Nature of foulants and fouling mechanism in the Protein A MabSelect resin cycled in a monoclonal antibody purification process.

The composition and origin of foulants and their spatial distribution within the particles of the Protein A MabSelect resin cycled in a mAb purification process are determined using electron and confocal microscopy techniques with gold and fluorescently labeled protein probes that associate with the foulants. The results show that the foulants are primarily related to the mAb product, are heterogeneously dispersed both on the outer surface and in the interior of the resin beads, and accumulate only when loading the conditioned CHO cell culture supernatant. Insignificant accumulation is seen if the process is run with purified mAb or with the null cell culture supernatant. When bound to the Protein A ligand, the mAb responsible for the observed fouling behavior is shown to associate with BSA and α-lactalbumin. This property is exploited using labeled versions of these lipophilic proteins to assess the effectiveness of improved resin cleaning processes and to elucidate the fouling mechanism. Resin fouling for this mAb appears to be consistent with the occurrence of conformational changes that occur upon binding, which, in turn, facilitate association of lipophilic proteins with the mAb. Upon desorption at low pH, these destabilized mAb complexes are deposited on and within the resin growing with each cycle and eventually leading to significant degradation of process performance.