Fragments of protein A eluted during protein A affinity chromatography.
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.
Carter-Franklin
Jayme N
JN
Early Stage Purification, Bioprocess Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. franklin.jayme@gene.com
Victa
Corazon
C
McDonald
Paul
P
Fahrner
Robert
R
D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay; D010446:Peptide Fragments / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry*
D010446:Peptide Fragments; D013205:Staphylococcal Protein A