Complete mapping of disulfide linkages for etanercept products by multi-enzyme digestion coupled with LC-MS/MS using multifragmentations including CID and ETD

Complete mapping of disulfide linkages for etanercept products by multi-enzyme digestion coupled with LC-MS/MS using multifragmentations including CID and ETD

The disulfide linkages of two etanercept products, Enbrel® (innovator drug) and TuNEX®,were characterized and compared using a multi-fragmentation approach consisting ofelectron transfer dissociation (ETD) and collision induced dissociation (CID) in combinationwith multi-enzyme digestion protocols (from Lys-C, trypsin, Glu-C, and PNGase F). Multifragmentationapproach allowed multi-disulfide linkages contained in a peptide to beun-ambiguously assigned based on the cleavage of both the disulfide and the backbonelinkages in a MS3 schedule. New insights gained using this approach were discussed. Atotal of 29 disulfides, Cys18-Cys31, Cys32-Cys45, Cys35-Cys53, Cys56-Cys71, Cys74-Cys88,Cys78-Cys-96, Cys98-Cys104, Cys112-Cys121, Cys115-Cys139, Cys-142-Cys157, Cys163-Cys178 in TNFR portion and Cys240-Cys240, Cys246-Cys246, Cys249-Cys249, Cys281-Cys341, Cys387-Cys445 in IgG1 Fc domain, were completely assigned with the demonstrationof the same disulfide linkages between the Enbrel® and TuNEX® products. Thedata showed the higher order structure was preserved throughout the recombinantmanufacturing processes and consistent between the two products.