\n| 82 – 97<\/td>\n | 95<\/td>\n | 5<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n Flow rate 0.3 mL\/min.<\/p>\n Detection: Fluorimeter at 360 nm for excitation and 425 nm for emission.<\/p>\n Autosampler Set at 4 \u00b0C.<\/p>\n Injection 50 \u03bcL.<\/p>\n Identification of peaks: Use the chromatogram in Figure 3103.-1 to identify the 6 peaks corresponding to fucosylated (peaks 1, 2 and 3), other neutral (peak cluster 4), mono-sialylated (peak 5) and di-sialylated (peak 6) glycans; record the retention time of each peak.<\/p>\n System suitability:<\/p>\n \u2014 the chromatogram obtained with reference solution (a) is qualitatively similar to the chromatogram supplied with golimumab CRS and peaks 1 to 6 are clearly visible;<\/p>\n \u2014 no significant peaks are observed in the chromatogram obtained with the blank solution.<\/p>\n Results:<\/p>\n \u2014 the profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with reference solution (b);<\/p>\n \u2014 the retention times of the peaks in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with reference solution (b);<\/p>\n \u2014 no additional peaks are observed in the chromatogram obtained with the test solution in comparison with the chromatogram obtained with reference solution (b).<\/p>\n Calculate the relative peak areas of the individual peaks corresponding to fucosylated, other neutral, mono- and di- sialylated glycans with reference to the sum of the areas of all retained glycan peaks.<\/p>\n Calculate the percentage contents of other neutral and sialylated glycans, using the following expressions:<\/p>\n A\/A+B x 100<\/p>\n B\/A+B x 100<\/p>\n A = sum of the areas of the peaks due to fucosylated and other neutral glycans;<\/p>\n B = sum of the areas of the peaks due to mono- and di-sialylated glycans.<\/p>\n NOTE: other neutral and mono-sialylated glycans elute as peak clusters and are integrated as such.<\/p>\n Limits:<\/p>\n \u2014 percentage of neutral glycans: as authorised by the competent authority;<\/p>\n \u2014 percentage of sialylated glycans: as authorised by the competent authority.<\/p>\n ![\"Golimumab](\"https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/1-152-300x163.jpg\") <\/p>\n
Figure 3103.-1. \u2013 Chromatogram for glycan analysis of golimumab<\/p>\n Charged variants<\/p>\n Capillary electrophoresis (2.2.47).<\/p>\n The following procedure is given as an example.<\/p>\n Solution B: Use pI marker 7.6 and pI marker 9.5, prepared according to the manufacturer’s instructions. Mix 1400 \u03bcL of a 1 per cent m\/m solution of methyl cellulose, 20 \u03bcL of pI marker 7.6, 40 \u03bcL of pI marker 9.5, 40 \u03bcL of carrier ampholyte solution pH 3-10, 120 \u03bcL of carrier ampholyte solution pH 8-10.5 and 400 \u03bcL of a 1 per cent V\/V solution of tetramethylethylenediamine R.<\/p>\n Test solution: Dilute the preparation to be examined with water R to obtain a concentration of 10 mg\/mL. To 10 \u03bcL of this solution, add 1 \u03bcL of a 5 mg\/mL solution of carboxypeptidase B R. Incubate at 25 \u00b0C for 30 min and cool on ice for at least 30 min. To 5 \u03bcL of the digested solution, add 94 \u03bcL of water R and 101 \u03bcL of solution B. Use this solution within 3 h of preparation.<\/p>\n Reference solution (a): Dissolve the contents of a vial of golimumab CRS in water R to obtain a concentration of 10 mg\/mL. Proceed in the same manner as for the test solution.<\/p>\n Reference solution (b): Use a suitable golimumab in-house reference preparation shown to be representative of batches tested clinically and batches used to demonstrate consistency of production. Dilute with water R to obtain a concentration of 10 mg\/mL. Proceed in the same manner as for the test solution.<\/p>\n Reference solution (c) Use pI marker 8.7 and pI marker 9.0, prepared according to the manufacturer’s instructions. Mix 2 \u03bcL of each pI marker, 95 \u03bcL of water R and 101 \u03bcL of solution B.<\/p>\n Blank digest: Prepare at the same time and in the same manner as for the test solution but using water R instead of the preparation to be examined.<\/p>\n Blank solution: Mix 99 \u03bcL of water R and 101 \u03bcL of solution B.<\/p>\n Focusing:<\/p>\n \u2014 catholyte: mix 5 mL of a 0.1 per cent m\/m solution of methyl cellulose and 26 \u03bcL of a 500 g\/L solution of sodium hydroxide R (100 mM solution of sodium hydroxide R in a 0.1 per cent m\/m solution of methyl cellulose);<\/p>\n \u2014 anolyte: mix 5 mL of a 0.1 per cent m\/m solution of methyl cellulose and 270 \u03bcL of an 85 per cent m\/m solution of phosphoric acid R (80 mM solution of phosphoric acid R in a 0.1 per cent m\/m solution of methyl cellulose).<\/p>\n Capillary:<\/p>\n \u2014 material: fluorocarbon-coated;<\/p>\n \u2014 size: total length = 5 cm, \u00d8 = 100 \u03bcm;<\/p>\n \u2014 temperature: 25 \u00b0C.<\/p>\n Detection At 280 nm.<\/p>\n Autosampler Set at 4 \u00b0C.<\/p>\n Sample volume 160 \u03bcL.<\/p>\n Focusing Apply a field strength of 3000 V for 8 min.<\/p>\n Identification of peaks: Use the electropherogram in Figure 3103.-2 to identify the 4 major peaks C, 1, 2 and 3, and the 2 minor peaks A and B. Additional peaks may be present.<\/p>\n System suitability:<\/p>\n \u2014 in the electropherogram obtained with reference solution (a), the peaks corresponding to pI marker 7.6 and pI marker 9.5 are clearly visible;<\/p>\n \u2014 in the electropherogram obtained with reference solution (a), 6 peaks (4 major and 2 minor) in the pI region 8.5-9.0 are clearly visible;<\/p>\n \u2014 in the electropherogram obtained with reference solution (c), the peaks corresponding to pI marker 7.6, pI marker 8.7, pI marker 9.0 and pI marker 9.5 are clearly visible;<\/p>\n \u2014 resolution: minimum 5.0 between the peaks corresponding to pI marker 8.7 and pI marker 9.0;<\/p>\n \u2014 in the electropherogram obtained with the blank digest, the peaks corresponding to pI marker 7.6 and pI marker 9.5 are clearly visible; no additional peaks are observed between pI marker 7.6 and pI marker 9.5;<\/p>\n \u2014 in the electropherogram obtained with the blank solution, the peaks corresponding to pI marker 7.6 and pI marker 9.5 are clearly visible; no additional peaks are observed between pI marker 7.6 and pI marker 9.5.<\/p>\n Results:<\/p>\n \u2014 the electropherogram obtained with the test solution is similar to the electropherogram obtained with reference solution (b). Plot the migration distances of the relevant pI markers versus their pI and determine the apparent isoelectric points of the principal components of the test solution and reference solution (b); they do not differ by more than 0.05 pI units;<\/p>\n \u2014 no additional peaks are observed in the electropherogram obtained with the test solution in comparison to the electropherogram obtained with reference solution (b).<\/p>\n Calculate the relative peak areas of the individual peaks due to isoforms with reference to the total area of all peaks.<\/p>\n Limits:<\/p>\n \u2014 sum of isoforms C, 1, 2 and 3: as authorised by the competent authority;<\/p>\n \u2014 isoform 3: as authorised by the competent authority;<\/p>\n \u2014 isoform 2: as authorised by the competent authority;<\/p>\n \u2014 isoform 1: as authorised by the competent authority;<\/p>\n \u2014 isoform C: as authorised by the competent authority;<\/p>\n \u2014 isoform B: as authorised by the competent authority.<\/p>\n ![\"Golimumab](\"https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/2-151-300x163.jpg\") <\/p>\n
Figure 3103.-2. \u2013 Electropherogram for the test for charged variants of golimumab<\/p>\n CHARACTERS<\/h2>\nAppearance<\/h3>\nColourless or light yellow liquid.<\/p>\n IDENTIFICATION<\/h2>\nA. It complies with the limits of the assay (potency).<\/p>\n B. Peptide mapping (2.2.55).<\/p>\n SELECTIVE CLEAVAGE OF THE PEPTIDE BONDS<\/h4>\nDilution buffer: Dilute 500 \u03bcL of 1 M tris-hydrochloride buffer solution pH 8.0 R to 10 mL with water R.<\/p>\n Test solution: Dilute the preparation to be examined with the dilution buffer to obtain a concentration of about 10 mg\/mL.<\/p>\n Desalt 100 \u03bcL by a suitable procedure. Transfer a volume of the desalted solution containing 200 \u03bcg of protein to a polypropylene tube and evaporate to dryness.<\/p>\n Reference solution: Dissolve the contents of a vial of golimumab CRS in the dilution buffer to obtain a concentration of about 10 mg\/mL. Desalt a volume of this preparation at the same time and in the same manner as for the test solution.<\/p>\n Reduction and alkylation To 5 mL of water R, add 11.5 g of guanidine hydrochloride R. Dilute to 15 mL with water R. To 1500 \u03bcL of this solution, add 100 \u03bcL of 1 M tris-hydrochloride buffer solution pH 8.0 R, 20 \u03bcL of a 186 g\/L solution of sodium edetate R, 20 \u03bcL of a 154 g\/L solution of dithiothreitol R and 360 \u03bcL of water R. Dilute to 2.0 mL with water R. Add 150 \u03bcL to the desalted protein and incubate at 37 \u00b0C for 60 min. Add 3.0 \u03bcL of a freshly prepared 184 g\/L solution of iodoacetamide R and incubate at room temperature for 60 min protected from light. Add 1 \u03bcL of a 154 g\/L of solution of dithiothreitol R.<\/p>\n Digestion Add 350 \u03bcL of dilution buffer and 400 \u03bcL of a 0.01 mg\/mL solution of lysyl endopeptidase R in dilution buffer. Incubate a 37 \u00b0C for 60 min. Add another 4 \u03bcL of a 1 mg\/mL solution of lysyl endopeptidase R, mix gently and spin down. Incubate at 37 \u00b0C for 3 hours. Add 5 \u03bcL of trifluoroacetic acid R and mix gently.<\/p>\n NOTE: a protease\/protein ratio of 1:25 (m\/m) is used.<\/p>\n Carry out the reduction\/alkylation and digestion steps for the reference solution in the same manner as for the test solution.<\/p>\n CHROMATOGRAPHIC SEPARATION [Store the solutions at 2-8 \u00b0C]<\/h4>\nLiquid chromatography (2.2.29). Store the solutions at 2-8 \u00b0C.<\/p>\n Column:<\/p>\n \u2014 size: l = 0.15 m, \u00d8 = 2.1 mm;<\/p>\n \u2014 stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 \u03bcm) with a pore size of 30 nm;<\/p>\n \u2014 temperature: 40 \u00b0C.<\/p>\n Mobile phase:<\/p>\n \u2014 mobile phase A: to 1000 mL of water for chromatography R, add 1 mL of trifluoroacetic acid R and mix;<\/p>\n \u2014 mobile phase B: to 1000 mL of acetonitrile R1, add 1 mL of trifluoroacetic acid R and mix;<\/p>\n \n\n\nTime
\n(min)<\/td>\n | Mobile phase A
\n(per cent V\/V)<\/td>\n | Mobile phase B
\n(per cent V\/V)<\/td>\n<\/tr>\n | \n| 0 – 5<\/td>\n | 100<\/td>\n | 0<\/td>\n<\/tr>\n | \n| 5 – 10<\/td>\n | 100 \u2192 98<\/td>\n | 0 \u2192 2<\/td>\n<\/tr>\n | \n| 10 – 15<\/td>\n | 98 \u2192 95<\/td>\n | 2 \u2192 5<\/td>\n<\/tr>\n | \n| 15 – 60<\/td>\n | 95 \u2192 80<\/td>\n | 5 \u2192 20<\/td>\n<\/tr>\n | \n| 60 – 95<\/td>\n | 80 \u2192 78<\/td>\n | 20 \u2192 22<\/td>\n<\/tr>\n | \n| 95 – 130<\/td>\n | 78 \u2192 67<\/td>\n | 22 \u2192 33<\/td>\n<\/tr>\n | \n| 130 – 150<\/td>\n | 67 \u2192 65<\/td>\n | 33 \u2192 35<\/td>\n<\/tr>\n | \n| 150 – 155<\/td>\n | 65 \u2192 30<\/td>\n | 35 \u2192 70<\/td>\n<\/tr>\n | \n| 155 – 160<\/td>\n | 30 \u2192 0<\/td>\n | 70 \u2192 100<\/td>\n<\/tr>\n | \n| 160 – 165<\/td>\n | 0<\/td>\n | 100<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n Flow rate 0.2 mL\/min.<\/p>\n Detection Spectrophotometer at 214 nm.<\/p>\n Autosampler Set at 4 \u00b0C.<\/p>\n Injection 100 \u03bcL.<\/p>\n Identification of peaks: Use the chromatogram supplied with golimumab CRS to identify peaks 1 to 40.<\/p>\n System suitability Reference solution:<\/p>\n \u2014 the chromatogram obtained is qualitatively similar to the chromatogram supplied with golimumab CRS and peaks 1 to 40 are clearly visible;<\/p>\n \u2014 peaks 26 and 27 are separated as shown in the chromatogram supplied with golimumab CRS.<\/p>\n Results:<\/p>\n \u2014 the profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution except for peaks 12, 13, 29 and 30 that show known variability from batch to batch;<\/p>\n \u2014 no additional peak in the chromatogram obtained with the test solution has a peak area ratio of more than 0.08 with reference to peak 20.<\/p>\n TESTS<\/h2>\npH (2.2.3)<\/h3>\nAs approved by the competent authority.<\/p>\n Deamidation analysis<\/h3>\nProceed as described for peptide mapping, with the following modifications.<\/p>\n NOTE: because of the instability of the solutions, analysis is limited to 3 samples within a run.<\/p>\n Column:<\/p>\n \u2014 size: l = 0.25 m, \u00d8 = 2.1 mm;<\/p>\n \u2014 stationary phase: end-capped octadecylsilyl silica gel for chromatography R (3.5 \u03bcm) with a pore size of 30 nm;<\/p>\n \u2014 temperature: 50 \u00b0C.<\/p>\n Mobile phase:<\/p>\n \u2014 mobile phase C: mix 700 mL of mobile phase A and 300 mL of mobile phase B;<\/p>\n \u2014 mobile phase D: mix 300 mL of mobile phase A and 700 mL of mobile phase B;<\/p>\n \n\n\nTime
\n(min)<\/td>\n | Mobile phase C
\n(per cent V\/V)<\/td>\n | Mobile phase D
\n(per cent V\/V)<\/td>\n<\/tr>\n | \n| 0 – 15<\/td>\n | 100<\/td>\n | 0<\/td>\n<\/tr>\n | \n| 15 – 75<\/td>\n | 100 \u2192 87.5<\/td>\n | 0 \u2192 12.5<\/td>\n<\/tr>\n | \n| 75 – 80<\/td>\n | 87.5 \u2192 0<\/td>\n | 12.5 \u2192 100<\/td>\n<\/tr>\n | \n| 80 – 85<\/td>\n | 0<\/td>\n | 100<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n Identification of peaks: Use the chromatogram supplied with golimumab CRS to identify peaks 1-8 corresponding to native and deamidated isoforms at LC Asn93 and HC Asn43.<\/p>\n System suitability Reference solution:<\/p>\n \u2014 the chromatogram obtained is qualitatively similar to the chromatogram supplied with golimumab CRS and peaks 1 to 7 are clearly visible;<\/p>\n \u2014 resolution: minimum 1.1 between peaks 6 and 7.<\/p>\n Result In the chromatogram obtained with the test solution, peaks 1 to 7 are clearly visible; the retention times of peaks 1-7 in the chromatogram obtained with the test solution correspond to those of peaks 1-7 in the chromatogram obtained with the reference solution.<\/p>\n Calculate the relative peak areas of the individual peaks corresponding to native and deamidated isoforms with reference to the sum of the areas of peaks 1-8.<\/p>\n Calculate the percentage contents of total deamidated LC Asn93 and total deamidated HC Asn43, using the following expressions:<\/p>\n ![\"Golimumab](\"https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/3-141-300x163.jpg\") <\/p>\n
APi<\/sub> = area of peaks 1-8.<\/p>\nNOTE: peak 8 elutes as a cluster and is integrated as such.<\/p>\n Limits:<\/p>\n \u2014 total deamidated LC Asn93: maximum 5.8 per cent;<\/p>\n \u2014 total deamidated HC Asn43: maximum 79 per cent;<\/p>\n \u2014 sum of peaks 4 and 5: maximum 30 per cent.<\/p>\n Related proteins<\/h3>\nCapillary electrophoresis (2.2.47) under both reducing and non-reducing conditions.<\/p>\n Sample buffer (reducing conditions): Dissolve 1 g of sodium dodecyl sulfate R in tris(hydroxymethyl)aminomethane buffer solution pH 9.0 R1 and dilute to 100.0 mL with the same solution.<\/p>\n Sample buffer (non-reducing conditions): To 80 mL of water R, add 523 mg of bis-tris methane R. Add 10 mL of a 100 g\/L solution of sodium dodecyl sulfate R. Adjust to pH 7.0 with a 210 g\/L solution of citric acid monohydrate R and dilute to 100 mL with water R.<\/p>\n Test solution: Dilute the preparation to be examined with water R to obtain a concentration of 10 mg\/mL:<\/p>\n \u2014 reducing conditions: mix 25 \u03bcL of the solution and 68 \u03bcL of sample buffer (reducing conditions). Add 2 \u03bcL of a 5 mg\/mL solution of 10 kDa internal standard and 5 \u03bcL of 2-mercaptoethanol R. Incubate at 80 \u00b0C for 2 min;<\/p>\n \u2014 non-reducing conditions: mix 30 \u03bcL of the solution and 155 \u03bcL of sample buffer (non-reducing conditions). Add 5 \u03bcL of a 5 mg\/mL solution of 10 kDa internal standard and 10 \u03bcL of a 16 g\/L solution of N-ethylmaleimide R. Incubate at 75 \u00b0C for 5 min.<\/p>\n Reference solution (a) Dissolve the contents of a vial of golimumab CRS in water R to obtain a concentration of 10 mg\/mL:<\/p>\n \u2014 reducing conditions: mix 2.5 \u03bcL of the solution and 75.5 \u03bcL of sample buffer (reducing conditions). Add 2 \u03bcL of a 5 mg\/mL solution of 10 kDa internal standard, 5 \u03bcL of a 5 mg\/mL solution of carbonic anhydrase, 5 \u03bcL of a 5 mg\/mL solution of bovine albumin R and 5 \u03bcL of a 5 mg\/mL solution of \u03b2-galactosidase. Add 3 \u03bcL of a 46 g\/L solution of iodoacetamide R. Incubate at 80 \u00b0C for 2 min;<\/p>\n \u2014 non-reducing conditions: mix 5 \u03bcL of the solution and 150 \u03bcL of sample buffer (non-reducing conditions). Add 5 \u03bcL of a 5 mg\/mL solution of 10 kDa internal standard, 10 \u03bcL of a 5 mg\/mL solution of carbonic anhydrase, 10 \u03bcL of a 5 mg\/mL solution of bovine albumin R and 10 \u03bcL of a 5 mg\/mL solution of \u03b2-galactosidase. Add 10 \u03bcL of a 16 g\/L solution of N-ethylmaleimide R. Incubate at 75 \u00b0C for 5 min.<\/p>\n Reference solution (b): Dissolve the contents of a vial of golimumab CRS in water R to obtain a concentration of 10 mg\/mL:<\/p>\n \u2014 reducing conditions: mix 75 \u03bcL of the solution and 204 \u03bcL of sample buffer (reducing conditions). Add 6 \u03bcL of a 5 mg\/mL solution of 10 kDa internal standard and 15 \u03bcL of 2-mercaptoethanol R. Incubate at 80 \u00b0C for 2 min;<\/p>\n \u2014 non-reducing conditions: mix 90 \u03bcL of the solution and 465 \u03bcL of sample buffer (non-reducing conditions). Add 15 \u03bcL of a 5 mg\/mL solution of 10 kDa internal standard and 10 \u03bcL of a 16 g\/L solution of N-ethylmaleimide R. Incubate at 75 \u00b0C for 5 min.<\/p>\n Capillary:<\/p>\n \u2014 material: uncoated fused silica;<\/p>\n \u2014 size: total length = about 30.2 cm, effective length = 20 cm, \u00d8 = 50 \u03bcm.<\/p>\n Temperature 25 \u00b0C.<\/p>\n Gel buffer Use a formulation suitable for a sieving range of approximately 10-225 kDa.<\/p>\n Acidic wash solution dilute hydrochloric acid R1.<\/p>\n Basic wash solution 4 g\/L solution of sodium hydroxide R.<\/p>\n Detection Spectrophotometer at 220 nm.<\/p>\n Autosampler Set at 25 \u00b0C.<\/p>\n Injection Electrokinetically at 5 kV reversed polarity for 20 s.<\/p>\n Migration: Apply a voltage of 15 kV reversed polarity for 35 min using the gel buffer as the electrolyte in both buffer reservoirs.<\/p>\n Migration time:<\/p>\n \u2014 reducing conditions: light chain = about 15 min; heavy chain = about 19 min;<\/p>\n \u2014 non-reducing conditions: intact IgG = about 27 min.<\/p>\n Calculate corrected areas of all peaks with a migration time greater than 11 min, using the following expression:<\/p>\n Ld<\/sub>\u00d7A\/t<\/p>\nLd<\/sub> = capillary length to detector;<\/p>\nA = uncorrected peak area;<\/p>\n t = migration time.<\/p>\n System suitability:<\/p>\n \u2014 in the electropherogram obtained with reference solution (a), all 5 peaks corresponding to the molecular weight markers and golimumab are clearly visible; plot the migration times of the markers versus their logarithmic molecular weight; the coefficient of determination (R ) calculated for the regression line is not less than 0.98;<\/p>\n \u2014 reducing conditions: the electropherogram obtained with reference solution (b) is qualitatively similar to the electropherogram supplied with golimumab CRS;<\/p>\n \u2014 non-reducing conditions: the electropherogram obtained with reference solution (b) is qualitatively similar to the electropherogram supplied with golimumab CRS.<\/p>\n Determine the molecular weight of the principal components of the test solution.<\/p>\n Result:<\/p>\n \u2014 the profile of the electropherogram obtained with the test solution is qualitatively similar to that of the
\nelectropherogram obtained with reference solution (b), except for minor peaks, that may be absent in the
\nelectropherogram obtained with the test solution. Calculate individual peak areas expressed as a percentage of the sum of all corrected peak areas.<\/p>\n Limits:<\/p>\n Reducing conditions:<\/p>\n \u2014 sum of all peaks other than the peaks due to heavy and light chains: maximum 1.7 per cent;<\/p>\n Non-reducing conditions:<\/p>\n \u2014 sum of all peaks other than the principal peak: maximum 1.7 per cent.<\/p>\n Impurities with molecular masses differing from that of golimumab<\/h3>\nSize-exclusion chromatography (2.2.30): use the normalisation procedure.<\/p>\n Test solution: Dilute the preparation to be examined with the mobile phase to obtain a concentration of 10 mg\/mL.<\/p>\n Reference solution (a): Dissolve the contents of a vial of golimumab CRS in the mobile phase to obtain a concentration of 10 mg\/mL.<\/p>\n Reference solution (b): Dilute 50 \u03bcL of reference solution (a) with the mobile phase to obtain a concentration of 0.5 mg\/mL.<\/p>\n Reference solution (c): Reconstitute a mixture of thyroglobulin, gamma-globulin, ovalbumin, myoglobin and vitamin B12 with 1 mL of water for chromatography R. Further dilute to 10 mL with water for chromatography R.<\/p>\n Precolumn:<\/p>\n \u2014 size: l = 0.04 m, \u00d8 = 6 mm;<\/p>\n \u2014 stationary phase: hydrophilic silica gel for chromatography R (7 \u03bcm).<\/p>\n Column:<\/p>\n \u2014 size: l = 0.30 m, \u00d8 = 7.8 mm;<\/p>\n \u2014 stationary phase: hydrophilic silica gel for chromatography R (5 \u03bcm) with a pore size of 25 nm and of a grade suitable for fractionation of globular proteins in the relative molecular mass range of 10 000 to 500 000 Da.<\/p>\n Mobile phase: Dissolve 11.5 g of sodium dihydrogen phosphate monohydrate R and 31.2 g of disodium hydrogen phosphate heptahydrate R in 800 mL of water for chromatography R. Dilute to 1000.0 mL with water for chromatography R; filter and degas.<\/p>\n Flow rate 1.0 mL\/min.<\/p>\n Detection: Spectrophotometer at 214 nm and at 280 nm.<\/p>\n Autosampler Set at 4 \u00b0C.<\/p>\n Injection 20 \u03bcL.<\/p>\n Relative retention: With reference to golimumab monomer (retention time = about 9 min): aggregates = about 0.84.<\/p>\n NOTE: protein species that elute before the monomer peak are classified as aggregates, while those that elute after the monomer peak are classified as fragments.<\/p>\n System suitability:<\/p>\n \u2014 the chromatogram obtained with reference solution (a) is qualitatively similar to the chromatogram supplied with golimumab CRS;<\/p>\n \u2014 ratio (R) of the sum of the areas of the peaks at 214 nm over the sum of the areas of the peaks at 280 nm in the chromatograms obtained with reference solution (b): minimum 10;<\/p>\n \u2014 ratio of the area of the monomer peak at 214 nm over the area of the monomer peak at 280 nm in the
\nchromatograms obtained with reference solution (b): minimum 10;<\/p>\n \u2014 resolution at 214 nm: minimum 1.2 between the peaks due to gamma-globulin and ovalbumin in the chromatogram obtained with reference solution (c);<\/p>\n \u2014 number of theoretical plates: minimum 3750, calculated for the peak due to vitamin B12 in the chromatogram obtained with reference solution (c) at 214 nm.<\/p>\n Results:<\/p>\n \u2014 the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the principal peak in the chromatogram obtained with reference solution (a).<\/p>\n Calculate the relative area (in per cent) for the sum of peaks due to aggregates and for the sum of peaks due to fragments at 280 nm using the chromatograms obtained at 214 nm and R. Calculate the relative peak area (in per cent) for the sum of all peaks other than the monomer peak in the chromatogram obtained at 280 nm.<\/p>\n Limits:<\/p>\n \u2014 sum of all peaks other than the monomer peak: maximum 0.40 per cent;<\/p>\n \u2014 aggregates: maximum 0.22 per cent;<\/p>\n \u2014 fragments: maximum 0.15 per cent.<\/p>\n ASSAY<\/h2>\nProtein (2.5.33, Method 1)<\/h3>\nTest solution Dilute the preparation to be examined with a 9 g\/L solution of sodium chloride R to obtain a concentration of about 0.5 mg\/mL. Prepare and analyse each preparation in triplicate.<\/p>\n Record the UV spectrum at 280 nm Measure the value at the absorbance maximum of 280 nm.<\/p>\n Calculate the protein content, taking the specific absorbance to be 14.0.<\/p>\n Potency<\/h3>\nThe potency of golimumab is determined by comparison of dilutions of the test preparation with dilutions of
\ngolimumab BRP using a suitable cell-based assay based on the inhibitory action of golimumab on the biological activity of TNF-\u03b1 with a suitable readout for assessing this inhibitory effect.<\/p>\n The following procedure is given as an example.<\/p>\n Carry out a cell proliferation assay based on the ability of golimumab to block TNF-induced inhibition of murine fibrosarcoma WEHI-13VAR cell proliferation. The WEHI-13VAR cells (ATCC CRL-2148) are incubated with varying dilutions of test and reference preparations of golimumab in the presence of TNF-\u03b1. Cell growth is assessed by a staining method using a tetrazolium salt (MTS), which is converted by cellular dehydrogenases to a coloured formazan product. The amount of released formazan is then measured spectrophotometrically and is directly proportional to the number of living cells.<\/p>\n Assay medium: To 450 mL of RPMI 1640, add 50 mL of heat-inactivated foetal bovine serum, 5.0 mL of 29.24 g\/L solution of L-glutamine R (0.2 M), 5.0 mL of 11 g\/L solution of sodium pyruvate R (100 mM) and 5.0 mL of non-essential amino acids solution (100 \u00d7).<\/p>\n Modified D-PBS: Dulbecco’s phosphate buffered saline (D-PBS), without calcium, or magnesium.<\/p>\n Test solution: Dilute the preparation to be examined with water R to obtain a concentration of about 10 mg\/mL. Further dilute with assay medium to obtain a concentration of about 500 ng\/mL. Prepare in triplicate.<\/p>\n Reference solution: Dissolve the contents of a vial of golimumab BRP in assay medium to obtain a concentration of about 500 ng\/mL. Prepare in triplicate.<\/p>\n TNF-\u03b1 working solutions Dissolve the contents of a vial of TNF-\u03b1 according to the manufacturer’s instructions to obtain a concentration of 500 ng\/mL (working solution A). Dilute 50 \u03bcL of this solution with 450 \u03bcL assay medium to obtain a concentration of 50 ng\/mL (working solution B). Dilute 336 \u03bcL of this solution with 10 mL of assay medium to obtain a concentration of 1.68 ng\/mL (\u2018TNF-\u03b1 for neutralisation’). As the biological activity of TNF-\u03b1 is likely to vary between different suppliers and also between different batches from the same supplier, this should be controlled by use of an appropriate standard (e.g. WHO International Standard for TNF-\u03b1).<\/p>\n Tetrazolium salt solution Dissolve 500 mg of tetrazolium salt R in modified D-PBS. Adjust to pH 7.0-7.5 with a 150 g\/L solution of sodium hydroxide R. Further dilute to 250 mL with modified D-PBS to obtain a concentration of 2 mg\/mL. To 8 mL of this solution, add 400 \u03bcL of a 0.96 mg\/mL solution of phenazine methosulfate R and mix thoroughly.<\/p>\n Method.<\/p>\n Plate preparation Each replicate dilution of the test and reference solutions is placed in duplicate on the plate. Add 75 \u03bcL of assay medium to the wells designated for \u2018TNF-\u03b1 cytotoxicity curve’ (columns 1-12, row A) on a 96-well microplate. Add 90 \u03bcL of TNF-\u03b1 working solution B to well A1. Transfer 15 \u03bcL from A1 through to A12. Discard 15 \u03bcL of solution from A12. Add 75 \u03bcL of assay medium to the wells designated for \u2018cells only’ control (columns 1-6, row H). Add 50 \u03bcL of assay medium and 25 \u03bcL of TNF-\u03b1 for neutralisation to the wells designated for \u2018cells + TNF-\u03b1’ control (columns 7-12, row H). Add 50 \u03bcL of assay medium to the sample wells (columns 2-12, rows B-G) and 100 \u03bcL of the test or reference solutions (column 1, rows B-G). Further prepare a series of 2-fold dilutions (columns 2-12, rows B-G), by removing 50 \u03bcL from column 1 and transferring to the adjacent well in column 2, repeating for subsequent wells. Then, add 25 \u03bcL of TNF-\u03b1 for neutralisation (columns 1-12, rows B-G). Incubate at 36-38 \u00b0C for 1 h in an incubator using 4-7 per cent CO2<\/sub>.<\/p>\nCell preparation Prepare a suspension of WEHI-13VAR cells containing 2 \u00d7 10 cells per millilitre, using assay medium containing 8 \u03bcg\/mL of actinomycin D.<\/p>\n Plating cells Add 25 \u03bcL of the cell suspension to each well maintaining the cells in a uniform suspension during addition.<\/p>\n Incubate at 36-38 \u00b0C for 20-24 h in an incubator using 4-7 per cent CO2<\/sub>.<\/p>\nAddition of tetrazolium salt Add 20 \u03bcL of tetrazolium salt solution to each well and re-incubate at 36-38 \u00b0C for 105-135 min using 4-7 per cent CO2<\/sub>. Add 25 \u03bcL of a 100 g\/L solution of sodium dodecyl sulfate R. Estimate the quantity of formazan produced using a microtitre well plate reader at 490 \u00b1 5 nm, 10-30 min after addition of SDS.<\/p>\nCalculate the potency of the preparation to be examined using the four-parameter logistic curve model (see general chapter 5.3).<\/p>\n System suitability:<\/p>\n \u2014 the TNF-\u03b1 cytotoxicity curve corresponds to a sigmoid curve;<\/p>\n \u2014 the coefficient of determination calculated for the TNF-\u03b1 control cytotoxicity curve (R ) is not less than 0.97;<\/p>\n \u2014 the standard curve is a sigmoid curve with well-defined upper and lower asymptotes and linear part;<\/p>\n \u2014 the values of the upper and lower asymptotes of the standard curve are within the pre-defined range established from the minimum and maximum values of the corresponding controls;<\/p>\n \u2014 the coefficient of determination calculated for the standard curve (R ) is not less than 0.97;<\/p>\n \u2014 maximum (\u2018cells only’ control) value to minimum (\u2018cells + TNF-\u03b1’ control) value ratio: minimum 3.0.<\/p>\n Result: The estimated potency is not less than 80 per cent and not more than 130 per cent relative to the reference solution.<\/p>\n In addition, the WEHI-164 cytotoxicity assay (2.7.26, Procedure B) has been found suitable. Carry out the assay as described with the following modifications.<\/p>\n Reference solution: Dissolve the contents of a vial of golimumab BRP in sterilised water for injections R to obtain a concentration of about 640 ng\/mL. Analyse 2 independent dilutions per plate.<\/p>\n STORAGE<\/p>\n In an airtight container, under approved conditions.<\/p>\n LABELLING<\/p>\n The label states the content in milligrams of protein per millilitre.<\/p>\n","protected":false},"excerpt":{"rendered":" (Ph. Eur. monograph 3103) C6530H10068N1752O2026S44 (dimer without glycosylation) Mr approx. 147 kDa (dimer without glycosylation) Action and use Monoclonal antibody (TNF); treatment of arthritis. DEFINITION Solution of a monoclonal antibody consisting of a bisdisulfide dimer of 1342 amino acid residues with a molecular weight of approximately 150 kDa, which binds with high affinity to both…<\/p>\n","protected":false},"author":3,"featured_media":13434,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[174],"tags":[],"class_list":["post-13369","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-medicinal-substances"],"acf":[],"_links":{"self":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/13369","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/comments?post=13369"}],"version-history":[{"count":4,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/13369\/revisions"}],"predecessor-version":[{"id":13436,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/13369\/revisions\/13436"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media\/13434"}],"wp:attachment":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media?parent=13369"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/categories?post=13369"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/tags?post=13369"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}} | | |