Antibody capture by mixed-mode chromatography: A comprehensive study from determination of optimal purification conditions to identification of contaminating host cell proteins

► Mixed mode resins PPA HyperCel, MEP HyperCel, HEA HyperCel and Capto adhere are effective as a capture step for antibody purification from a crude culture supernatant of CHO. ► Optimal conditions of antibody purification were defined using a microplate approach and validated in column. ► Interactions between ligand and proteins in these mixed mode resins were identified. ► HCPs that are potentially coeluted with the antibody were identified by mass spectrometry. We evaluated mixed mode chromatography for the capture of recombinant antibodies from CHO cell culture supernatants. We studied PPA HyperCel, HEA HyperCel, MEP HyperCel and Capto adhere resins, which all contain hydrophobic and cationic groups. A microplate approach combined with DoE modeling allowed the exploration of the complex behaviors of these mixed mode resins. Optimal conditions for antibody purification and host cell proteins (HCPs) elimination were determined and then directly up-scaled to laboratory columns. Then we used mass spectrometry to identify the major HCPs potentially coeluted with the antibody. Differences between the four resins in terms of amount, complexity and identity of the HCPs present in the elution fractions were investigated.