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• mAb structural stability decreases when lowering the solution pH from 6.0 to 4.5 • mAb structural stability further decreases when bound on CEX surfaces • mAb-accessible hydrophobicity of CEX resins affects structural stability of bound mAb • A fluorescence-based method is used to quantitatively characterize CEX resin hydrophobicity • Overall mAb/resin interactions and inherent resin hydrophobicity dictate mAb aggregation in CEX step High molecular weight (HMW) aggregate formation of therapeutic monoclonal antibodies (mAbs) during cation-exchange chromatography (CEX) has been frequently observed, and can be a challenge for downstream purification. To gain mechanistic understanding of this phenomenon, aggregate formation in bind-elute CEX for two therapeutic mAbs (IgG1 and IgG4) was examined on three CEX resins (Capto SP ImpRes, Fractogel EMD SE Hicap, and POROS XS). First, mAb structural stability was studied in solutions under CEX load conditions. Using differential scanning fluorimetry (DSF), the measured melting temperature (Tm DSF (Unbound)) decreased from 60.7 to 52.4°C for mAb1 and 51.5 to 45.2°C for mAb2 when lowering pH from 6.0 to 4.5. Then, mAb structural stability was further investigated in the bound state on CEX surfaces. Using differential scanning calorimetry (DSC), the measured melting temperature of the bound mAbs (Tm DSC (Bound)) was 4.5 - 6.5°C lower than that for the unbound mAbs (Tm DSC (Unbound)) in the same solutions. The Tm differences (∆Tm DSC (Unbound minus Bound)) between the two states correlated with the severity of mAb aggregation in CEX operations, indicating the importance of both intrinsic mAb stability and resin properties. In particular, resin hydrophobicity was shown to have a critical impact. The interplay among these protein- and resin-related factors, together with solution conditions, ultimately dictates the aggregate formation observed. Finally, the hydrophobicity of the CEX resins (Capto SP ImpRes < Fractogel EMD SE Hicap < POROS XS) was measured using a fluorescence-based method to quantitatively characterize this resin property. Results suggest that the mAb-accessible hydrophobic regions of the CEX resins affect the structural stability of the bound mAbs to various degrees, leading to differences in aggregate formation upon mAb elution. In summary, this study offers insight into the mechanism of mAb aggregation in bind-elute CEX operations, and the in-depth understanding facilitates the development of robust CEX conditions for mAb purification. [ABSTRACT FROM AUTHOR]