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Journal of chromatography. A

Unfolding and aggregation of a glycosylated monoclonal antibody on a cation exchange column. Part II. Protein structure effects by hydrogen deuterium exchange mass spectrometry.


PMID 25011681

Abstract

Hydrogen-deuterium exchange mass spectrometry (HX-MS) with proteolytic fragmentation is used to determine patterns of unfolding, as measured by increased solvent exposure, with peptide-level resolution for a glycosylated monoclonal antibody both when eluted from a tentacle-type cation exchange column (Fractogel EMD SO3-) and while bound to the resin. Two peaks are obtained when the bound protein is eluted with either a NaCl gradient or with two NaCl steps at increasing concentration. The first, early eluting peak contains only monomeric species whose structure is consistent with the native monomer. The second, late eluting peak contains a mixture of monomeric and aggregated species. The monomeric species in this mixture is also found to have a structure consistent with that of the native mAb, showing no evidence of increased solvent exposure. The aggregated species show instead significant unfolding in areas of the protein structure contained within the Fc region. The same peptides that exhibit the greatest level of solvent exposure in the aggregated species are also found in the fraction of protein that elutes from the resin only at high salt concentration, indicating that the aggregates are formed when the strongly-bound unfolded intermediate is desorbed at high salt. There is no evidence that the unfolded intermediate, formed while the protein is bound on the resin, is present in any of the eluted fractions indicating that, upon desorption from the resin, the intermediate either quickly refolds or forms aggregates which end-up co-eluting with the refolded protein at high salt concentrations.

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