Development of a multi-layering protein grafting process on miniaturized monolithic columns for weak affinity nano liquid chromatography application purposes.

Affinity chromatography is a powerful technique to identify and quantify weak ligand-protein interactions (Kd in the range of mM to 0.1µM). In some fields such as Fragment Based Drug Discovery, the detection of very weak affinities (mM) is of utmost importance since weak ligands can be good starting points for the conception of high affinity ligands. However, the identification of such weak ligands can be hampered by the limited bulk density of active target grafted onto the support. At the same time, downscaling the chromatographic column is of utmost interest when scarce and/or expensive proteins are targeted. In this context, we herein present a novel approach of protein immobilization to improve the bulk density of active protein grafted onto organic capillary monolithic columns. The proposed approach is based on the streptavidin-biotin interaction and consists of successive grafting steps of biotinylated target protein onto streptavidin layers through a multi-layering process. Concanavalin A was used as model protein. The study focuses on the optimization of the grafting conditions to maximize the amount of active protein during the multi-layering process and highlights the impact of the biotinylation ratio of the protein. It is demonstrated that a 3-layer grafting process allows to improve the bulk density of active sites by a 2-fold factor compared to a single layer. This improvement in protein density allows to increase the affinity range of this technique to the millimolar range.