Optimization And Validation Of Detection Methods For Biological Activity Of Recombinant Humanized Anti-CD52 Monoclonal Antibody

The evaluation of the biological activity of drugs is an important part of the drug research and development process,and is the key evaluation point to determine whether a drug has research value and determines the research direction.The evaluation of the in vitro biological activity of the recombinant antibody can provide direction for optimization of the process research of the product and ensure the quality control in the production process.In this dissertation,a recombinant humanized anti-CD52 monoclonal antibody was used as the target drug to establish and optimize the detection method of ATP assay for drug-complement dependent cytotoxicity.Recombinant humanized anti-CD52 monoclonal antibody was cloned andfrom Campath,an existing marketed drug abroad.When the antibody CDC activity was measured,Campath was used as a reference substance,and the relative CDC activity of both antibodies was used to evaluate the biological activity of the antibody.Determination of antibody CDC activity by ATP method is based on the determination of adenosine triphosphate(ATP)concentration using the luciferase system Since ATP rapidly degrades after cell death,its concentration is related to the number of cells,and the intensity of fluorescein light is linear to the ATP concentration.Since the bias of each level in biometrics can have a huge impact on the measurement results,limiting the trend of each factor’s deviation will help improve the accuracy of the method.Through the selection and screening of target cells,the target cells most suitable for the detection will be selected.,Optimize key factors such as reaction system,standard curve,and complement to obtain stable and accurate detection methods and verified.Results After the monoclonal antibody was digested,the peptides were separated and detected by liquid chromatography.The liquid chromatograms of the two antibodies were consistent,and the retention times of the CDRs were almost the same.The monoclonal antibody glycosyl group was separated from the antibody by glycosidase and labeled with 2-AB fluorescence.The identity of the sugar component was determined by HPLC retention time,and the content of each sugar component was calculated based on the peak area.The two monoclonal antibodies were glycosylated.The modification pattern is basically the same,and the content of each component is basically similar.It can be confirmed that the recombinant humanized anti-CD52 monoclonal antibody is confirmed by the structure and commercially available Campath and the same peptide map and glycosylation modification pattern have similar antibody structure and function.Commercial Campath can be used as a relative activity reference product for CDC.CD52-FITC labeled HUT78,MC/CAR and Ramos cells were detected by flow cytometry to compare the expression and stability of CD52 antigen on the cell surface.HUT78,MC/CAR and Ramos cells all expressed high levels of CD52 antigen,and the expression levels of CD52 antigen were MC/CAR,HUT-78 and Ramos cells in descending order.The stability of CD52 antigen expression from high to low was Ramos,MC/CAR and HUT78 cells.The optimization and validation test of the recombinant humanized CD52 monoclonal antibody CDC cytotoxicity showed that: Ramos cells are the optimal target cells for CDC cytotoxicity detection;Specificity verification of isotype control antibody human IgG has no reaction with target cells and no killing effect.The reference product Campath was prepared into 50%,75%,100%,125% and 150% of the theoretical titer samples.After the test,the relative activity was calculated and compared with the theoretical titer.The parallel experiment was conducted 3 times to obtain the sample.The recovery rate of the method was between 90% and 115%.Between the three parallel tests,the theoretical titer of the 5 groups was relatively within 6% relative to the CDC activity and the recovery RSD;the relative CDC activity was calculated by repeating the experiment 6 times with the reference product Campath.Between 94% and 113%,RSD values were less than 7%;daytime precision and personnel precision were less than 15%.Conclusion Through the optimization and verification of the recombinant humanized anti-CD52 monoclonal antibody CDC activity method,it is shown that the method has high accuracy and precision,and the specificity is good.The linearity and durability are in line with the requirements.The method is simple and easy to use,can provide fast and accurate evaluation of anti-CD52 antibody activity in vitro,provide powerful data basis for the development of anti-CD52 antibody drugs,and clear the direction of process research and development to promote the early development and application of drugs.