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FILGRASTIM CONCENTRATED SOLUTION


Category Granulocyte-colony stimulating agent.

       Filgrastim Concentrated Solution is a solution of a protein, produced by a method based on recombinant DNA (rDNA) technology, containing 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. huG-CSF is produced and secreted by endothelial cells, monocytes and other immune cells. The protein stimulates the differentiation and proliferation of leucocyte stem cells into mature granulocytes. It contains not less than 0.9 mg of the protein per mL.

       The tests for host-cell-derived proteins and host-cell- or vector-derived DNA are carried out on each batch of filgrastim concentrated solution, unless exemption has been granted by a competent authority. The limit is established by each laboratory and approved by the regulatory authority.

Strength available Not less than 0.9 X 108 IU per mg of protein.

Description Clear, colourless or slightly yellowish liquid.

Storage Filgrastim Concentrated Solution shall be stored according to the conditions approved by the regulatory authority.

Labelling Complies with the “General Information for Biological Products”. In addition, the label on the container states (1) the content, in mg of protein per mL; (2) the potency, in IU per mg of protein; (3) the storage condition.

Identification
       A. The assay or, where applicable, the biological activity may serve as an identification test.
       B. Examine the electropherograms obtained in the test for Impurities with charges differing from that of filgrastim: the principal band in the electropherogram obtained from the test solution is similar in position to the principal band in the electropherogram obtained from reference solution (a).
       C. Examine the chromatograms obtained in the test for Impurities with molecular weights higher than that of filgrastim: the principal peak in the chromatogram obtained from the test solution is similar in retention time to the principal peak in the chromatogram obtained from the reference solution.
       D. Examine the electropherograms obtained under both reducing and non-reducing conditions in the test for Impurities with molecular weights differing from that of filgrastim: the principal band in the electropherogram obtained from test solution (a) is similar in position to the principal band in the electropherogram obtained from reference solution (b).
       E. 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 the reference solution.
       F. Peptide mapping Carry out the test as described in the “Liquid Chromatography” (Appendix 3.5).
       (a) Selective cleavage of the peptide bonds
       Standard solution Transfer about 25 μg of Filgrastim RS, accurately weighed, into a polypropylene tube. Add 25.0 μL of a 0.1 mg per mL solution of glutamyl endopeptidase for peptide mapping. Dilute to 100.0 μL with 0.02 M sodium phosphate buffer solution pH 8.0, stopper the tube and incubate at about 37° for 17 hours. Cool to 2° to 8° until analysis.
       Test solution Transfer an accurately measured volume of the sample containing about 25 μg of protein into a polypropylene tube. Prepare at the same time and in the same manner as for the standard solution but using the sample instead of Filgrastim RS.
       (b) Separation by chromatography
       CHROMATOGRAPHY
       Mobile phase
       MOBILE PHASE A Trifluoroacetic acid, water, acetonitrile for chromatography (0.5:950:50)
       MOBILE PHASE B Trifluoroacetic acid, water, acetonitrile for chromatography (0.5:50:950)
       The step gradient of mobile phases is as follows:

Time
(Minutes)
Mobile Phase A
(Per Cent V/V)
Mobile Phase B
(Per Cent V/V)
0-8 97→94 3→6
8-25 94→66 6→34
25-40 66→10 34→90
40-45 10 90

       Chromatographic system
       DETECTOR Ultraviolet light (215 nm).
       COLUMN A stainless steel column (10 cm × 2.1 mm), packed with octadecylsilyl bonded to porous silica microparticles, compatible with 100 per cent aqueous mobile phases (5 μm) with a pore size of 20 nm, end-capped.
       COLUMN TEMPERATURE 60°
       FLOW RATE 0.2 mL per minute
       System suitability
       SAMPLE Standard solution
       Suitability requirements The chromatogram obtained from the standard solution is qualitatively similar to the chromatogram of filgrastim digest supplied with Filgrastim RS.
       Procedure Inject a portion (about 10 µL) of Standard solution and Test solution into the chromatograph. Measure the areas of the principal peaks in each chromatogram. Identify the peaks due to filgrastim in the test solution using the retention time of filgrastim in the standard solution.
       Results The profile of the chromatogram obtained from the test solution corresponds to that of the chromatogram obtained from the standard solution.

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 Dissolve 4.1 g of sodium acetate in 400 mL of water, adjust to pH 4.0 with acetic acid and dilute to 500 mL with water.
       Standard solution Dilute Filgrastim RS with Diluent to obtain a concentration of 0.4 mg per mL.
       Test solution Dilute Filgrastim Concentrated Solution with Diluent to obtain a concentration of 0.4 mg per mL.
       Resolution solution Mix a suitable portion of Standard solution for about 30 seconds using a vortex mixer.
       Mobile phase Dissolve 7.9 g of ammonium hydrogen carbonate in 1000 mL of water and adjust to pH 7.0 with phosphoric acid; dilute to 2000 mL with water.
       Chromatographic system
       DETECTOR Ultraviolet light (215 nm)
       COLUMN A column (30 cm × 7.8 mm), packed with spherical silica (5 m) with hydrophilic coating (suitable for fractionation of globular proteins in the relative molecular mass range of 10,000 to 500,000), equipped with a similarly packed guard column of a suitable size.
       COLUMN TEMPERATURE 30°
       FLOW RATE 0.5 mL per minute
       System suitability
       SAMPLE Resolution solution
       (Note The relative retention with reference to the filgrastim monomer: aggregates, filgrastim oligomer 1, filgrastim oligomer 2, and filgrastim dimer are about 0.60, 0.75, 0.80, and 0.85, respectively, where the retention time for filgrastim monomer is about 19 minutes.)
       Suitability requirements
       RETENTION TIME 17 to 20 minutes for filgrastim monomer.
       RESOLUTION Not less than 3 between filgrastim dimer and filgrastim monomer peaks.
       Procedure Separately inject equal volumes (about 20 μL) of Standard solution and Test solution into the chromatograph, record the chromatograms and measure the responses for the major peaks.
       Calculation Calculate the percentage content of the dimer, oligomers and aggregates.
       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 2 per cent.

Impurities with molecular weights differing from that of filgrastim Carry out the test as described in the “Electrophoresis” (Appendix 3.7) under both reducing and non-reducing conditions.
       Sample buffer (non-reducing conditions) Mix equal volumes of water and concentrated SDS-PAGE sample buffer.
       Sample buffer (reducing conditions) Mix equal volumes of water and concentrated SDS-PAGE sample buffer for reducing conditions containing 2-mercaptoethanol as the reducing agent.
       Reference solution (a) Solution of molecular weight markers suitable for calibrating SDS-polyacrylamide gels in the range of 14.4 to 94 kDa.
       Reference solution (b) Dilute Filgrastim RS with Sample buffer to obtain a concentration of 100 μg per mL.
       Test solution (a) Dilute Filgrastim Concentrated Solution with Sample buffer to obtain a concentration of 100 μg per mL.
       Test solution (b) To 200 μL of Test solution (a) add 200 μL of Sample buffer.
       Test solution (c) Dilute 200 μL of Test solution (b) to 1.0 mL with Sample buffer.
       Test solution (d) Dilute 200 μL of Test solution (c) to 1.0 mL with Sample buffer.
       Test solution (e) To 200 μL of Test solution (d) add 200 μL of Sample buffer.
       Electrophoresis system
       SAMPLE TREATMENT Boil for 5 minutes.
       GEL DIMENSIONS 1 mm thick.
       RESOLVING GEL 13 per cent acrylamide.
       APPLICATION 20 μL.
       DETECTION Silver staining.
       System suitability
       SAMPLE Reference solution (a) the validation criteria are met;
       SUITABILITY REQUIREMENTS
       - a band is seen in the electropherogram obtained from the test solution (e);
       - a gradation of intensity of staining is seen in the electropherograms obtained from the test solutions (a) to (e).
       Limit Test solution (a)
       - impurities with molecular weights lower or higher than that of filgrastim: no band is more intense than the principal band in the electropherogram obtained from Test solution (d) (2.0 per cent).

Impurities with charges differing from that of filgrastim Carry out the test as described in the “Isoelectric Focusing” (Appendix ==).
       Reference solution (a) Dilute Filgrastim RS with water to obtain a concentration of 0.3 mg per mL.
       Reference solution (b) Dilute Filgrastim RS with water to obtain a concentration of 30 μg per mL.
       Reference solution (c) Use an isoelectric point (pI) calibration solution, in the pI range of 2.5 to 6.5, prepared according to the manufacturer’s instructions.
       Test solution Dilute Filgrastim Concentrated Solution with water to obtain a concentration of 0.3 mg per mL.
       Focusing system
       pH GRADIENT 4.5 to 8.0
       CATHOLYTE 1 M sodium hydroxide
       ANOLYTE 0.04 M glutamic acid in a 0.0025 per cent v/v solution of phosphoric acid
       APPLICATION 20 μL
       Detection as described in the “Isoelectric Focusing” (Appendix ==).
       System suitability
       SAMPLE Reference solutions (a) and (c)
       SUITABILITY REQUIREMENTS
       - in the electropherogram obtained from reference solution (c), the relevant isoelectric point markers are distributed along the entire length of the gel;
       - in the electropherogram obtained from reference solution (a), the pI of the principal band is 5.7 to 6.3.
       Limit
       - any impurity: no band is more intense than the principal band in the electropherogram obtained from reference solution (b) (10 per cent).
Bacterial endotoxins When tested as described in the “Test for Bacterial Endotoxins” (Appendix 8.5), it contains not more than 2 Endotoxin Units in the volume that contains 1.0 mg of protein.

Related proteins Carry out the test as described in the “Liquid Chromatography” (Appendix 3.5), use the normalization procedure.
       Reference solution (a) Dilute the content of a vial of Filgrastim RS with water to obtain a concentration of 0.5 mg per mL.
       Reference solution (b) To 250 μL of Reference solution (a), add 2.5 μL of a 4.5 g per L solution of hydrogen peroxide. Mix and incubate at 25º±2º for 30 minutes, then add 1.9 mg of L-methionine.
       Reference solution (c) To 250 μL of Reference solution (a), add 0.25 mg of dithiothreitol. Mix and incubate at 35º±2º for 60 minutes.
       Test solution Dilute Filgrastim Concentrated Solution with water to obtain a concentration of 0.5 mg per mL.
       Mobile phase
       
MOBILE PHASE A Trifluoroacetic acid and water (1:1000)
       MOBILE PHASE B Trifluoroacetic acid, water, acetonitrile for chromatography (1:100:900) The step gradient of mobile phases is as follows:

Time
(Minutes)
Mobile Phase A
(Per Cent V/V)
Mobile Phase B
(Per Cent V/V)
0-30 60→20 40→80
30-35 20 80
35-45 20→60 80→40

       Chromatographic system
       DETECTOR Ultraviolet light (215 nm).
       COLUMN A stainless steel column (15 cm × 4.6 mm), packed with butylsilyl chemically bonded to porous silica or ceramic microparticles (5 μm) with a pore size of 30 nm, end-capped.
       COLUMN TEMPERATURE 60°
       FLOW RATE 0.8 mL per minute
       Procedure Separately inject equal volumes (about 50 μL) of Reference solutions (b) and (c) and Test solution into the chromatograph, record the chromatograms and measure the responses for the major peaks.
       System suitability
       SAMPLE Reference solution (b)
       (Note The relative retention with reference to filgrastim: oxidized filgrastim (form 1), oxidized filgrastim (form 2), and reduced filgrastim are about 0.84, 0.98 and 1.04, respectively, where the retention time for filgrastim is about 23 minutes.)
       SUITABILITY REQUIREMENTS
       Symmetry factor Not more than 1.8 for the peak due to filgrastim;
       Peak-to-valley ratio Not less than 2.0, where Hp = height above the baseline of oxidized filgrastim (form 2) peak and Hv = height above the baseline of the lowest point of curve separating this peak from the filgrastim peak.
       System suitability
       SAMPLE Reference solution (c)
       SUITABILITY REQUIREMENTS
       Resolution Not less than 1.5 between the filgrastim and reduced filgrastim peaks.
       Symmetry factor Not more than 1.8 for the filgrastim peak.
        Limits
       - any impurity: for each impurity, not more than 1.0 per cent;
       - total: not more than 2.0 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 Solution taken, using the declared content of C845H1339N223O243S9 in Filgrastim RS.
       FOR POTENCY The potency of the Solution 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 dehydrogenases 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 Solution 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.

TP SUPPLEMENT 2022 • FILGRASTIM CONCENTRATED SOLUTION
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