Static and dynamic binding capacities of human immunoglobulin G on polymethacrylate based mixed-modal, thiophilic and hydrophobic cation exchangers

The aim of this study was to investigate functional increments of ion exchange type ligands, which may improve the performance of mixed-modal ligands for antibody capture out of feed solutions with pH above 6.0 and containing sodium chloride concentrations of 150 mM and higher. For this purpose several functional groups such as sulfonyl, sulfanyl, amide, methoxy, short alkyl and aromatic moieties were tested in combination with a strong sulfonic acid and/or a weak carboxylic acid group. Therefore a series of ligands were synthesized and subsequently coupled onto epoxide activated Fractogel® EMD. In the first instance, all materials were tested by static binding capacity measurements (SBC) under test conditions, comprising a wide variety of different sodium chloride concentrations and differing pH values ranging from 4.5 to 7.5. From these preliminary experiment it was found that especially the aromatic groups improved the binding of human immunoglobulin G (h-IgG) under isotonic conditions, while other increments, e.g. thiophilic or amide groups, were not able to increase the capacity significantly. Taking the SBC results into account, the most promising materials were investigated under dynamic binding conditions (DBC) with a reduced selection of test conditions (pH 5.5, 6.5 and 7.4 at 75 and 150 mM NaCl). N-benzoyl-homocysteine (material J) and 3,5-dimethoxybenzoyl-homocysteine (material K) showed 100% DBCs of 37 mg/mL and 32 mg/mL in the presence of 75 mM NaCl and pH 6.5. Material L carrying mercaptobenzoic acid as a ligand and tested with the same solution provided a 100% DBC of 68 mg/mL. The influence of Pluronic F68 in a mock feed solution as well as in cell culture supernatant was investigated with the best performing bio-affinity type adsorbent, material L. For the real sample feed subsequent SDS-PAGE was conducted for the collected fractions.