Preparation of Protein A Membrane Adsorbers Using Strain-Promoted, Copper-Free Dibenzocyclooctyne (DBCO)-Azide Click Chemistry

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.

Keywords

• Protein A
• membrane chromatography
• alkyne–azide
• copper-free click chemistry
• antibody purification