Development and Characterization of High Performance Functionalized Membranes for Antibody Adsorption

Microfiltration (220 nm pore size) polyethersulfone membranes containing hydrolyzed poly(styrene-b-ethoxystyrene) (poly(styrene-b-HS)) grafts have been developed for adsorption of immunoglobulin G (IgG) under convective flow conditions. Styrene-b-ethoxystyrene (post hydrolysis) block copolymer mimics phenylalanine-132/tyrosine-133 dipeptide structure of ligand protein A that specifically binds with the Fc region of IgG. A surface-initiated living sequential cationic polymerization technique was used to immobilize polymeric grafts containing multiple binding sites using solid-phase sulfuric acid as an initiator. The dynamic binding capacity (DBC) which also happens to be equilibrium binding capacity from breakthrough curve is as high as 95 mg/ml of membrane. The Langmuir adsorption model correlated well (R² = 0.99) with the experimental adsorption data. Control experiments showed that the DBC was independent of flow rate indicating that the binding of IgG to the adsorptive sites is predominantly by convection and not diffusion. Additionally, the DBC peaked at 120 minutes of polymerization reaction time and then decreased at longer times. Bind and elute experiments showed that there was no significant loss of DBC over a period of five cycles and the average recovery of antibody was >94%.

Poly(chloromethylstyrene) grafts were incorporated into the functionalized membrane containing poly(styrene-b-HS) grafts by sequential addition of 4-chloromethystyrene monomer. This was subsequently followed by functionalization of chloromethyl groups with glycine to impart an overall negative charge to the grafted chain. Competitive sorption studies in presence of bovine serum albumin (BSA) using negatively charged functionalized membrane showed high selectivity of membrane for IgG. The concentration of IgG was measured by performing an overall material balance on feed and permeate solutions using UV-VIS spectroscopy. Size exclusion chromatography was used to measure the amount of IgG and BSA adsorbed during competitive sorption studies. Finally, ¹H NMR characterization confirmed incorporation of poly(styrene-b-HS), poly((styrene-b-HS)-b-chloromethylstyrene), and subsequent glycine functionalization of chloromethyl moiety. Furthermore, ¹H NMR results also showed stability of functional moieties without any side reactions. The contributions from this research provide insights into applications of synthetic affinity ligand membrane adsorbers for low-cost and large scale purification of antibodies in downstream biotechnology industry.