Aggregate formation of therapeutic IgG mAbs during Cation Exchange Chromatography (CEX) has been frequently observed as a downstream purification challenge. To gain a thorough mechanistic understanding, this study examined the on-column aggregation behavior of two therapeutic proteins (IgG1 and IgG4) on three CEX resins of different properties and structures (Capto SP ImpRes, Fractogel EMD SE Hicap, and POROS XS), and assessed the formation of high molecular weight (HMW) species in CEX step when operated in bind/elute mode. In addition, protein melting temperatures before (TmSolution) and after chromatographic adsorption on the resins (TmResin) were measured by various techniques such as differential scanning fluorimetry (DSF) and differential scanning calorimetry (DSC) to characterize the structural stability of the adsorbed proteins. Results showed that both IgG1 and IgG4 mAbs have lower TmResin than TmSolution, indicating partial unfolding of the adsorbed proteins, to a various extent for the CEX resins evaluated here. Our data also indicated that TmResin is correlated reasonably well to the level of HMW formation on each CEX resin. Furthermore, utilizing a novel fluorescence-based approach, the overall hydrophobicity of these CEX resins were quantified and manifested to be useful in correlating the TmResin and HMW data. This study provides insight into the on-column aggregation behaviors for different mAb subclasses on CEX resins of various properties, and the improved understanding can facilitate the development of efficient and robust CEX processes.

Hydrophobicity of CEX resins and Its Impact on mAb aggregation

2019 Spring National Meeting

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