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SOMATROPIN BIOIDENTITY TESTS
Somatropin is a protein hormone that contains the same amino acid sequence as the human growth hormone produced by the pituitary gland. A robust and precise physicochemical chromatographic procedure is used in the Assay to assign potency on a mass basis. Bioidentity is still required, and two procedure options are presented here: an in vivo bioassay procedure based on somatropin-induced weight gain in rats and a more precise, rat cell line-based approach that measures production of ATP as a direct indicator of cell growth. [Note—The bioidentity test may be performed either on the somatropin bulk drug substance or on the drug product. ]
PROCEDURE
• In Vivo Bioidentity Test
Buffer solution:
0.1 M ammonium bicarbonate. Adjust with sodium hydroxide to a pH of 8.0.
Standard solutions:
10–100 µg/mL of USP Somatropin RS in Buffer solution
Sample solutions:
10–100 µg/mL of Somatropin in Buffer solution. [Note—Do not agitate while mixing; swirl gently. ]
Control:
Buffer solution
Test animals:
Select an appropriate number of only female or only male Sprague Dawley rats hypophysectomized at 25–30 days of age. After hypophysectomization, feed the rats on rat chow and 5% dextrose water for at least 72 h. After 72 h, feed the rats on rat chow and filtered and deionized water adjusted with 1 N hydrochloric acid to a pH of 3.0 ± 0.25. Weigh the rats when they are 37–44 days old, and retain only healthy rats. Reweigh the remaining rats 7 days later, and use only those rats that are in good health and have not gained or lost more than 10% of their body weight in the previous 7-day period.
Analysis:
Randomly divide the rats into control, standard, and test groups, each group containing approximately 10 rats. Each day for 10 days inject subcutaneously 0.1 mL of the Control, Standard solutions, and Sample solutions to the control, standard, and test groups, respectively. Record the body weight of each animal at the start of the test and at approximately 18 h after the 10th injection.
Calculations:
Determine the change in body weight for each rat during the 10-day period, and compute the potency of the Sample solution relative to that of the Standard solution using appropriate statistical analysis. Calculate the mean potency in USP Somatropin Units/mg and, using appropriate statistical methods, calculate the width, L, of a 95% confidence interval for the estimated base 10 logarithm of the relative potency. If L is more than 0.40, repeat the test until the results from two or more tests, combined by appropriate statistical methods, produce an L of NMT 0.40, corresponding to confidence limits of 63%–158% of the calculated potency.
Acceptance criteria:
NLT 2 USP Somatropin Units/mg when L is NMT 0.40
• In Vitro Cell-Based Bioidentity Test
Medium B:
Fischer's medium containing 1% horse serum, 1% sodium bicarbonate, and 0.05 mM 2-mercaptoethanol. Filter sterilize. [Note—Store for up to 2 weeks at 2
–8. ]
–8. ]
Phosphate buffered saline:4
Calcium- and magnesium-free phosphate buffered saline containing 1.5 mM monobasic potassium phosphate, 155 mM sodium chloride, and 3 mM dibasic sodium phosphate, pH 7.2–7.4
Cell culture preparation:
Prepare cell suspension cultures of Nb2-115 cells in Medium A in a humidified incubator at 37 and containing 5% carbon dioxide. Cells should be passaged twice/week and reseeded at a density of 1 × 105 cells/mL for 2 days, 2 × 104 cells/mL for 3 days, and 1 × 104 cells/mL for 4 days in Medium A. [Note—Seeding densities may need adaptation when analysts qualify new lots of fetal bovine serum and horse serum. ] On the day of an assay, cells are harvested from flasks and are pelleted by centrifugation for 7 min at about 218 × g. The supernatant is discarded, and the cells are washed twice with Phosphate buffered saline followed by centrifugation. The cells are resuspended in Medium B and are counted, and the cell concentration is adjusted to 1 × 105 cells/mL with Medium B. Except for the wells of the first column, transfer 50 µL of cell suspension into each well of a 96-well black tissue culture plate with a clear bottom.6 Pipet 50 µL of Medium B into each well of the first column of the plate. [Note—Cover the plate, and incubate at 37 for up to 1 h while preparing the Standard solutions and Test solutions. ]
and containing 5% carbon dioxide. Cells should be passaged twice/week and reseeded at a density of 1 × 105 cells/mL for 2 days, 2 × 104 cells/mL for 3 days, and 1 × 104 cells/mL for 4 days in Medium A. [Note—Seeding densities may need adaptation when analysts qualify new lots of fetal bovine serum and horse serum. ] On the day of an assay, cells are harvested from flasks and are pelleted by centrifugation for 7 min at about 218 × g. The supernatant is discarded, and the cells are washed twice with Phosphate buffered saline followed by centrifugation. The cells are resuspended in Medium B and are counted, and the cell concentration is adjusted to 1 × 105 cells/mL with Medium B. Except for the wells of the first column, transfer 50 µL of cell suspension into each well of a 96-well black tissue culture plate with a clear bottom.6 Pipet 50 µL of Medium B into each well of the first column of the plate. [Note—Cover the plate, and incubate at 37 for up to 1 h while preparing the Standard solutions and Test solutions. ]
Standard solutions:
Reconstitute USP Somatropin RS in 1 mL of Phosphate buffered saline, and then dilute this material further with Medium B to a concentration of 2.0 ng/mL. [Note—Use polypropylene test tubes to make dilutions. For each plate approximately 1 mL of this solution is needed. Do not use single step dilutions of more than 1:100 or smaller transfer volumes than 40 µL. ]
Test solutions:
For each test solution two results must be obtained by two independent preparations of somatropin. Reconstitute two independent preparations of somatropin in Water for Injection to a concentration of 5 mg/mL. Prepare test solutions by diluting this material further with Medium B to a concentration of 2.0 ng/mL. [Note—Use polypropylene test tubes to make dilutions. For each plate approximately 1 mL of this solution is needed. Do not use single step dilutions of more than 1:100 or smaller transfer volumes than 40 µL. ] One preparation is Test solution A, and the other preparation is Test solution B.
Positive control solution:
Starting with the stock concentration of 5 mg/mL of USP Somatropin RS reconstituted in Water for Injection, dilute with Medium B to a concentration of 20 ng/mL. [Note—Use polypropylene test tubes to make dilutions. Do not use single step dilutions of more than 1:100 or smaller transfer volumes than 40 µL. ]
Procedure:
Dispense 100 µL of Medium B into each well of a U-bottom, clear 96-well plate,7 except those wells in column 12 (wells A12–H12 = positive control) and wells A2–A10. [Note—For each black plate seeded with cells as described in Cell culture preparation, one separate U-bottom plate is used for preparation of the Standard solution and Test solution dilutions. ] Next, dispense 200 µL of 2.0 ng/mL Standard solution into wells A3, A6, and A9 of the dilution plate. Dispense 200 µL of 2.0 ng/mL Test solution A into wells A2, A5, and A8 of the dilution plate. Dispense 200 µL of 2.0 ng/mL Test solution B into wells A4, A7, and A10 of the dilution plate. Dispense 100 µL of 20 ng/mL Positive control solution into the last plate column (wells A12–H12) of the dilution plate. Using a 12-channel pipet, perform serial two-fold dilutions on the plate by aspirating 100 µL from the first row (A2–A10), transfer to the second row, and mix three times. Then aspirate 100 µL from the second row, transfer to the third row, and mix three times, etc. Repeat this procedure across the whole plate up to row H. Discard the 100 µL aspirated from the last row. [Note—The wells of column 11 (wells A11–H11) are negative controls containing 100 µL of Medium B; the wells of column 1 (wells A1–H1) are blanks. ]
Starting with the lowest concentration and using a 12-channel pipet, transfer 50 µL of each solution in the dilution plate to the respective well of the black plate containing the cells. [Note—During the transfer, immerse the pipet tips into the cell solution. This transfer yields an additional 1:2 dilution of somatropin, or a final concentration of 1.0 ng/mL for the highest dose added to the cells and 10 ng/mL for the Positive control solution. A plate layout is shown in Table 1. ] Following all transfers, gently agitate the black plate for 30 s, and then incubate it in a humidified 37 incubator containing 5% carbon dioxide for 30 ± 2 h. Next, add 100 µL of reconstituted luminescent substrate solution8 to all wells. Incubate the plates for 15 min at room temperature while gently agitating the plates on an appropriate plate shaker, protected from light. Incubate the plates for an additional 15 min at room temperature without shaking, protected from light. Read the luminescence from the plate wells in a suitable plate reader with a luminescence detection mode.
incubator containing 5% carbon dioxide for 30 ± 2 h. Next, add 100 µL of reconstituted luminescent substrate solution8 to all wells. Incubate the plates for 15 min at room temperature while gently agitating the plates on an appropriate plate shaker, protected from light. Incubate the plates for an additional 15 min at room temperature without shaking, protected from light. Read the luminescence from the plate wells in a suitable plate reader with a luminescence detection mode.
Table 1. Schematic Representation of the Final Assay Plate
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | BL | A,#1 | St,#1 | B,#1 | A,#1 | St,#1 | B,#1 | A,#1 | St,#1 | B,#1 | NEG | POS |
| B | BL | A,#2 | St,#2 | B,#2 | A,#2 | St,#2 | B,#2 | A,#2 | St,#2 | B,#2 | NEG | POS |
| C | BL | A,#3 | St,#3 | B,#3 | A,#3 | St,#3 | B,#3 | A,#3 | St,#3 | B,#3 | NEG | POS |
| D | BL | A,#4 | St,#4 | B,#4 | A,#4 | St,#4 | B,#4 | A,#4 | St,#4 | B,#4 | NEG | POS |
| E | BL | A,#5 | St,#5 | B,#5 | A,#5 | St,#5 | B,#5 | A,#5 | St,#5 | B,#5 | NEG | POS |
| F | BL | A,#6 | St,#6 | B,#6 | A,#6 | St,#6 | B,#6 | A,#6 | St,#6 | B,#6 | NEG | POS |
| G | BL | A,#7 | St,#7 | B,#7 | A,#7 | St,#7 | B,#7 | A,#7 | St,#7 | B,#7 | NEG | POS |
| H | BL | A,#8 | St,#8 | B,#8 | A,#8 | St,#8 | B,#8 | A,#8 | St,#8 | B,#8 | NEG | POS |
|
LEGEND:
BL = Blank: no somatropin, no cells. A = Dilution series of Test solution A (with 1.0 ng/mL as starting concentration). St = Dilution series of Standard solution (with 1.0 ng/mL as starting concentration). B = Dilution series of Test solution B (with 1.0 ng/mL as starting concentration). NEG = Negative control: no somatropin, but cells are present. POS = Positive control solution: maximum somatropin concentration (10 ng/mL). |
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Calculations:
A minimum of two independent Test solution preparations must be used for each test sample. The Test solutions and Standard solutions are normalized by protein content before calculation of the relative potency. [Note—One mg of anhydrous somatropin is equivalent to 3.0 USP Somatropin Units. ] Technical outliers (but NMT 4/curve) are omitted, and then the same number of Standard solution and Test solution dose responses, including the 50% response (EC50) of the standard/test sample within this range, are used to calculate the relative potency of each somatropin sample using statistical methods for parallel-line analysis. For each individual Test solution compared to the Standard solution, the statistical tests for linearity, slope, and parallelism must pass at the 95% level. The confidence limit for each calculated relative potency must be within 75%–133%. The relative potency of a Test solution must be within the validated assay range (50%–200% relative potency) of the Standard solutions. The unweighted mean log relative potency is calculated from valid individual samples, and the potency is then calculated in USP Units/mg relative to the USP Somatropin RS.
System suitability criteria:
The signal-to-noise ratio of the mean chemiluminescence signal of the Positive control solution to the mean chemiluminescence signal of the negative control wells must be NLT 3. NMT 4 technical outliers may be omitted per standard curve. Any plate that fails one or more of these criteria is rejected and must be repeated.
Acceptance criteria:
NLT 2 USP Somatropin Units/mg
• USP Reference Standards 11
11
USP Somatropin RS
1
Quality Biological, catalog #112-032-101, or equivalent.
2
Gibco, catalog #10082-147, or equivalent.
3
Gibco, catalog #26050-088, or equivalent.
4
Amimed catalog #8-05F29I, Gibco catalog #20012-019, or equivalent.
5
HPA Culture Collections or Sigma-Aldrich catalog #97041101.
6
Costar catalog #3904, or equivalent.
7
Greiner #650 160 plate or equivalent.
8
CellTiterGlo-Luminescence Kit, e.g., Promega #G7573, or equivalent.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| General Chapter | Maura C Kibbey, Ph.D.
Senior Scientific Liaison, Biologics & Biotechnology (301) 230-6309 |
(BIO12010) Monographs - Biologics and Biotechnology 1 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP38–NF33 Page 202
Pharmacopeial Forum: Volume No. 39(5)