Abstract：OBJECTIVE To establish a mass spectrometry method for the qualitative analysis of anti-CTLA4 monoclonal antibody and its N-linked glycosylation. METHODS The anti-CTLA4 monoclonal antibody was characterized by liquid-mass technique from the aspects of intact molecular weight, subunit molecular weight, amino acid sequence coverage, disulfide bond, N-linked glycosylation site and glycoform. RESULTS The molecular weight of anti-CTLA4 mAb (A2G0F/A2G0F) is 147 992; the deglycosylation molecular weight of anti-CTLA4 mAb is 145 103; the molecular weight of light chain is 23 450; the molecular weight of heavy chain (A2G0F) is 50 548; the heavy chain Fd segment has a molecular weight of 25 355 and the heavy chain sFc segment (A2G0F,1xK_Loss) has a molecular weight of 25 189. Peptide mapping was performed with Trypsin & Chymotrypsin, and the coverage of the amino acid sequence was 100%. The peptide containing the N-linked glycosylations site is EEQYNSTYR, and the N-glycosylation site is located at Asn298 of the heavy chains. Thirteen glycoform were characterized including A2G0F(59.8%), A2G1Fa(18.66%), A2G1Fb(7.28%), A2G2F(3.2%), M5(1.66%), A2S1G0F(1.17%), A1G0F (1.16%), A3G1F(0.95%), A2G0(0.94%), A2S2F(0.78%), A2S1G1F(0.67%), A3G0F(0.53%)and A1G1F(0.51%). CONCLUSION A method for qualitative and quantitative analysis of monoclonal antibody is established.
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