Recent evidence suggests that certain cation-exchangers can cause unfolding and aggregation of antibodies. For a mAb on the resin Fractogel SO3 (EMD Millipore), two distinct peaks appeared when the protein was bound at low ionic strength, held on the resin for a period of time, and then eluted with a salt gradient. The effects of hold time before elution, protein mass load, and loading salt concentration were determined. Size exclusion chromatography confirmed the presence of both high molecular weight species and monomers in the late eluting peak. Reversibility of the aggregation was indicated by dynamic light scattering measurements showing decreasing size of the isolated aggregates with time. Hydrogen-deuterium exchange mass spectrometry (HXMS) was used to determine, at the peptide level, solvent accessibility of each eluted species relative to the native monomer. Early-eluted and late-eluted monomer had the same pattern of solvent accessibility suggesting that they have the same conformation. On the other hand, a significant number of residues in the aggregates showed greater solvent exposure relative, suggesting that the aggregates contain partially unfolded regions. The residues with greatest increased solvent exposure were located in the Fc region of the mAb. On-column HXMS was further used to determine the unfolding regions while the protein was bound to the resin. These HXMS results showed a pattern of increased solvent accessibility similar to that of the aggregates in solution with the most unfolded peptides located in the Fc region. Overall, the results suggest that the CEX resin studied has a catalytic effect on unfolding the mAb. The unfolded species is held more tightly by the resin than the native monomer requiring a higher salt concentration for elution. Upon reaching a higher salt concentration, this species elutes, in part as a refolded monomer with the native conformation, and in part as aggregates.

Antibody aggregation and unfolding on cation exchange chromatography media

247th National Meeting (2014)

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