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       Filgrastim Injection is a sterile solution for injection of a protein prepared from Filgrastim concentrated solution having the primary structure of the 174-amino-acid isoform of human granulocyte colony-stimulating factor (huG-CSF) plus 1 additional amino acid, an N-terminal methionine. In contrast to its natural counterpart, the protein is not glycosylated. It contains not less than 90.0 per cent and not more than 110.0 per cent of the amount of filgrastim stated on the label. Its potency is not less than 80 per cent and not more than 125 per cent of the potency stated on the label when determined using the conditions described under Assay.

Description Clear, colourless or slightly yellowish liquid.

Strengths available  300 μg per 0.5 mL, 300 μg per mL, 480 μg per 0.8 mL, and 480 μg per 1.6 mL.

Dose Intravenous, intramuscular or subcutaneous, as directed by a physician.

Other relevant information
       1. It may cause cough, trouble breathing; nosebleeds; bone pain, muscle or joint pain; diarrhea; headache; numbness; or rash, thinning hair.
       2. It should not be diluted with 0.9 per cent w/v sodium chloride injection because precipitation may occur.
       3. Caution should be exercised if it is to be used in children, pregnant and nursing women.

Packaging and storage Filgrastim Injection shall be stored in a sterile, tightly closed, tamper-evident container, protected from light, at a temperature between 2º and 8º.

Identification
       A. The assay or, where applicable, the biological activity, may serve as an identification test.
       B. Examine the chromatograms obtained in the test for Related proteins: the principal peak in the chromatogram obtained from the test solution is similar in retention time and shape to the principal peak in the chromatogram obtained from reference solution (a).

pH As approved by the regulatory authority (Appendix 4.11).

Impurities with molecular weights higher than that of filgrastim Carry out the test as described in the “Size-Exclusion Chromatography” (Appendix 3.6). Use the normalization procedure.

       Diluent, Standard solution, Resolution solution and Mobile phase, Chromatographic system, Procedure, System suitability, and Calculation Proceed as directed in the test for Impurities with molecular weights higher than that of filgrastim under Filgrastim Concentrated Solution.
       Test solution Dilute Filgrastim Injection with Diluent to obtain a concentration of 0.4 mg per mL.
       Limits
        - impurities with molecular weights higher than that of filgrastim, other than the dimer: not more than 0.5 per cent;
        - total of impurities with molecular weights higher than that of filgrastim: not more than 1.0 per cent.

       Impurities with charges differing from that of filgrastim Carry out the test as described in the “Isoelectric Focusing” (Appendix ==), with the following modifications. (Note Prepare reference solutions (a), (b) and (c) immediately before loading on the gel.)
       Reference solution (a) Dilute Filgrastim RS with water to obtain a concentration of 0.6 mg per mL.
       Reference solution (b) To 10 μL of Reference solution (a) add 190 μL of water to obtain a concentration of 30 μg per mL.
       Reference solution (c) To 10 μL of Reference solution (a) add 490 μL of water to obtain a concentration of 12 μg per mL.
       Reference solution (d) Use an isoelectric point (pI) calibration solution, in the pI range of 3 to 10, prepared according to the manufacturer’s instructions.
       Test solution Dilute the Filgrastim Injection with water to obtain a concentration of 0.6 mg per mL.
       Gel concentration 5 per cent; commercially available gels may be used.
       Focusing system
       pH GRADIENT 5.0 to 7.0
       CATHOLYTE a 2 per cent w/v solution of sodium hydroxide
       ANOLYTE a 3 per cent w/v solution of glacial acetic acid
       APPLICATION 15 μL
       Procedure Proceed with the isoelectric focusing by applying the electrical parameters according to the manufacturer’s instructions. The following parameters have been found suitable for gels of dimensions 250 mm X 110 mm X 0.43 mm:
       PRE-FOCUSING 700 V, 12 mA, 8 W, 20 minutes;
       POST-APPLICATION 500 V, 8 mA, 8 W, 20 minutes;
       FOCUSING 2000 V, 14 mA, 14 W, 90 minutes;
       FINAL FOCUSING 2500 V, 14 mA, 14 W, 10 minutes.
       Detection Silver staining
       Fixing solution A solution containing 3.6 per cent w/v solution of sulfosalicylic acid and 11.6 per cent w/v solution of trichloroacetic acid.
       Sensitizing solution Dissolve 17 g of sodium acetate in about 100 mL of water. Add 75 mL of ethanol, 10 mL of a 5 per cent w/v solution of anhydrous sodium thiosulfate and 1.25 mL of a 25 per cent w/v solution of glutaraldehyde and dilute to 250 mL with water.
       Silver solution Mix 100 μL of formaldehyde solution and 25 mL of a 2.5 per cent w/v solution of silver nitrate and dilute to 250 mL with water.
       Developing solution Dissolve 6.25 g of sodium carbonate in about 200 mL of water, add 50 μL of formaldehyde solution and dilute to 250 mL with water.
       Stopping solution Dissolve 3.65 g of sodium edetate in 250 mL of water. Alternatively, commercially available staining kits may be used.
       System suitability
       SAMPLE Reference solutions (c) and (d)
       SUITABILITY REQUIREMENTS
       - in the electropherogram obtained from reference solution (d), the relevant isoelectric point markers are distributed along the entire length of the gel;
       - in the electropherogram obtained from reference solution (c), the principal band is clearly visible.
       Limits
       - maximum seven bands of an intensity between that of the principal band in the electropherogram obtained from reference solution (c) (2 per cent) and that of the principal band in the electropherogram obtained from reference solution (b) (5 per cent) are present;
       - no band has an intensity greater than that of the principal band in the electropherogram obtained from reference solution (b) (5 per cent).

Bacterial endotoxins When tested as described in the “Test for Bacterial Endotoxins” (Appendix 8.5), it contains not more than 10 Endotoxin Units in the volume that contains 1.0 mg of protein.

Osmolality As approved by the competent authority (Appendix 4.35).

Related proteins Carry out the test as described in the “Liquid Chromatography” (Appendix 3.5), use the normalization procedure.
       Reference solution (a), Reference solution (b), Reference solution (c), Mobile phase Proceed as directed in the Related proteins under Filgrastim Concentrated Solution.
       Chromatographic system, Procedure and System suitability Proceed as directed in the Related proteins under Filgrastim Concentrated Solution.
       Test solution Dilute the Injection with water to obtain a concentration of 0.5 mg per mL.
       Limits
       - any impurity: for each impurity, not more than 3.0 per cent;
       - total: not more than 6.5 per cent.
Assay
       FOR PROTEIN Carry out the test as described in the “Liquid Chromatography” (Appendix 3.5).
       Reference solution (a), Test solution, Mobile phase, Chromatographic system, Procedure and System suitability Proceed as directed in the test for Related proteins.
       Calculation Calculate the content of C₈₄₅H₁₃₃₉N₂₂₃O₂₄₃S₉ in the Injection taken, using the declared content of C₈₄₅H₁₃₃₉N₂₂₃O₂₄₃S₉ in Filgrastim RS.
       FOR POTENCY The potency of the Injection is determined by comparison of the dilutions for the test solution with the dilutions of the International Standard of filgrastim or with a reference solution calibrated in International Units.
        The International Units (IU) is the activity contained in a stated amount of the appropriate International Standard. The equivalence in International Units of the International Standard is stated by the World Health Organization.
       Carry out the assay by using a suitable method such as the following, which uses the conversion of a tetrazolium salt (MTS) as a staining method. Alternative methods of quantifying cell proliferation, such as measurement of intracellular ATP by luciferase bioluminescence, have also been found suitable, and may be used as the assay readout, subject to appropriate validation. The assay conditions (for example, cell concentration, incubation time and dilution steps) are then adapted accordingly.
       Use an established cell line responsive to filgrastim. M-NFS-60 cells (ATCC No. CRL-1838) that have been made sensitive to G-CSF have been found suitable. Incubate with varying dilutions of test and reference solutions of filgrastim. Then incubate with a solution of tetrazolium salt. This cytochemical stain is converted by cellular dehydrogenase to a coloured formazan product. The formazan is then measured spectrophotometrically.
       Add 50 μL of dilution medium to all wells of a 96-well microtitre plate. Add an additional 50 μL of this solution to the wells designed for the blanks. Add 50 μL of each solution to be tested in triplicate (test solution and reference solution at a concentration of about 800 IU per mL, plus a series of 10 twofold dilutions to obtain a standard curve). Prepare a suspension of M-NFS-60 cells containing 7 X 10⁵ cells per mL. Immediately before use, add 2-mercaptoethanol to a final concentration of 0.1 mM, and add 50 μL of the prepared cell suspension to each well, maintaining the cells in a uniform suspension during addition.
       Incubate the plate at 36.0º to 38.0º for 44 to 48 hours in a humidified incubator using 6±1 per cent CO2. Add 20 μL of a 0.5 per cent w/v sterile solution of tetrazolium salt to each well and re-incubate for 4 hours. Estimate the quantity of formazan produced using a microtitre well plate reader at 490 nm.
       Calculation Calculate the potency of the Injection using a suitable statistical method as described in the “Statistical Analysis of Results of Biological Assays and Tests” (Appendix 9), for example the parallel line assay.
       The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency. The confidence limits (P = 0.95) are not less than 74 per cent and not more than 136 per cent of the estimated potency.

Other requirements Complies with the requirements described under “Parenteral Preparations” (Appendix 1.16).