Establishment Of Bioactivity Detection Methods For The Bevacizumab Biosimilars And Detection Of Fermentation Process Products With These Methods

Vascular endothelial growth factor（VEGF）is one of the important regulating angiogenesis factor,its expression in tumor tissues with high level.Tumor blood vessels are the important factors in tumorigenesis,development and metastasis.If no new blood vessels are generated,the primary tumor growth will not exceed 2 mm,and the tumor metastasis will not be realized.Therefore,the blocking of VEGF has become a research hotspot in tumor therapy.Bevacizumab is a humanized monoclonal antibody against the target of VEGF.It can bind to VEGF specifically and inhibit the downstream signal pathway,so that VEGF loses its biological activity and inhibits the growth of tumor blood vessels.With the advent of the bevacizumab patent period,and clinical demand continues to improve,a worldwide trend of bevacizumab is being developed.In the development and production of monoclonal antibiotics,the similarity evaluation of candidate drugs and original research drugs is an important criterion to evaluate the quality,efficacy and safety of monoclonal antibodies.Biological activity is one of the key quality attributes of biosimilars,and a stable and reliable biological activity detection method should be established to control the quality of biosimilars.The purpose of this study is to establish the bioactivity detection methods for bevacizumab biosimilars.In this study,cell proliferation inhibition test of human umbilical vein endothelial cells（HUVEC）and surface plasmon resonance（SPR）were used to optimize the detecting conditions,including the optimal concentration of VEGF,initial concentration of samples,the suitable dilution ratio,gradient of samples and the best concentration of 1igand and analyte.The detection methods have been verified and we used the methods to detect bioactivity of candidate drug samples.The results showed as follows:1.The optimal conditions of cell proliferation inhibition test were 30 ng/ml VEGF,3μg/ml samples,3×dilution and 8 gradient.This method was validated possesses excellent specificity,as only bevacizumab biosimilars showed typical dose-response curve with more than 97%of R²value from six repeated tests.The relative standard deviation（RSD）of the six repeated test results for EC₅₀0 were all less than 25%.The specific activity of accuracy and intermediate precision validation test within the specific activity range of 70%¹30%.2.In the assay of SPR,the optimized concentrations of ligand and analyte were 0.5μg/ml and2.3μg/ml.3.The optimum culture conditions were determined by optimizing the culture conditions,including the culture temperature,PH value and rotational speed in the preparation process of the bevacizumab biosimilars.The culture temperature was determined to be 37℃,pH value was7.1,the initial rotation speed was 200 rpm,and the seventh day was adjusted to 300 rpm.4.Using HUVEC proliferation inhibition method,the activity detection of the fermentation optimization process of bevacizumab anti-generic drugs was carried out,and the specific activity of the candidate drug was 98.47%.At the same time,according to the activity detection method based on SPR technology,this candidate drug was tested,and the combination rate constant ka（1/Ms）value was 8.46E+05,the dissociation rate constant kd（1/s）was 1.61E-04,and the affinity KD value was 1.90E-10.In summary,The specificity,reproducibility,accuracy and intermediate precision of the HUVEC proliferation inhibitory assay were in conformity with the relevant requirements.The affinity value of candidate drug was very close to that of standard substance,and the kinetics curves of candidate drug and standard substance were almost the same by SPR.These two methods we developed were suitable for bioactivity detection of bevacizumab biosimilars and with potential clinical application values.