Toward improving selectivity in affinity chromatography with PEGylated affinity ligands: The performance of PEGylated protein A

Abstract

Chemical modification of macromolecular affinity chromatography ligands with polyethylene glycol chains or "PEGylation" can potentially improve selectivity by sterically suppressing non-specific binding interactions without sacrificing binding capacity. For a commercial protein A affinity media and with yeast extract (YE) and fetal bovine serum (FBS) serving as mock contaminants, we found that the ligand accounted for more than 90% of the media-associated non-specific binding, demonstrating an opportunity for improvement. The IgG static binding affinity of protein A mono-PEGylated with 5.0 and 20.7 kDa poly(ethylene glycol) chains was found to be preserved using a biomolecular interaction screening platform. Similar in situ PEGylations of the commercial protein A media were conducted and the modified media was functionally characterized with IgG solutions spiked with YE and FBS. Ligand PEGylation reduced the mass of media-associated contaminants by a factor of two to three or more. Curiously, we also found an increase of up to 15% in the average recovery of IgG on elution after PEGylation. Combined, these effects produced an order of magnitude increase in the IgG selectivity on average when spiked with YE and a two- to three-fold increase when spiked with FBS relative to the commercial media. Dynamic binding capacity and mass-transfer resistance measurements revealed a reduction in dynamic capacity attributed to a decrease in IgG effective pore diffusivity and possibly slower IgG association kinetics for the PEGylated protein A ligands. Ligand PEGylation is a viable approach to improving selectivity in affinity chromatography with macromolecular ligands.

Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies

“…Despite the great performance of Protein A for purification of mAbs, González‐Valdez et al. demonstrated that the ligand accounted for >90% of the media‐associated non‐specific binding. This finding was evident after testing a commercial Protein A media including yeast extract and fetal bovine serum as contaminants.…”

Preparation of Protein A Membranes Using Propargyl Methacrylate-Based Copolymers and Copper-Catalyzed Alkyne–Azide Click Chemistry

“…Protein A contains 55 lysine residues and one N-terminal. It is unclear how many of these are surface residues, but 30 are in the X domain, originally where the protein was covalently bound to the staphylococcal bacteria cell wall [ 18 ]. Azide functionality was introduced into the Protein A structure via NHS ester chemistry involving these lysine groups.…”

Perspectives, Tendencies, and Guidelines in Affinity-Based Strategies for the Recovery and Purification of PEGylated Proteins

“…In one of these works, the purification of 20 kDa mono-PEGylated lysozyme with heparin affinity chromatography was optimized through an experimental design [32], and the elution curves for the operation were later simulated [33]. e other studies deal with the PEGylation of ligands in AC supports or resins, with the aim of improving their stability using as model concanavalin and protein A [34,35].…”