Anti-CD52 Antibodies: The Study Of Post-Translational Modification And Process Optimization

Monoclonal antibody?mAb?is widely used as therapeutic protein for its high specificity,low toxicity and long half-time in serum.Compared with chemical drugs,mAb is synthesized and produced in a complex way,which may contribute to PTMs,including glycosylation,deamidation,oxidation,isomerization,C-terminal lysine clipping and N-terminal pyroglutamate formation.These PTMs will attribute to charge heterogeneity as well as a changed structure,therefore,a lower affinity with Fc receptors and targeted antigen.In a word,PTMs have effects on the stability,efficacy and safety of antibodies.PTM is closely related to charge heterogeneity.To analyze the factors of charge heterogeneity,acid/main/basic peak of anti-CD52 antibody was separated by CEX-HPLC and analyzed by UPLC-MS.Resluts showed that PTMs on anti-CD52 antibody included N-297 glycosylation,Asn59/Asn384/Asn325/Asn159 deamidation,Met68/Met252/Met428oxidation,C-terminal lysine clipping and N-terminal pyroglutamate formation.Besides,deamidation attributed to acid variants;oxidation and C-terminal lysine clipping partly attributed to basic variants.Stability studies were critical evidence of product's shelf-time.The stability study was conducted under 25?for 6 months,and an increase in acid variants level was observed.Based on the analysis of PTM and potential influence of pH on PTM,we optimized the storage pH of anti-CD52 antibody:in pH 6.6,the level of acid peak was 20.9%;in pH 7.4,the level of acid peak was 49.3%.Therefore,anti-CD52 antibody has a more stable profile in pH 6.6.Glycosylation is another important PTM,which had no influences on charge heterogeneity during storage.However,glycosylation was flexible during manufactory.Anti-CD52 antibody has one glycosylation site at N-297,the main glycans include G0F,G1F,G0,Man5 and so on.In this study,we used different cell retention devices in perfusion process.The results showed antibodies from these two processes have different Man5 level?9.3±0.47%vs.1.8±0.058%?,which may due to a higher NH₄⁺level in the process with ATF.Besides,a higher level of acid variant was observed in the process of ATF.However,no difference was observed in bioactivities of antibodies from two processes in vitro.Becacuse of complex considerations in process optimation and the lack of in vivo data,further studies were needed for the selection of cell retention device.In this study,we analyzed the PTM of anti-CD52 antibody using UPLC-MS.Besides,we optimized the storage pH and production process based on the characterization of PTM.Our studies will lead to a better understanding of antibody,and lay a basis for the development of antibody in GMP manufactory and clinical studies.