Improved adsorption reactions, kinetics and stability for model and therapeutic proteins immobilised on affinity resins

Abstract

Protein adsorption on solid state media is important for the industrial affinity chromatography of biotherapeutics and for preparing materials for self-interaction chromatography where fundamental protein solution thermodynamic properties are measured. The adsorption of three model proteins (lysozyme, catalase and BSA) and two antibodies (a monoclonal and a polyclonal antibody) have been investigated on commercial affinity chromatography media with different surface functionalities (Formyl, Tresyl and Amino). Both the extent of protein immobilised (mg protein/ml media) and the reaction kinetics are reported for a range of reaction conditions, including pH, differing buffers as well as the presence of secondary reactants (glutaraldehyde, sodium cyanoborohydride, EDC and NHS). Compared to the reaction conditions recommended by manufacturers as well as those reported in previous published work, significant increases in the extent of protein immobilisation and reaction kinetics are reported here. The addition of glutaraldehyde or sodium cyanoborohydride was found to be especially effective even when not directly needed for the adsorption to happen. For mAb and pIgG, immobilisation levels of 50 and 31 mg of protein/ml of resin respectively were achieved, which are 100% or more than previously reported. Enhanced levels were achieved for lysozyme of 120 mg/ml with very rapid reaction kinetics (< 1 h) with sodium cyanoborohydride. It can be concluded that specific chromatography resins with Tresyl activated support offered enhanced levels of protein immobilisation due to their ability to react to form amine or thio-ether linkages with proteins. Additionally, glutaraldehyde can result in higher immobilisation levels whilst it can also accelerate immobilisation reaction kinetics.

Removal of anthracycline cytostatics from aquatic environment: Comparison of nanocrystalline titanium dioxide and decontamination agents

“…Wastewater cytotoxicity is also increased when active cytostatic metabolites are excreted [3]. An effective way of wastewater treatment from organic micro-pollutants can be achieved by using adsorption on a variety of nanomaterials including metal nanostructures [4], nanocarbons [5], graphene nanosheets [6,7] or hybrid composites [8] have been explored for the effective removal of organic pollutants. These materials are characterized by high surface area, significant porosity, small particle size, and mechanical strength, as well as the occurrence of a significant number of defects in the crystal lattice and surface functional groups [9].…”