Fragments of protein A eluted during protein A affinity chromatography.

Abstract

Protein A affinity chromatography is a common method for process scale purification of monoclonal antibodies. During protein A affinity chromatography, protein A ligand co-elutes with the antibody (commonly called leaching), which is a potential disadvantage since the leached protein A may need to be cleared for pharmaceutical antibodies. To determine the mechanism of protein A leaching and characterize the leached protein A, we fluorescently labeled the protein A ligand in situ on protein A affinity chromatography media. We found that intact protein A leaches when loading either purified antibody or unpurified antibody in harvested cell culture fluid (HCCF), and that additionally fragments of protein A leach when loading HCCF. The leaching of protein A fragments can be reduced by EDTA, suggesting that proteinases contribute to the generation of protein A fragments. We found that protein A fragments larger than about 6000 Da can be measured by enzyme linked immunosorbent assay, and that they can be more difficult to clear than whole protein A by cation-exchange chromatography.

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@article{CarterFranklin2007FragmentsOP, title={Fragments of protein A eluted during protein A affinity chromatography.}, author={Jayme N Carter-Franklin and Corazon Victa and Paul J McDonald and Robert L Fahrner}, journal={Journal of chromatography. A}, year={2007}, volume={1163 1-2}, pages={105-11} }