Protein A affinity chromatography is widely used as a capture step for monoclonal antibodies (mAb) and molecules that possess an Fc-domain, such as fusion proteins and bispecific antibodies. The use of low pH (3.0-4.0) to elute the molecule and achieve acceptable yield (&gt;85%) can lead to product degradation (e.g. fragmentation, aggregation) for low pH-sensitive molecules. Elution of the product into a neutral pH buffer solution is a valid alternative but results in lower manufacturing flexibility.<br/>In this study, two protein A resins that elute at a mild pH were evaluated and compared against two conventional protein A resins using five mAbs (four glycosylated, one aglycosylated) and an Fc-fusion protein that formed aggregate at low pH. All mAbs showed higher elution pH on the novel resins compared to conventional resins. One novel resin is a universal mild pH elution resin, with elution pH at or above 4.6 for glycosylated mAbs and Fc-fusion protein, with glycosylation contributing to binding affinity. The other novel resin elutes at a pH above those of the commercially available protein A resins for all molecules evaluated, and the elution pH of the antibodies is dependent on their VH sequences. Additionally, the Fc-fusion protein which did not possess Fab domains showed an even larger difference in elution pH between novel and conventional resins, indicating that interaction between the Fab and protein A ligand contributes to retention.  <br/>Both novel resins had similar dynamic binding capacities to next-generation protein A resins for all molecules evaluated. Purification runs with cell culture material were performed on all resins with one mAb and the Fc-fusion protein, using optimized elution pH and wash conditions. Compared to the conventional resins, the novel resins had improved HCP and aggregate removal, and a milder native eluate pool pH for both molecules. For the low pH sensitive Fc-fusion protein, product eluate from the conventional resins had higher starting aggregate levels which also increased significantly with time. In contrast, product eluate from the novel resins had lower starting levels of aggregate which remained constant with time, demonstrating that the novel mild elution pH protein A resins provide a good alternative to conventional resins for low pH-sensitive molecules.

Evaluation of mild pH elution protein A resins for purification of antibodies and Fc-fusion proteins

ACS Fall 2021

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