Host-cell proteins (HCPs) and monoclonal antibody (mAb) aggregates are mostly removed during Protein A affinity and anion-exchange chromatography in downstream platform processes for mAbs. The remaining trace amounts of HCPs and aggregates are, however, difficult to remove in a typical cation-exchange (CEX) chromatography polishing step. HCP removal in CEX has not been studied in detail and depends largely on the pH of the wash buffers and the choice of resin chemistry. Aggregate clearance is achieved mainly by peak cutting and peak pooling in gradient elution, leading to yield loss. In the present work, we demonstrate the use of multi-column displacement chromatography for removal of HCPs and mAb aggregates. HCPs can be placed into two broad categories based on their isoelectric points (pI): HCPs with pI values higher than that of the mAb will flow through during product loading, while HCPs with pI values higher than that of the mAb will bind more strongly and displace the mAb. The displacement profiles of HCP versus mAb samples were constructed using analysis of elution fractions at different axial positions in the column. Based on ELISA analysis and capacity calculations for HCPs, it was found out that a column of bed height ~ 15 % of the main column prior to the main column would be required to displace the mAb sufficiently so as to obtain less than 10 ppm of HCP in the final elution pool. In a similar way, mAb aggregates, owing to their higher binding affinity, also showed displacement of monomer on the CEX column. The displacement profile was obtained by analyzing fractions of breakthrough load on a single column at different pH values. The optimal displacement condition was obtained at a pH where the mAb did not bind too weakly or too strongly to the column. To this end, a multi-column set-up was used to obtain product with less than 0.2% aggregate and 90% yield. These multi-column displacement methods can easily be incorporated into a continuous chromatography system to obtain product of high purity without compromising product recovery.

Removal of host-cell proteins and mAb aggregates using cation-exchange multi-column displacement chromatography

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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