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,
so a▲ (USP 1-Dec-2022)rat-cell-line-based approach that measures production of ATP as a direct indicator of cell growth ▲is presented here.▲ (USP 1-Dec-2022)[Note—The bioidentity test may be performed either on the somatropin bulk drug substance or on the drug product.]
PROCEDURE
•In Vitro Cell-Based Bioidentity Test
Medium A:Fischer's medium1 containing 10% heat-inactivated fetal bovine serum,2 10% heat-inactivated horse serum,3 0.075% sodium bicarbonate, and 0.05 mM 2-mercaptoethanol. Filter sterilize. [Note—Store for up to 2 weeks at 2°–8°.]
Medium B:Fischer's medium containing 1% horse serum, 0.075% sodium bicarbonate, and 0.05 mM 2-mercaptoethanol. Filter sterilize. [Note—Store for up to 2 weeks at 2°–8°.]
Phosphate buffered saline4: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 per 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.]
Source form USP:https://online.uspnf.com/uspnf/document/2_GUID-EA0131B6-8C2D-4525-8FAF-70BC080E5D58_10101_en-US
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