of substance to be examined in 0.05 M Tris- hydrochloride buffer solution of pH 7.0 and transfer 500 µL of it to a clean sample container. Add 10 µL of 1 mg / mL trypsin solution (in Water R) and incubate for 24 h at 37 °C. Stop the reaction by freezing at or below  20 C.

Reference solution Prepare 1 mg / mL solution of internal reference standard in 0.05 M Tris buffer solution of pH 7.0 and transfer 500 µL of it to a clean sample container. Add 10 µL of 1 mg / mL trypsin solution (in Water R) and incubate for 24 hr at 37 °C. Stop the reaction by freezing at or below  20 C.

CHROMATOGRAPHIC SEPARATION

Liquid chromatography

Column

– Stationary phase: octadecylsilyl silica gel for chromatography R (3 µm),

– Size: l = 0.15 m, Ø = 4.6 mm, A = 20 nm.

Mobile phase

– Mobile phase A: 0.05 per cent V / V solution of trifluoroacetic acid R.

– Mobile phase B: To 800 mL of acetonitrile add 0.5 mL of trifluoroacetic acid R and 200 mL of acetonitrile for chromatography.

Time Mobile phase A Mobile phase B (min) (per cent V / V) (per cent V / V) 0  30 95  45 5  55

30  30.01 45 0 55 100

30.01  35 0 100

35  35.01 0  95 100  5

35.01  45 95 5

Flow rate 1 mL / min.

Equilibration At initial conditions for at least 15 min.

Injection 50 µL.

System suitability

The chromatogram obtained with the reference solution should appear with eight prominent peaks as shown below 

Results

The profile of the chromatogram obtained with test solution corresponds to that of the chromatogram obtained with the reference solution.

Method 2

Peptide mapping using S.aureus V-8 Protease-

Sampe preparation: Dissolve rh-PTH( 1-34) in Dilution Buffer to a concentration of 1.5mg/ml. Add 0.090ml of the 0.25mg/ml enzyme solution to 0.150ml of the 1.5mg/ml rh-PTH solution. Mix and incubate at 37ºC for 18-24 hours. Quench the digest by adding 0.660ml of Mobile phase A to reach a final digested protein concentration of 0.25mg/ml

Reference standard preparation: Dissolve rh-PTH( 1-34) reference standard in Dilution Buffer to a concentration of 1.5mg/ml. Add 0.090ml of the 0.25mg/ml enzyme solution to 0.150ml of the 1.5mg/ml rh-PTH solution. Mix and incubate at 37ºC for 18-24 hours. Quench the digest by adding 0.660ml of Mobile phase A to reach a final digested protein concentration of 0.25mg/ml

Blank preparation: Combine 0.150ml of dilution buffer to 0.09ml of enzyme solution. Mix and incubate at 37% for 18-24 hours. Quench the digest by adding 0.660ml of Mobile phase A.

Dilution Buffer: 20mM Sodium Phosphate,pH 7.8. Adjust the pH using Phosphoric acid reagent Grade

Mobile phase A: 0.1% TFA in water

Mobile phase B: 0.1% TFA in 40% water/60% ACN

All buffers are stable for two weeks at ambient temperature and light.

Column

Stationary phase: octadecylsilyl silica gel for chromatography

– Size: l =15cm, Ø = 4.6 mm, 3.5µm

– Flow rate: 1ml/min

– Detection: UV at 214nm

– Injection Volume : 20µl

– Run time: 35 min ( 10 min re equilibration)

– Gradient:

Time Mobile phase A ( %) Mobile phase B ( %)

0 96 4

6 96 4

20 45 55

25 0 100

System suitability

The chromatogram obtained with the reference solution should appear with five prominent peaks as shown.

Results

The profile of the chromatogram obtained with test solution corresponds to that of the chromatogram obtained with the reference solution showing five major fragments.

Fig: Typical Chromatogram of a S.aureus V-* Protease Digest of rh-PTH (1-34)

D. Examine by Capillary Isoelectric Focusing

Ampholyte gel solution pI range 3.0 to 10.0 Prepare and use according to the manufacturer’s instructions.

Test solution Dilute the preparation to be examined with water R, if required to a protein concentration of 1 mg / mL. To the ampholyte gel solution, add 5 µL of sample to be examined. Centrifuge the mixture at 7000g × 2 min.

Reference solution To the ampholyte gel solution add pI marker(s) suitable for calibrating in pI range of 3 – 10 as per manufacturer’s instructions. Centrifuge the mixture at 7000g × 2 min.

Capillary

– Material: Neutral capillary

– Size: Effective length = 20 cm, Ø = 50 µm

Detection Spectrophotometer at 214 nm

Prewashing Rinse the capillary with 10 mM phosphoric buffer for 1 min, with water R for 1 min under pressure / vacuum.

Injection Fill the capillary with reference solution / test solution for 1.5 min under pressure or vacuum.

Prefocusing Apply a voltage of 15 kV for 6 min with 0.17 min ramping time, using anolyte (containing cIEF gel and 91 mM phosphoric acid as per manufacturers’ instructions) and 20 mM NaOH as catholyte in both buffer reservoirs.

Migration Apply a voltage of 21 kV for 64 min at 0.5 psi using above electrolytes.

Rinse with water R for 1 min under pressure / vacuum

Limits Isoelectric point of the test solution should appear between 8.2 and 8.4.

E. Polyacrylamide gel electrophoresis

The test is performed under non-reducing conditions, using 20 percent acrylamide. The gel is developed by silver staining.

Gel dimensions 1.5 mm thick, 10 cm × 10.5 cm.

Sample buffer stock solution 0.2 M Tris-hydrochloride buffer solution pH 6.8 containing 48 % glycerol, 2 % sodium dodecyl sulfate, 0.4 mg / mL coomassie G250.

Test solution Dilute the preparation to be examined in water to obtain a concentration of 0.25 mg / mL. To 38.4 L of this solution add 15 L of sample buffer. Dilute to 60 µL with water R.

Reference solution (a) A solution of protein molecular weight markers suitable for calibrating SDS-polyacrylamide gels.

†Reference solution (b) Dilute the Reference Product in water R to obtain a concentration of 0.25 mg / mL. To 38.4 L of this solution add 15 L of sample buffer. Dilute to 60 µL with Water R. († The innovators’ product FORTEOTM can be used as Reference Product).

Reference solution (c) Dilute the test solution in water R to obtain a concentration of 0.025 mg / mL. To 3.84 L of this solution add 15 L of sample buffer. Dilute to 60 µL with water R.

Sample treatment Boil for 2 min.

Tris-SDS solution Mix 0.3 per cent (final) sodium dodecyl sulfate in 3.0 M Tris- hydrochloride buffer solution of pH 8.45.

Resolving gel In a conical flask, prepare appropriate volume of solution containing final 20 per cent acrylamide for the resolving gel, using the values given in below table. Mix the components in the order shown.

Solution components Component volume (mL) per gel volume of 7.5 mL Water R 1.20

50 % acrylamide solution* 3.00 Tris-SDS solution (pH 8.45) 2.49 Glycerol R 0.79 10 % APSφ 0.025

TEMED† 0.007

* 50 % acrylamide solution: 50 per cent acrylamide/bisacrylamide (50:1) solution R φ 10 % APS: a 100 g / L solution of ammonium persulfate R. Prepare freshly.

†TEMED: tetraethylethylenediamine R

Stacking gel In a conical flask, prepare appropriate volume of solution containing final 4 per cent acrylamide for the stacking gel, using the values given in below table. Mix the components in the order shown.

Solution components Component volume (mL) per gel volume of 5.0 mL Water R 3.05 30 % acrylamide solution* 0.65 Tris SDS solution (pH 8.45) 1.25 10 % APSφ 0.05

TEMED† 0.01

*30 % acrylamide solution: 30 per cent acrylamide / bisacrylamide (29.2:0.8) solution R φ 10 % APS: a 100 g / L solution of ammonium persulfate R. Prepare freshly. †TEMED: tetraethylethylenediamine R

Application 50 L in the following order: Reference solution (a), Reference solution (b), Test solution and Reference solution (c).

Cathode buffer 0.1 M Tris and 0.1 M Tricine and 0.1 per cent sodium dodecyl sulfate. Do not adjust pH.

Anode buffer 0.2 M Tris-hydrochloride buffer solution pH 8.9.

System suitability The test is not valid unless; a band is seen in the electropherogram obtained with reference solution (c) in the electropherogram.

Results The electropherogram obtained with the test solution shows a single broad band corresponding in position and intensity to the single broad band obtained with the reference solution (b).

F. N-terminal sequencing

The first 15 amino acids are: Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly- Lys-His- Leu.

Perform the Edman degradation using an automated solid-phase sequencer, operated in accordance with manufacturer’s instruction

Identify the phenylthiohydantoin (PTH) – amino acids released at each sequencing cycle by reverse-phase liquid chromatography. The procedure may be carried out using the column and reagents recommended by the manufacturer of the sequencing equipment for the separation PTH- amino-acids.

The separation procedure is calibrated using:

– the mixture of PTH-amino acids provided by the manufacturer of the sequencer, with the gradient conditions adjusted as indicated to achieve optimum resolution of all amino acids,

– a sample obtained from a blank sequencing cycle obtained as recommended by the equipment manufacturer.

Liquid chromatography

Test solution Dilute the preparation to be examined with mobile phase A to a protein concentration of 0.25 mg / mL.

Reference solution To a volume of the test solution, add a suitable volume of hydrogen peroxide and acetic acid to give final protein concentration of 0.88 mg / mL, 3 mM acetic acid and 0.06 per cent hydrogen peroxide. Allow to stand at room temperature for 1 hour. Add ~ 30 mg of L-methionine R per milliliter of solution and mix. Allow to stand at room temperature for 2-3 minutes.

Column

– Stationary phase: octadecylsilyl silica gel for chromatography R (3 µm),

– Size: l = 0.15 m, Ø = 4.6 mm, A = 20 nm.

Mobile phase

– Mobile phase A: 0.1 per cent V / V solution of trifluoroacetic acid R.

– Mobile phase B: To 800 mL of acetonitrile add 1.0 mL of trifluoroacetic acid R and 200 mL of acetonitrile for chromatography R.

Time Mobile phase A Mobile phase B (min) (per cent V / V) (per cent V / V) 0 - 26 76  66.5 24  33.5

26 - 26.01 66.5  60 33.5  40 26.01 - 30 60 40

30 - 30.01 60  76 40  24 30.01 - 40 76 24

Flow rate 1 mL / min.

Equilibration At initial conditions for at least 15 min.

Injection 40 µL.

System suitability

In the chromatogram obtained with the reference solution three peaks corresponding to oxidized rhPTH134 appear at retention time of 0.79, 0.66 and 0.46 relative to principal peak.

Results

In the chromatogram obtained with the test solution, the sum of the area of any peaks other than the principal peak is not greater than 3.0 per cent of the total area of the peaks. The sum of the area of peaks corresponding to the oxidized form is not greater than 2.0 per cent of the total area of the peaks.

Bacterial endotoxins

Less than 10 IU in the volume that contains 1 mg of protein.

ASSAYS

Content

80 percent to 120 percent of the stated concentration

Test Solution Dilute the preparation to be examined in acetate buffer solution pH 4.0.

Record the absorbance spectrum between 250 nm and 400 nm. Measure the value at the absorbance maximum (276  280 nm), after correction for any light scattering, measured up to 400 nm. Calculate the concentration of rhPTH134 by taking the specific absorbance to be 13.3.

Potency

The biological activity of rhPTH 1-34 is estimated to be equipotent to that of innovator based on its comparative ability to stimulate the accumulation of cAMP in UMR-106 cells with respect to the innovators product, FORTEOTM.

Determination of the biological activity of rhPTH 1-34 is based on the stimulation of adenylate cyclase activity in the rat osteosarcoma cell line UMR 106. Activation of the PTH receptor initiates a cascade event which culminates in an intracellular rise in cAMP concentration. The method uses cAMP end-point measurement by ELISA. Incubate UMR-106 cells in tissue culture plate until an even monolayer is observed. Maintain the cells in serum free media for 5-6 hours in order to decrease the levels of endogenous cAMP. Suitably wash the cells and incubate with varying dilutions of test and reference preparations for up to 8 dilutions for the suitable time. Collect the cell lysate containing the cAMP by freeze-thaw cycle. Determine the cAMP accumulation by using a suitable ELISA or detection assay. Analyze the data by fitting a sigmoidal dose – response curve to the data obtained and by using a suitable statistical method, for example the 4- parameter or parallel line model. Report the relative potency. The estimated potency is not less than 60 percent and not more than 180 percent of the stated potency. The confidence limits (P = 0.95) of the estimated potency are not less than 64 percent and not more than 156 percent of the stated potency.

STORAGE

Store in sterile container at or below –20 °C.

LABELLING

The label states:

– The rhPTH134 content in milligrams per milliliter,

– The name and the concentration of any other excipients.

Reference Product: Reference product refers to in-house developed reference standard or Commercial FORTEOTM.

Version 4 dated 26th Sep 2012 14/14