Monolith weak affinity chromatography for μg-protein-ligand interaction study

Lucile Lecas 1 Jérôme Randon 1 Alain Berthod 2 Vincent Dugas 1 Claire Demesmay 1, *
* Corresponding author
1 Separative Methods - Techniques séparatives
ISA - Institut des Sciences Analytiques
Abstract : Affinity monolith columns of 375 nL (effective length 8.5 cm, internal diameter 75 μm) were developed for protein-ligand affinity investigations needing only 3 μg of human serum albumin (HSA). To promote specific interactions and avoid non-specific ones, different combinations of monolithic supports and bio-functionalization pathways were evaluated. Silica and glycidylmethacrylate based monoliths were in-situ synthesized and grafted with HSA. Two direct grafting methods epoxy-amine and Schiff Base plus the streptavidin-biotin method were compared. The columns were evaluated by frontal analysis with ligands of known affinity for HSA. It is shown that a classical capillary electrophoresis instrument equipped with an external pressure device can be used to do weak affinity chromatography at low pressure (less than 1.2 MPa) in a fully automated way and with very low reagent consumption. The grafting pathways were compared in terms of (i) total and active amounts of immobilized protein, (ii) non-specific interactions, (iii) protein denaturation. According to these criteria, the organic monoliths combined with the streptavidin-biotin approach provided the best results. This immobilization pathway led to the highest active protein content (40 pmol of HSA per 8.5-cm column) with less than 10% non-specific interactions and 84% protein activity. The target grafting step lasts only 10 min and is UV-monitored, the UV breakthrough curve giving the exact amount of bound protein. This novel approach was validated by Kd measurements of 3 known ligands of HSA. Streptavidin generic monolith columns could be stored at 4 °C for 3 months maintaining activity. μg of a biotin modified sensitive protein could be attached to a stable streptavidin monolith for immediate interaction studies avoiding stability problems. This development was subsequently extended to another protein of higher pharmaceutical interest: the N-terminal domain of HSP90. Affinity was measured for two known ligands and determined Kd values were in accordance with the literature, proving that our technique is applicable to other proteins.
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Submitted on : Tuesday, February 12, 2019 - 3:08:59 PM
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  • HAL Id : hal-02015987, version 1



Lucile Lecas, Jérôme Randon, Alain Berthod, Vincent Dugas, Claire Demesmay. Monolith weak affinity chromatography for μg-protein-ligand interaction study. Journal of Pharmaceutical and Biomedical Analysis, Elsevier, 2019, 166, pp.164-173. ⟨hal-02015987⟩



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