Strategy to Rapidly and Comprehensively Characterize Innovator Biologics and Their Biosimilars

摘要

Extensive primary sequence and PTM analysis was performed on 5 lots of bevacizumab: commercial Avastin from the U.S., commercial Avastin from the E.U., and 3 experimental biosimilar lots. Byonic and Byologic software tools were employed to perform this analysis and produce automated reports. PTMs reported here included: (1) oxidation, (2) deamidation, (3) glycosylation, and (4) glycation. Relative baseline oxidation and deamidationratios at certain sites on the antibodies were high. Storage at 4°C for 12 months does not seem to increase the PTMs significantly. Significant differences were seen in glycosylation, and in relative degree of glycation; glycation was considerably higher in Avastin. Absolute quantification of glycationwill require a follow-on study with a digestion enzyme that does not cut at lysine because glycationstrongly affects trypsin digestion efficiency. Major surprises were observed for sequence variants, notably the substitution of cysteine by tyrosine (C to Y). This will disrupt disulfide bonding and three-dimensional structure. Although some very high amounts were found in the experimental biosimilar material, as much as 3% variant was found in Avastin. Our integrated software workflow allows exhaustive analysis of these proteins in a way that makes the data analysis easily manageable. The comprehensive analysis also revealed unexpected information.