Síntese e caracterização de criogéis de poliacrilamida-quitosana com íons metálicos Cu2+ e Ni2+ imobilizados para a purificação de imunoglobulina G humana por IMAC

Abstract

Antibodies or immunoglobulins are amongst the proteins that require the most degree of purity for their applications, such as in the diagnostic and treatment of diseases. Current purification methods, in which allow high yields and degree of purity, for those biomolecules can involve affinity chromatography, a technique that is based on the selective interaction between an immobilized ligand and the product of interest. The utilization of those procedures leads to the reduction of the number of additional steps, costs and processing time when compared to other protein purification methods. This work aimed to synthetize and characterize a monolithic stationary phase with adequate properties to be used in immobilized metal ion affinity chromatography to separate immunoglobulin G (hIgG) from human serum. Different conditions of cryopolimerization of the acrylamide, bisacrylamide, allyl-glycidyl-ether in the presence of chitosan chains and the crosslinking agent glutaraldehyde were tested and the cryogel that proved more suitable was selected. The chelating agent iminodiacetic acid (IDA) was covalently immobilized onto the cryogel, which was named as PAAm-CS-GA-AGE-IDA. Then, the adsorption and purification capacities of hIgG by the cryogel in the presence or not of chelated Cu 2+ and Ni 2+ ions were investigated. In the cryogel with chelated Ni 2+ , the sodium phosphate buffer system at pH 7,0 (25 mmol L -1 ) with imidazole (2 mmol L -1 ) proved to be the most promising condition for providing yield of 55% and purity of 88% for hIgG. The breakthrough curve revealed that the cryogel continued to be selective for hIgG under saturation conditions, resulting in 96% of purity. The kinetics experiments evidenced that the equilibrium time of the adsorption is quickly reached. Finally, adsorption isotherms were built and the parameters of the Langmuir and Langmuir-Freundlich models were adjusted by non-linear regression.The Langmuir-Freundlich model represented well the adsorption experimental data, resulting in the maximum capacity equal to 31,19 mg hIgG/g dry cryogel and a dissociation constant in the order of Kd = 10 -7 mol L -1 . The obtained results proved the potential of the cryogel of polyacrylamide and chitosan with immobilized metal ions to separate and purify hIgG from human serum.