| Monoclonal antibodies are widely used in the treatment of tumors,autoimmune diseases,organ transplantation rejection,cardiovascular diseases,infections and other diseases due to their low immunogenicity,weak side effects and significant therapeutic effects.However,different glycation and oxidation modifications are occurred during production and clinical use by different physical and chemical factors,and these modifications may change the physicochemical properties of antibodies drugs,as well as may change the biological function.Therefore,it is of great significance to study the relationship between the glycation and oxidation modification of therapeutic monoclonal antibody drugs and their biological functions for the process development,preparation production and quality control.The glycation quantitative analysis method established and validated for recombinant humanized monoclonal antibody in this study was of good precision.The RSD values of 6 samples measured respectively by two experimenters were 0.5%,1.2%,and 0.9% for total 12 samples.There was a good linearity in the range from 0.3125 % to 20%,with the linear correlation coefficient r=0.9999,and the ratio between the absolute value of the y-axis intercept and the response value of 100% glycation content was 0.7%.It also had a good accuracy that the recovery for each level solution were(95.9±1.355)%,(100.3±0.402)%,(102.0±0.336)%,(102.6±0.569)%,(103.7±1.000)%,(106.6±2.369)%,(108.6±1.212)%,and the RSD of the recovery was between 0.3% to 2.2%.The oxidation quantitative analysis method established for recombinant humanized monoclonal antibody in this study was of good precision.The RSD values of 6 samples measured respectively by two experimenters were 4.5%,3.0%,and 6.5% for total 12 samples.There was a good linearity in the range from 3.6% to 28.4%,with the linear correlation coefficient r=1.0000,and the ratio between the absolute value of the y-axis intercept and the response value of 100% glycation content was 2.4%.It also had a good accuracy,that the recovery for each level solution were(100.8±0.413)%,(99.3±1.632)%,(98.4±0.721)%,(99.8±1.0)%,and the RSD of the recovery was between 0.4% to 1.6%.The biological activity assay method for recombinant humanized monoclonal antibody established in this study had strong selectivity that there was dose-curve relationship with VEGF-110,VEGF-121 factors but no dose-curve relationship with VEGF-B,VEGF-C,VEGF-D,PLGF factors,anti-TNF-? monoclonal antibody drugs and samples destroyed by high temperature.The method was of good precision that the RSD value of 6 samples detected respectively by two experimenters were 4.5% and 3.0%,and 6.4% for total 12 samples.There was a good linearity that the linear equation y=1.0973x-0.1055,and the linear correlation coefficient r=0.9950,slope was 1.0973.And the recovery for each level were(106.00±3.46)%,(99.98±6.08)%,(102.27±1.53)%,(91.03±4.58)%,(99.52±5.86)%,and The RSD value of the recovery was between 1.7% to 7.7%.And the method had good durability in the cell number from 8000 cell/m L to 12000 cell/m L,cell generation from 4 to 11 generation,as well as in the different cell batch.This study compared the differences in glycation contents,oxidation contents and biological activity between commercial and self-developed monoclonal antibodies,and the results showed that self-developed monoclonal antibodies was slightly higher in glycation contents than commercial monoclonal antibodies,while the oxidation contents and biological activity were both similar.By measuring the biological activity of recombinant humanized monoclonal antibodies with different glycation contents and different oxidation contents,the relationship between glycation modification and biological function and the relationship between oxidative modification and biological function were established.The results showed that both high contents of glycation and oxidation modification can reduce the biological activity of recombinant humanized monoclonal antibodies,especially,when the glycation increases to more than 20.7% and the oxidation increases to more than 7.8%,the biological activity results will be reduce to below 80%. |
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