Effects of salt-induced reversible self-association on the elution behavior of a monoclonal antibody in cation exchange chromatography

Some monoclonal antibodies (mAbs) are reported to display concentration-dependent reversible self-association (RSA). There are multiple studies that investigate the effect of RSA on product characteristics such as viscosity, opalescence, phase separation and aggregation. This work investigates the effects of RSA on a bind-and-elute mode cation exchange chromatography (CEX) unit operation. We report a case study in which the RSA of an IgG2 (mAb X) resulted in significant peak splitting during salt gradient elution in CEX. Multiple factors including resin type, load challenge, residence time and gradient slope were evaluated and demonstrated little effect on the peak splitting of mAb X. It was determined that high NaCl concentrations in combination with high protein concentrations induced mAb X to form one RSA species that binds more strongly to the column, resulting in a large second elution peak. The finding of NaCl-induced RSA suggested that lower NaCl elution concentrations and different types of salts could mitigate RSA and thus eliminate peak splitting. Different salts were tested, showing that chaotropic salts such as CaCl2 reduced the second elution peak by inducing less RSA. The addition of a positively charged amino acid (such as 50 mM histidine) into the CEX elution buffer resulted in elution at lower NaCl concentrations and also effectively reduced peak splitting. However, experiments that were intended to reduce salt concentration by increasing the elution buffer pH did not significantly mitigate peak splitting. This is because higher pH conditions also increase RSA. This work identifies salt-induced RSA as the cause of peak splitting of a mAb in CEX and also provides solutions to reduce the phenomenon.