The disulfide linkages of two etanercept products, Enbrel® (innovator drug) and TuNEX®, were characterized and compared using a multi-fragmentation approach consisting of electron transfer dissociation (ETD) and collision induced dissociation (CID) in combination with multi-enzyme digestion protocols (from Lys-C, trypsin, Glu-C, and PNGase F). Multi-fragmentation approach allowed multi-disulfide linkages contained in a peptide to be un-ambiguously assigned based on the cleavage of both the disulfide and the backbone linkages in a MS 3 schedule. New insights gained using this approach were discussed. A total 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 demonstration of the same disulfide linkages between the Enbrel® and TuNEX® products. The data showed the higher order structure was preserved throughout the recombinant manufacturing processes and consistent between the two products.

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