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 Nterminal 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 
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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
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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
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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 Trishydrochloride 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.
–
Temperature: 40 °C
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
(min)
Mobile phase A
(per cent V / V)
Mobile phase B
(per cent V / V)
95  45
5  55
30  30.01
45 0
55 100
30.01  35
0
100
0  30
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35  35.01
0  95
100  5
35.01  45
95
5
Flow rate 1 mL / min.
Detection Spectrophotometer at 214 nm.
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 
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Recombinant Human Parathyroid Hormone (rhPTH134)
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 ProteaseSampe 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
– Temperature: 40 °C
– Flow rate: 1ml/min
– Detection: UV at 214nm
– Injection Volume : 20µl
– Run time: 35 min ( 10 min re equilibration)
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Recombinant Human Parathyroid Hormone (rhPTH134)
– 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)
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Recombinant Human Parathyroid Hormone (rhPTH1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
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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 Trishydrochloride 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
Water R
Component volume (mL) per gel
volume of 7.5 mL
1.20
50 % acrylamide solution*
3.00
Tris-SDS solution (pH 8.45)
2.49
Glycerol R
0.79
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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
Water R
Component volume (mL) per gel
volume of 5.0 mL
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).
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F. N-terminal sequencing
The first 15 amino acids are: Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-GlyLys-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.
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TESTS
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.
–
Temperature: 40 °C
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
(min)
Mobile phase A
(per cent V / V)
Mobile phase B
(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.
Detection Spectrophotometer at 214 nm.
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Recombinant Human Parathyroid Hormone (rhPTH134)
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
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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.
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