Characterization of charge variants in anti-CGRP monoclonal antibodies
Objective To perform full characterization of an anti-CGRP monoclonal antibody and its charge variants.Methods The charge variants of the anti-CGRP mAb were separated and collected through ion exchange chromatography(IEC),and then the anti-CGRP mAb and the components of collected charge variants were characterized in terms of molecular weight,light and heavy chain subunits,and post-translational modification by LC-MS/MS.Results The charge variants of the anti-CGRP mAb collected by IEC were divided into main peak,acidic peaks(A1-A3)and basic peaks(B1-B4).The relative molecular weight of the anti-CGRP mAb(mainly N-glycoform A2G1F and A2G0F)was 147 103 by LC-MS.Detailed glycopeptide analysis revealed that the peptide containing the N-linked glycosylation site was TKPREEQFNSTYR,and the N-linked glycosylation site was located at N296.Additionally,A2G0F(＞30％)and A2G1F(＞35％)accounted for the largest proportion of N-glycans of the charge variants,and all the acidic charge variants exhibited sialic acid modifications.Deamidation was detected across all charge variants,with a higher incidence in the acidic variants.The oxidation level at Trp109 within the FR region of the heavy chain was the highest,and the acidic variants typically exhibited a higher oxidation proportion compared to the basic variants.Furthermore,the occurrence of pyroglutamate cyclization was significantly higher in acidic variants.The glycation sites of lysine of the anti-CGRP mAb were relatively abundant,with notable glycation propor-tions at light chain K1 88 and heavy chain K149 and K245.Conclusion The comprehensive characterization analysis shows that the acidic variants of the anti-CGRP mAb exhibit a higher proportion of sialic acid modification,oxidation,and pyroglutamate cyclization compared to the basic variants,and the sites and relative abundance of each modification are successfully identified.These information helps to assess the necessity for further forced drug degradation studies and develop the rational approach for drug quality control.
migrainecalcitonin gene related peptidecharge variantsmass spectral characterizationpost-translational modification
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维普
