Modulation Of Monoclonal Antibody Charge Variants By CHO Cell Culture Process

Objective:Chinese hamster ovary（CHO）cells were cultured with different metal ions and antioxidants to compare their regulatory effects on the charge variants of monoclonal antibody,and to comprehensively evaluate the feasibility of the process in terms of cell growth performance,protein expression and other quality attributes.The optimal culture conditions were selected.Methods:（1）Fed-Batch culture:The effects of three metal ions(Cu²⁺,Zn²⁺,Fe²⁺)and three antioxidants（catechin,epicatechin,romatinic acid）on the growth of CHO cells were evaluated by cell viability and cell density.（2）Affinity chromatography purification method:the harvested cell supernatant was purified by affinity chromatography for subsequent quality detection.（3）Detection of protein expression and charge heterogeneity:protein expression of CHO cells was determined by Protein A affinity chromatography.The content of charge variants of monoclonal antibody was detected by cation exchange chromatography（CEX）,and the effects of metal ions and antioxidants on the charge variants of monoclonal antibody were investigated.4)Effects of antioxidants on physicochemical properties of monoclonal antibody:1)Using size exchange chromatography（SEC）and capillary electrophoresis-sodium dodecyl sulfate（CE-SDS）was used to detect and analyze the molecular size purity of monoclonal antibody.2)The glycoform distribution and content of the monoclonal antibody were detected by hydrophilic interaction chromatography（HILIC）;3)qualitative and quantitative analysis of the relevant post-translational modifications of monoclonal antibody using tryptic peptide mapping（5）Effect of antioxidants on the relative binding activity of monoclonal antibody:Indirect enzyme linked immunosorbent assay（ELISA）was used to detect the binding activity of monoclonal antibody to antigens.Results:（1）In terms of CHO cell growth and protein expression,all experimental groups grew well during the whole Fed-batch culture period,and the growth curves showed a similar trend.120μM Zn²⁺in metal ions significantly inhibited the expression of protein,while 50μM epicatechin,25μM and 50μM romatinic acid in antioxidants significantly reduced the expression of protein.（2）In terms of charge heterogeneity,CEX-HPLC results showed that 120μM Zn²⁺in metal ions could reduce the content of acid charge variants,but its content was still more than 20%.Acidic charge variants were reduced by all three antioxidants,with 50μM rosmarinic acid having the most significant effect.（3）In terms of molecular size purity,SEC-HPLC results showed that the purity of mabs in the three antioxidant groups was above 98.0%.The results of CE-SDS showed that the purity of the heavy chain of monoclonal antibody was more than 95.0%,and the non-glycosylated heavy chain was about 1.0%at the reduction level.Except for 50μM epicatechin,the purity of mabs in the other experimental groups was above 93.0%at the non-reducing level.（4）In terms of the distribution and proportion of glycoforms,the results of HILIC-UPLC showed that the distribution and proportion of glycoforms in the experimental group and the control group were basically the same.There were five main glycoforms,among which G0F was the most dominant glycoform,the content was about 75%,G1F was about 10%,and G0F-Glc NAc was more than 6.0%.The content of Man5 was about 5.0%and G0 was about 2.0%.（5）Except for 10μM catechin,10μM and 25μM rosmarinic acid,the relative binding activities of other experimental groups were in the range of 70%-150%.（6）In terms of the types,sites and proportions of post-translational modifications,the results of trypsin enzymolysis peptide mapping showed that the post-translational modifications in monoclonal antibody included glutamine cyclization,methionine oxidation,asparagine deamidation and related glycosylation.Methionine at positions 256 and 432 of the heavy chain were modified by about 8%oxidation,respectively.The asparagine residue at position 301 of the heavy chain has a glycosylation site,and the predominant glycoform is A2G0F,accounting for about 67%.Conclusion:In the process of CHO cell culture,the addition of 50μM catechin at Day0 in the early stage of culture can effectively modulate the charge variants of monoclonal antibody while meeting the requirements of other quality attributes of monoclonal antibody,which has certain reference value for improving the quality of antibody-based drugs and can provide a reference for other animal cells expressing antibody-based drugs.