Sargramostim for Injection

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION 

Sargramostim for Injection is a sterile, lyophilized preparation of Sargramostim. Its biological activity is NLT 73.0% and NMT 146.0% of that stated on the label in USP Sargramostim Units. It contains NLT 90.0% and NMT 110.0% of the total protein content stated on the label. 

2 IDENTIFICATION 

A. The retention times of the peaks of the Sample solution do not differ by more than 0.5 min from those of the Standard solution, as obtained in the test for Chromatographic Purity. 

B. Peptide Mapping 

Solution A: trifluoroacetic acid and water (1:1000), filtered and degassed 

Solution B: trifluoroacetic acid and acetonitrile (1:1000), filtered and degassed 

Mobile phase: See Table 1. 

Table 1 

Digestion solution: Dissolve 29.4 mg of calcium chloride and 1.8 mg of β-alanine in 2 mL of water. Adjust with hydrochloric acid to a pH of 4.0. Add 0.4 mg of trypsin, and mix. 

Standard solution: 500 µg/mL of USP Sargramostim RS in water. Transfer 100 µL of this solution to a clean test tube, and add 11 µL of pH 7.6 buffer solution (see Reagents, Indicators, and Solutions—Buffer Solutions) containing 0.1 M tris(hydroxymethyl)aminomethane. Add 25 µL of Digestion solution, and incubate at 37° for 2 h. Quench the reaction by adding 3 µL of 20% trifluoroacetic acid. 

Sample solution: 500 µg/mL of sargramostim in water. Transfer 100 µL of this solution to a clean test tube, and add 11 µL of pH 7.6 buffer solution (see Reagents, Indicators, and Solutions—Buffer Solutions) containing 0.1 M tris(hydroxymethyl)aminomethane. Add 25 µL of Digestion solution, and incubate at 37° for 2 h. Quench the reaction by adding 3 µL of 20% trifluoroacetic acid. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 220 nm 

Column: 4.6-mm × 25-cm; 10-µm packing L1 

Column temperature: Ambient 

Flow rate: 1 mL/min 

Injection volume: 100 µL 

Analysis 

Samples: Standard solution and Sample solution 

Equilibrate the system with a Mobile phase consisting of 100% Solution A. Measure the responses for the eight major peaks as defined in the USP Sargramostim RS Data Sheet. 

Acceptance criteria: The retention times of the peak responses of the Sample solution correspond to those of the Standard solution if the retention times of the corresponding peaks do not differ by more than 0.3 min; the peak area ratios for peaks 4, 8, and 10 are between 0.7 and 1.3; and no additional significant peaks or shoulders are found. 

3 ASSAY 

Procedure 

Iscove's Modified Dulbecco's medium: Prepare a mixture of the ingredients in the quantities shown in Table 2 in sufficient water to obtain 1 L of medium, and sterilize by filtration. 

Table 2 

Medium A: Prepare a mixture of Iscove's Modified Dulbecco's medium with 10% heat-inactivated fetal bovine serum as described in Table 3.

Table 3

Prepare aseptically, sterilize by filtration, and store at 2°–8°. Use within 1 month. 

Medium B: 1 µg/mL of USP Sargramostim RS in Medium A. Prepare aseptically, sterilize by filtration, and store at 2°–8°. Use within 1 month. Standard solution: 100 ng/mL of USP Sargramostim RS in Medium A. Dispense aseptically in equal portions, and store at −60° or below. Use within 24 months. Store thawed portions at a temperature between 2° and 8°, and use within 1 month. At the time of use, dilute with Medium A to obtain a solution having a known concentration of 2 ng/mL. 

Sample solution: Reconstitute Sargramostim for Injection in water, then dilute to a concentration of 2 ng/mL of sargramostim in Medium A. Cell culture preparation: Prepare cell cultures of TF-1 cells (ATCC CRL-2003). Passage the cultures every 3–4 days, using a 1:10 subculture of the cells for up to 3 months. After 3 months, initiate a new culture. Use Medium A containing 0.5% Medium B for passage propagation and storage in the frozen state.

Cell suspension: Wash the cells three times in Medium A, and adjust the cell concentration to 5 × 104 cells/mL in Medium A. Tritiated thymidine solution: 1 mL of a solution of tritiated thymidine with a specific activity of 20 Ci/mmol in 49 mL of Medium A. Store at 2°–8°. Use within 2 weeks. 

Analysis: Use a 96-well, at-bottom microtitration plate with wells arranged in 8 rows (labeled A–H) with 12 wells (numbered 1–12) in each row. Place 50 µL of Medium A in wells 2–12. Place 100 µL of the Standard solution or each Sample solution or Medium A (negative control) in well 1. Make serial dilutions by transferring 50 µL from well 1 to well 2, and so on through well 12 (serial two-fold dilutions). Place 50 µL of the Cell suspension in each well, and incubate the microtitration plate for 72 h at 37° in a 10% carbon dioxide incubator. Following incubation, add 25 µL of Tritiated thymidine solution to each well, and return the plate to the same incubator for an additional 4–5 h. Before harvesting the cells on a filter mat, prewet the mat filter, using distilled water. [Note—The prewetting minimizes background radiation noise.] Using a multiple, automated sample harvesting system, place the incubated plate under the harvesting system. Fill the wells to the top with deionized water. Aspirate the water, and pass it through the collecting filter mat. Repeat the procedure at least ve times or until all the cells have been fully harvested. When all wells have been fully harvested, pour 5–10 mL of alcohol on the plate tray, and aspirate the methanol. Repeat the procedure if further drying of the filter mat is desired. [Note—The alcohol helps to dry out the filter mat by carrying away the wash uid.] Remove the filter mat, and repeat the procedure until all plates under test have been harvested. 

Dry the filter mat in a drying oven for about 30 min. Place the completely dry filter mats in a beta counter, and determine the amount of radioactivity in each cell well. 

Convert the amount of incorporated radioactivity in each well to a percentage of the maximum incorporated radioactivity. If fewer than ve values are between 3% and 97% of the maximum revision, repeat the Assay. Using the least-squares method of regression analysis, plot the slope of each test specimen versus the slope of the standard, excluding any values exceeding the maximum of each dilution set. Calculate the USP Sargramostim Units in each mL of the Sample solution in terms of the dilution that gives half-maximal activity. To convert this value to units of protein/mg, divide the USP Sargramostim Units/mL by the weight, in mg/mL, of protein in the initial undiluted solution. 

Acceptance criteria: The potency is 73.0%–146.0% of the potency stated on the label in terms of USP Sargramostim Units/mg of protein. 

4 PERFORMANCE TESTS 

Uniformity of Dosage Units 〈905〉: Meets the requirements for Content Uniformity 

5 SPECIFIC TESTS 

Chromatographic Purity 

Solution A: trifluoroacetic acid and water (1:1000), filtered and degassed 

Solution B: trifluoroacetic acid and acetonitrile (1:1000), filtered and degassed 

Solution C: Dissolve 116.9 g of sodium chloride in 2 L of water, adding 2 mL of trifluoroacetic acid. 

Mobile phase: See Table 4. Make adjustments if necessary (see Chromatography 〈621〉, System Suitability). 

Table 4 

Standard solution: 1 mg/mL of USP Sargramostim RS in water 

Sample solution: Reconstitute Sargramostim for Injection in water to obtain a 1 mg/mL solution of sargramostim. Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 220 nm 

Column: 4.6-mm × 25-cm; packing L1 

Column temperature: Ambient 

Flow rate: 1 mL/min 

Injection volume: 50 µL 

Analysis 

Samples: Standard solution and Sample solution 

Equilibrate the system with a Mobile phase consisting of 55% Solution A, 25% Solution B, and 20% Solution C. After injection of the solution under test, the composition is changed linearly. The major peaks are from hyperglycosylated sargramostim and from the three glycosylated forms of sargramostim, as indicated in the USP Sargramostim RS Data Sheet. 

Calculate the percentage of hyperglycosylated sargramostim in the Sample solution: 

Result = (r_U/r_T) × 100 

r_U = peak response of hyperglycosylated sargramostim 

r_T = sum of all the peak responses of sargramostim 

Calculate the percentage of each of the three glycosylated forms of sargramostim in the Sample solution: 

Result = (r_U/r_T) × 100 

r_U = peak response of each individual glycosylated form of sargramostim 

r_T = sum of all the peak responses of all three glycosylated forms of sargramostim 

Acceptance criteria: The peak responses of the Sample solution correspond to those of the Standard solution, and no peaks or shoulders are present in the chromatogram of the Sample solution that are not present in the chromatogram of the Standard solution. NMT 5.6% of hyperglycosylated sargramostim. The percentages of each of the three glycosylated forms of sargramostim, in order of elution, are 25%– 42%, 14%–32%, and 35%–53%. 

Constituted Solution: At the time of use, it meets the requirements in Injections and Implanted Drug Products 〈1〉. 

Bacterial Endotoxins Test 〈85〉: NMT 50 USP Endotoxin Units/mg 

Safety: Meets the requirements in Biological Reactivity Tests, In Vivo 〈88〉, Safety Tests—Biologicals 

Sterility Tests 〈71〉: It meets the requirements when tested as directed in Test for Sterility of the Product to Be Examined, Membrane Filtration. pH 〈791〉: 7.1–7.7, in the solution constituted as directed in the labeling 

Water Determination, Method I 〈921〉: NMT 2.0% 

6 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in hermetic containers at a temperature between 2° and 8°. 

Labeling: Label it to state the biological activity in USP Sargramostim Units/vial and the amount of protein/vial. 

USP Reference Standards 〈11〉 

USP Sargramostim RS