In the biotechnology industry, the generation of incorrectly folded recombinant proteins, either from an E.coli expression system or from an over-expressed CHO cell line (disulfide scrambling), is often a great concern as such incorrectly folded forms may not be completely removed in the final product. Thus, significant efforts have been devoted to map disulfide bonds to assure drug quality. Similar to ECD, disulfide bond cleavages are preferred over peptide backbone fragmentation in ETD. Thus, an on-line LC-MS strategy combining collision induced dissociation (CID-MS 2 ), electron transfer dissociation (ETD-MS 2 ), and CID of an isolated product ion derived from ETD (MS 3 ) has been used to characterize disulfide-linked peptides. Disulfide-linked peptide ions were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS 3 step. The on-line LC-MS approach is successfully demonstrated in the characterization of disulfide linkages of recombinant human growth hormone (Nutropin), a monoclonal antibody (Herceptin) and tissue plasminogen activator (Activase). The characterization of disulfide-dissociated or partially dissociated peptide ions in the MS 3 step is important to assign the disulfide linkages, particularly, for intertwined disulfide bridges and the unexpected disulfide scrambling of tissue plasminogen activator. The disulfide-dissociated peptide ions are shown to be obtained either directly from the ETD fragmentation of the precursors (disulfide-linked peptide ions) or indirectly from the charge-reduced species in the ETD fragmentation of the precursors. The simultaneous observation of disulfide-linked and disulfide-dissociated peptide ions with high abundance provided not only facile interpretation with high confidence but also simplified the conventional approach for determination of disulfide linkages, which often requires two separate experiments (with and without chemical reduction). The on-line LC-MS with ETD methodology represents a powerful approach to aid in the characterization of the correct folding of therapeutic proteins.

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Recombinant tissue plasminogen (rt-PA) with 35 cysteine residues has been completely assigned by mapping the 17 disulfide linkages and the unpaired cysteine. The result is consistent with the prediction from homology except for the unassigned cysteine, which was identified at Cys83. This cysteine was found to be blocked and paired with either a glutathione or cysteine residue in a ~ 60 : 40 ratio, respectively. The analysis was conducted using a multi-fragmentation approach consisting of ETD and CID, in combination with a multi-enzyme digestion strategy (Lys-C, trypsin, and Glu-C). The disulfide-linked peptides, even those containing N or O-linked glycosylation, could be assigned since the disulfide bonds were still preferably cleaved over the glycosidic cleavages under ETD fragmentation. The use of a multiple and sequential enzymatic digestion strategy was important in producing fragment sizes suitable for analysis. For the analysis of complex intertwined disulfides, the use of CID MS 3 to target partially disulfide dissociated peptides from the ETD fragmentation was necessary for linkage assignment. The ability to identify the exact location and status of the unpaired cysteine (free or blocked with a glutathione or cysteine) could shed light on the activation of rt-PA, upon stimulation by either oxidative or ischemic stress.

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Summary TNK-tPA products from the innovator and follow-on manufacturers were characterized and compared. All tryptic peptides including N-terminal, C-terminal and mutated peptides as well as the disulfide linked peptides were identified, with the demonstration of the same primary sequence and disulfide linkages between the innovator and follow-on products. The three N-linked and one O-linked fucose glycosylation sites were identified. The two N-linked (N103 and N448) and one O-linked fucose (T61) sites were fully glycosylated in both innovator and follow-on products. The other N-linked site (N184) was partially glycosylated and exhibited a ~2.5 fold difference between the innovator (60% occupancy) and follow-on (25% occupancy) products. Since the glycosylation occupancy at this site is known to affect biological activity in the clot lysis assay, this observed difference could cause a concern as to their bioequivalence. The cleavage site for the conversion of the zymogen form to active enzyme was also identified between R275 and I276, with a cleavage of 40% for the innovator and 10% for the follow-on products. Both the % glycosylation occupancy and the chain cleavage were determined by two independent approaches, starting from either the peptide or intact protein separation, with consistent results by both methods. Subtle differences of modifications such as deamidation and oxidation between innovator and biosimilar were shown at M207, M445, M490 and N58, N184. The observation of different extent of oxidation at M207 and deamidation at N184, which could influence the clot lysis activity, were also of potential concern in drug efficacy between the follow-on and innovator products.

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