Establishment of biological activity assay method of anti-CD3/CD20 bispecific antibody
Objective To establish a cell-based reporter gene assay(RGA)for bioactivity determination of anti-CD3/CD20 bispecific antibodies.Methods With WIL2-S cell line as target cells and Jurkat-NF-KB-Luc transgenic cell line as effector cells,the RGA for determining the biological activity of anti-CD3/CD20 bispecific antibodies was established by optimizing antibody concentration range,balance time,induction time,effector-to-target ratio,and cell seeding density.The specificity,the accuracy,the precision,the linearity,and the stability of RGA were comprehensively validated according to the guidelines of ICH Q2.Results A dose-response relationship-based assay method for determining the biological activity of anti-CD3/CD20 bispecific antibody was successfully established.The opti-mized conditions were as follows:the initial antibody concentration of anti-CD3/CD20 bispecific antibody of 500 ng/mL,the dilution rate of 1∶4,eight detected concentrations(500,125,31.25,7.81,1.95,0.49,0.12,0.031 ng/mL),the effector-to-target ratio of 4∶1,and the induction time of 4 h.The method possessed good specificity.The relative potency of bispecific anti-CD3/CD20 antibody was 59.63％±4.57％,75.54％±4.05％,99.98％±9.63％,123.90％±5.54％and 142.51％±12.82％for recovery samples under five dilute concentrations,and the corresponding recovery was 93.17％±7.15％,94.42％±5.06％,99.98％±9.63％,99.12％±4.43％and 91.21％±8.21％,respectively.The CV value was 7.67％,5.35％,9.63％,4.47％and 9.00％,respectively,and CV values of the above results were all lower than 10％.The measured potency to expected potency demonstrated good linearity(R2=0.982 3).The method established in this study was sensitive to detect the effect of structural changes of antibodies on biological activity,and had certain guiding significance for the stability test of bispecific antibody.Conclusion A biological activity assay method for anti-CD3/CD20 bispecific antibodies is successfully established using a transgenic cell-based approach.The method demonstrates strong specificity,high accuracy,and good repeatability,and might be used to evaluate the biological activity of anti-CD3/CD20 bispecific antibodies.
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