Evidence, Manipulation, and Termination of pH 'Nanobuffering' for Quantitative Homogenous Scavenging of Monoclonal Antibodies

This study demonstrates that pH-responsive polymers have a very high buffering capacity in their immediate vicinity, a phenomenon termed nanobuffering. This can be exploited to dissociate local nanoscale pH from bulk solution pH. Herein, a series of pH-responsive polymers were conjugated to Protein-A to rationally manipulate the latter's binding affinity toward antibodies via nanobuffering (i.e., this interaction is pH dependent), independently of bulk solution pH. Moreover, the nanobuffering effect could be terminated using low concentrations of strong ion pairing salts, to achieve quantitative release of the antibodies from the bioconjugate. These complementary discoveries are showcased in the context of the development of a homogeneous affinity precipitation agent (i.e., a scavenger) for the purification of polyclonal immunoglobulin G and two monoclonal antibodies from cell culture supernatant. Indeed, while bulk solution pH was used to induce precipitation of the scavenger, maintaining local nanoscale pH via nanobuffering maximized binding interaction with the antibodies. A 2:1 binding stoichiometry was observed, which was similar to that observed for native protein. The scavenger could be recycled multiple times, and the purification protocol circumvented lengthy/tedious physical purification processes typically associated with mAb manufacturing. Overall, this study provides perspectives on the local nanoscale pH near pH-responsive polymers and establishes lines of thought for predictably manipulating or even terminating nanobuffering, to control the activity of proteins.