(538a) Synthesis, Characterization and Application of Functionalized Membranes for Antibody Adsorption

This research focuses on synthesis and characterization of functionalized microfiltration membranes and evaluates their performance as membrane adsorbers for membrane chromatography. Functionalized membranes provide an excellent alternative to conventional packed bed chromatography for preparative separation of proteins and antibodies. This is due to realization of improved throughput because of dominance of convective mass transfer and active sites are located in the pores which allow easy access of sites to the target molecules. In this study functionalized membranes were synthesized by attachment of polymeric grafts with different functional groups or moieties within the pores of microfiltration polyethersulfone membranes. Living cationic polymerization technique was used to tether polymeric grafts that have active sites mimicking ligands in affinity chromatography. Functionalization of microfiltration membrane was achieved by a two step procedure. The first step was to introduce sulfonic acid initiator sites by mild sulfonation with 0.5N H₂SO₄. This was followed by cycling through copolymer mixture of styrene and 4-ethoxystyrne (ES). Styrene-co-ES (post hydrolysis) block copolymer graft resembles a synthetic analog of phenylalanine-tyrosine dipeptide structure of ligand protein A. This dipeptide structure has been shown in the literature to be very crucial for selective adsorption of antibody immunoglobulin G (IgG). 4-chloromethylstyrene (CMS) was used to introduce spacer units to prevent antibody denaturing and steric hindrance during adsorption. Characterization of polymeric grafts and stability of side groups without any side reactions was performed by ¹H NMR. Material balance calculations using analytical techniques like gas chromatography, atomic absorption and UV-Visible spectroscopy were performed on permeate solutions to quantify monomer reactivity ratios, estimate composition of grafts, understand kinetics and determination of polymer growth and graft length.

This study also aims at investigating the performance of functionalized membranes in terms of determination of antibody binding capacities and compares it with the commercially available ligand protein A. Competitive sorption studies using a mixture of bovine serum albumin (BSA) and IgG is studied to examine the selectivity of functionalized membrane. Selectivity based on size as well as electrical interaction between antibody and active sites (negatively charged) is also analyzed. The effect of initiator surface density, flow rates, polymer graft length and polymerization reaction time on antibody binding capacity is investigated. Additionally, the performance of the membrane in presence of the spacer units is also analyzed critically.