Preparation of miniaturized hydrophilic affinity monoliths: Towards a reduction of non-specific interactions and an increased target protein density

In affinity chromatography, non-specific interactions between the ligands and the affinity column may af-fect the results, leading to misinterpretations during the investigation of protein-ligand interactions (de-tection of false positives in ligand screening, lack of specificity in purification). Such non-specific inter-actions may arise both from the underlying support or from the target protein itself. If the second ones are protein-dependent (and cannot be studied in a general framework), the first ones occur in the same way regardless of the immobilized target. We propose a methodology to identify the origin of such non-specific interactions with the underlying material of the affinity column. This methodology relies on the systematic investigation of the retention behavior of a set of 41 low-molecular weight compounds cover-ing a wide chemical space (net charge, log D, functionality). We first demonstrate that the main source of non-specific interactions on the most commonly used GMA-co-EDMA monolith comes from hydrophobic effects. To reduce such non-specific interactions, we developed a new hydrophilic glycidyl methacrylate-based monolith by replacing the EDMA crosslinker by the more hydrophilic N -N' Methylenebisacrylamide (MBA). Optimization of the synthesis parameters (monomer content, initiation type, temperature) has fo-cused on the reduction of non-specific interaction with the monolithic support while maximizing the amount of protein that can be grafted onto the monolith at the issue of its synthesis. The retention data of the 41 test solutes on the new poly(GMA-co-MBA) monolith shows a drastic reduction of non-specific interactions except for cationic compounds. The particular behavior of cationic compounds is due to their electrostatic interactions with carboxylic groups resulting from the partial acidic hydrolysis of amide groups of MBA during the epoxide ring opening step. So, the ring opening step in acidic media was replaced by a hot water treatment to avoid side reaction on MBA. The new monolith poly(GMA-co-MBA) not only has improved hydrophilic surface properties but also a higher protein density (16 +/- 0.8 pmol cm -1 instead of 8 +/- 0.3 pmol cm -1). To highlight the benefits of this new hydrophilic monolith for affinity chromatographic studies, frontal affinity chromatography experiments were conducted on these monoliths grafted with con A.(c) 2022 Elsevier B.V. All rights reserved.

Automated summary

In affinity chromatography, non-specific interactions between ligands and the affinity column can cause misinterpretations and lack of specificity. A methodology was proposed to identify the origin of such interactions with the underlying material of the column. By investigating the retention behavior of different compounds, it was found that hydrophobic effects were the main source of non-specific interactions. To reduce these interactions, a new hydrophilic monolith was developed. It showed improved surface properties, higher protein density, and significantly reduced non-specific interactions.