Recombinant Chimeric Anti-cd20 Monoclonal Antibody Primary Structure Function Of Conclusive Evidence Of Its Sugar Chains Preliminary Study

Monoclonal antibody drugs show the tendency of biological products and are the most expected and potential industry in 21 century. Its global annual sales had increased from 310 million to 10.3 billion between 1997 and 2004, and it may fairly be predicted that it would be 30.3 billion in 2010. The share of monoclonal antibody drugs in biotech drug also had increased from 1/5 to 1/4 between 2000 and 2004, and is predicted to be 1/3 in 2010. Monoclonal antibody drugs possess enormous economic value and social value.Different from recombinant cytokines, monoclonal antibody drugs have high molecular mass, complicated conformation and frequent post-translational modification, which make the quality control more difficult. Thus it is both necessary and important to establish suitable quality control requirements for them. For that purpose, we selected a chimeric anti-CD20 monoclonal antibody as an example, identified its primary structure and researched the function of its oligosaccharides.In the first part of this paper, the primary structure of the chimeric anti-CD20 monoclonal antibody was identified. The exact molecular masses of its light and heavy chains with or without oligosaccharides were measured by LC-MS, all of the relative error were less than 0.01%. Results showed that the heavy chains are modified by N-linked core-fucosylated biantennary oligosaccharides, their major structures have three characteristics, all without N-acetylneuraminic acid, and their content is between 2.0%～2.4%. Being treated with pyroglutamate aminopeptidase, the N-terminals of light and heavy chains were sequenced by Edman degradation chemistry. By peptide mapping, the amino acid sequence was identified, the N-glycosylation site was found at Asn301, and 10 inter-chain disulfide bonds were identified.In the second part, the oligosaccharides' effects on structure of the monoclonal antibody were identified. Papain digesting test showed that cleavage sites changed after removing oligosaccharide, which indicated the alteration of structure in hinge region. Circular dichroism spectra also revealed that there were impalpable alterations in secondary structure.In the third part, the oligosaccharides' effects on biological function of the monoclonal antibody were identified. Results showed that the activity of antibodies" CDC disappeared after removing oligosaccharides, thus glycosylation is essential to it. CDC's activity depends on 2 kinds of protein-protein interactions: between antibody and CD20 antigen, and between antibody-antigen complex and complement. In following tests, mechanism of the effect was studied.The binding capabilities between CD20 antigen and antibodies with or without oligosaccharides were assessed by competition assy. Results showed that binding capability decreased 40% after removing oligosaccharides, which indicated that removing oligosaccharides influenced the binding capability between CD20 antigen and antibody. The binding capability between antibody and complement was assessed by BIAcore, they do not interact with each other obviously as complement binding site in free antibody is masking.In this study, we identified the primary structure of the antibody precisely, several methods were established and developed.This methods could be referenced for the quality control of recombinant monoclonal antibodies. The study on oligosaccharides shows the importance of the glycol-proteins either for antibodies' conformation or biological function, which could be referenced for the quality control of recombinant glycoproteins.