Affiliation:
1. Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, SC 29634, USA
Abstract
Protein A chromatography is the preferred unit operation for purifying Fc-based proteins. Convective chromatography technologies, like membrane adsorbers, can perform the purification rapidly and improve throughput dramatically. While the literature reports the preparation of Protein A membrane adsorbers utilizing traditional coupling chemistries that target lysine or thiol groups on the Protein A ligand, this study demonstrates a new approach utilizing copper-free dibenzocyclooctyne (DBCO)-azide click chemistry. The synthetic pathway consists of three main steps: bioconjugation of Protein A with a DBCO-polyethylene glycol (PEG) linker, preparation of an azide-functionalized membrane surface, and click reaction of DBCO-Protein A onto the membrane surface. Using polyclonal human immunoglobulins (hIgG) as the target molecule, Protein A membranes prepared by this synthetic pathway showed a flowrate-independent dynamic binding capacity of ~10 mg/mL membrane at 10% breakthrough. Fitting of static binding capacity measurements to the Langmuir adsorption isotherm showed a maximum binding (qmax) of 27.48 ± 1.31 mg/mL and an apparent equilibrium dissociation constant (Kd) of value of 1.72 × 10−1 ± 4.03 × 10−2 mg/mL. This work represents a new application for copper-less click chemistry in the membrane chromatography space and outlines a synthetic pathway that can be followed for immobilization of other ligands.
Funder
National Institute of General Medical Sciences of the National Institutes of Health
Subject
Filtration and Separation,Chemical Engineering (miscellaneous),Process Chemistry and Technology