Preparation of immunoglobulin adsorbents by radiation-induced grafting of cellulose fibers

GABRIELA TORCHIO; MARIA LAURA CARBAJAL; GRASSELLI, M.

Peninsula of Giens

Simposio; 12th meeting of ionizing radiation and polymers symposium, IRaP; 2016

CEA - France

Purification of biopharmaceuticals is generally more cost intensive than corresponding fermentation processes in the biotech industry, and can account for up to 80% of production costs. Affinity chromatography with Protein A is one of the most important and expensive step, industrially used for monoclonal antibody production. Novel process integration and intensification methods, based on the utilization of non-traditional chromatographic processes, or extractive methods, are highly required in order to reduce these costs. The application of aqueous two phase systems, expanded bead adsorptions, convective flow systems, and fiber-based adsorbents have been recently discussed to tackle bottlenecks [1]. Adsorbents fibers, prepared by radiation-induced and chemical grafting of cellulose fibers, have shown high capacity at higher flow rates for separation and purification of proteins [2]. In addition, the natural arrangement of cellulose into fibers allows the development of non-woven structures, which could be useful for adsorptive process of non-clarified biological samples.In this work are studied the preparation of modified cellulose fibers containing an Avimer [3], a similar Protein A ligand. This Avimer contain a Cysteine amino acid (with a thiol moiety) in the N-terminal tag of the protein, which can link to the epoxy group of glycidyl methacrylate (GMA) by a ?click? reaction. Radiation-induced graft polymerization has been used to prepare a composite material of poly(GMA)/cellulose fiber. Cellulose fibers have been previously treated with NaOH solution to improve their swelling. GMA 3 % in water/ethanol solution (1/1 v/v) was used for preparation of grafted fibers by simultaneous method, using a 60Co irradiation source (10 kGy at 1 kGy/h), yielding 20 % of grafting.Grafted fibers were modified with a thiol containing Green Fluorescent Protein, to follow the immobilization process by fluorescent methods. The optimized reaction for protein immobilization involves a pre-incubation of grafted fibers with DMSO followed by incubation with the Avimer solution in citrate buffer at pH 8.4 with EDTA 1 mM at 5 C during 72 h. The binding capacity of Avimer-fibers for immunoglobulins was 14.2 ± 1.3 mg/g and in dynamic conditions falls to 7.3 mg/g. This work demonstrated the preparation of advance affinity chromatography materials by radiation-induced modification of cellulose fibers.References [1] D'Souza, R.N., et al. Emerging technologies for the integration and intensification of downstream bioprocesses. Pharm. Bioproc. 1, (2013), 423-440.[2] Singh, N.K., et al. Preparation and characterization of grafted cellulosic fibers and their applications in protein purification. Sep. Purif. Technol. 143, (2015), 177-183.[3] Kangwa, M., et al., High-level fed-batch fermentative expression of an engineered Staphylococcal protein A based ligand in E. coli: purification and characterization. AMB Exp. 5, (2015), 1-10.