Mass Spectrometric Determination of Disulfide Linkages in Recombinant Therapeutic Proteins Using Online LC—MS with Electron-Transfer Dissociation.

In the biotechnology industry, the generation of incorrectly folded recombinant proteins, either from an E.coli expression system or from an overexpressed CHO cell line (disuffide 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 disuffide bonds to ensure drug quality. Similar to ECD, disuffide bond cleavages are preferred over peptide backbone fragmentation in ETD. Thus, an online LC-MS strategy combining collision-induced dissociation (CID-MS2), electron-transfer dissociation (ETDMS2), and CID of an isolated product ion derived from ETD (MS2) has been used to characterize disuffide-linked peptides. Disuffide-linked peptide ions were identified by CID and FTD fragmentation, and the disuffide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS2 step. The online LC-MS approach is successfully demonstrated in the characterization of disuffide linkages of recombinant human growth hormone (Nutropin), a therapeutic monoclonalantibody, and tissue plasminogen activator (Activase). The characterization of disuffide-dissociated or partially dissociated peptide ions in the MS2 step is important to assign the disuffide linkages, particularly, for intertwined disuffide bridges and the unexpected disuffide scrambling of tissue plasminogen activator. The disulfide-dissociated peptide ions are shown to be obtained either directly from the ETD fragmentation of the precursors (disuffide-linked peptide ions) or indirectly from the charge-reduced species in the ETD fragmentation of the precursors. The simultaneous observation of disuffide-linked and disuffide-dissociated peptide ions with high abundance not only provided facile interpretation with high confidence but also simplified the conventional approach for determination of disuffide linkages, which often requires two separate experiments (with and without chemical reduction). The online LC-MS with ETD methodology represents a powerful approach to aid in the characterization of the correct folding of therapeutic proteins. [ABSTRACT FROM AUTHOR]