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 50mM 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.

We have systemically investigated unusual elution behaviors of an IgG4 (mAb A) in cation exchange chromatography (CEX). This mAb A exhibited two elution peaks under certain conditions when being purified by several strong CEX columns. When either of the two peaks was isolated and re-injected on the same column, the similar pattern was observed again during elution. The protein distribution between the two peaks could be altered by NaCl concentration in the feed, or NaCl concentration in wash buffer, or elution pH, suggesting two pH-associated strong-and-weak binding configurations. The protein distributions under different pH values showed good correlation with protonated/un-protonated fractions of a histidine residue. These results suggest that the double-peak elution profile associates with histidine-protonation-based charge variants. By conducting pepsin digestion, amino-acid specific chemical modifications, peptide mapping, and measuring the effects of elution residence time, a histidine in the variable fragment (Fab) was identified to be the root cause. Besides double-peak pattern, mAb A can also exhibit peak-shouldering or single elution peak on different CEX resins, reflecting different resins' resolving capability on protonated/un-protonated forms. This work characterizes a novel cause for unusual elution behaviors in CEX and also provides alternative avenues of purification development for mAbs with similar behaviors.

In this paper, we discuss the optimization and implementation of a high throughput process development (HTPD) tool that utilizes commercially available micro-liter sized column technology for the purification of multiple clinically significant monoclonal antibodies. Chromatographic profiles generated using this optimized tool are shown to overlay with comparable profiles from the conventional bench-scale and clinical manufacturing scale. Further, all product quality attributes measured are comparable across scales for the mAb purifications. In addition to supporting chromatography process development efforts (e.g., optimization screening), comparable product quality results at all scales makes this tool is an appropriate scale model to enable purification and product quality comparisons of HTPD bioreactors conditions. The ability to perform up to 8 chromatography purifications in parallel with reduced material requirements per run creates opportunities for gathering more process knowledge in less time.

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