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

Abstract

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 ionpairing 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.

Nanobuffering of pH-Responsive Polymers: A Known but Sometimes Overlooked Phenomenon and Its Biological Applications

“…As demonstrated by Gauthier and coworkers, NBPRP requires intimate contact with the pH-responsive polymers ( i.e ., the pH-responsive polymers in the immediate vicinity) in any of the following forms: electrostatic interactions, covalent bonds, hydrogen bonding, or others. 41 Although it is a nanoscale phenomenon, NBPRP can generate larger-scale impacts. For example, other constituents such as degradable linkers, drug molecules, and biomolecules ( e.g ., therapeutic nucleic acids or proteins) in the immediate vicinity of pH-responsive polymers can be affected in terms of activity, release, conformation, or stability.…”

“…As discussed above, Gauthier and co-workers not only systematically defined the NBPRP effect and demonstrated its ability to dissociate local ( i.e ., in the immediate vicinity) nanoscale pH from bulk environmental pH but also applied this effect to develop a homogeneous scavenger for antibody purification. 41 Essentially, the final scavenger is a pH-responsive (co)polymer conjugated Protein-A ( i.e ., there is intimate contact between the pH-responsive polymer and the protein), screened from a library of six scavengers with a variety of polymer composition whose p K a values are between 6.3 and 7.8. The specific binding site of Protein-A is the constant region of antibodies of the immunoglobulin G family (IgG) at near-neutral pH.…”

“…In the February issue of ACS Nano , Gauthier and co-workers systematically define and describe nanobuffering in the context of rationally designed pH-responsive polymers and report a proof-of-concept application for antibody purification. 41 Although this is not the first report of nanobuffering, Gauthier and co-workers propose a specific definition and present the requirements for generation of this nanobuffering effect. In this Perspective, we summarize the concept and requirements of the nanobuffering of pH-responsive polymers (NBPRP), according to Gauthier’s report and previous studies.…”

Confined Microenvironments from Thermoresponsive Dendronized Polymers

“…[ 18 ] Furthermore, pH‐responsive polymers were found to be able to protect a local range of pH on nanoscale where they were composed. pH condition within the compartment can be different from the environmental pH to show appealing nano‐buffering effect, [ 23 ] which has been applied for proof‐of‐concept purification of antibody [ 24 ] or enzyme cascade reaction. [ 25 ]…”

The phage display of Bacillus subtilis Lipase A significantly enhances catalytic activity due to altered nanoscale distribution in colloidal solution

“…Indeed, because of its highly repetitive structure, the bacteriophage may create a local microenvironment that is different from bulk solution. For instance, our group and others have demonstrated that pH‐responsive polymers can alter the properties of proteins by locally altering solution pH (Anees et al, ; Zhang, Wang, & Hess, ). Moreover, the bacteriophage scaffold may itself be influenced by solution parameters such as salts, concentration, surfactants, or crowding agents, which may in turn influence the activity of the displayed protein.…”