Synthesis and evaluation of monolithic cryogel containing reactive group and o-phospho-l-tirosine for adsorption of IgG and its fragments

Author(s)

Camila Marcuz

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia Química
• Defense date: 2019-08-01

Examining board members

Sonia Maria Alves Bueno; Gisele Luiza Pavan Pereira; Everson Alves Miranda

• Advisor: Sonia Maria Alves Bueno

Abstract

Immunoglobulins G (IgG) are expressed by the immune system of vertebrate beings. Due to the high specificity of IgG and their fragments, these biomolecules are employed on a variety of medical, pharmaceutical and analytical applications. All of them require homogenous preparations of immunoglobulins and because these biomolecules are traditionally obtained from serum, plasma or cell culture supernatant, effective purification processes are fundamental, resulting in a high-cost bioproduct. Its high cost has driven advances in chromatographic techniques, especially in pseudobioaffinity chromatography that make possible to obtain the product with a high degree of purity, lower cost and a reduced number of steps. In addition, the introduction of polymeric monolithic beds as a solid support for the ligand immobilization, has added many advantages to the chromatographic purification process. These supports can be synthesized with different monomers and, when obtained by the cryopolymerization technique, they show interconnected macropores providing predominantly convective flow, which is their main advantage. Thereby, the aim of this work was to synthesize a monolithic cryogel based on acrylamide and sodium alginate monomers and allyl glycidyl ether and bisacrylamide crosslinking agents (PAAm-Alg-AGE) with the pseudobiospecific ligand o-phospho-L-tyrosine (OPT) immobilized (PAAm -Alg-AGE-OPT), and to test this adsorbent in the purification of IgG from the human serum as well as in the separation of its Fab and Fc proteolytic fragments. In IgG purification experiments, the selectivity of the adsorbent under different chromatographic conditions was evaluated by SDS-PAGE electrophoresis. For the separation of the fragments, the selectivity was evaluated by SDS-PAGE electrophoresis, Western Blot, and radial immunodiffusion. The results indicated that the adsorbent was selective for IgG present in human serum in the presence of Tris-HCl and Hepes, both at 10 and 25 mmol/L and pH 7.0. The breakthrough curves under the same conditions indicated a dynamic adsorption capacity of 24.91 ± 2.41 and 22.24 ± 0.21 mg total protein/g dry cryogel in Tris-HCl 10 mmol/L and Hepes 25 mmol/L, respectively. The cryogel was also effective in separating the proteolytic fragments Fab and Fc. Adsorption of 73.61% and 82.61% of the Fab fragments fed on Tris-HCl 10 mmol/L and Hepes 25 mmol/L, respectively, was obtained by radial immunodiffusion. All of these results evidenced the potential to use the ligand OPT immobilized to a monolithic cryogel of PAAm-Alg-AGE as a fixed bed for purification of human IgG and its Fab fragment by adsorption liquid chromatography (AU)

• FAPESP's process: 17/21751-5 - Adsorption of human IgG on polyacrylamide/alginate monolithic cryogel-Immobilized o-phosphotyrosine (OPT)
• Grantee: Camila Marcuz
• Support Opportunities: Scholarships in Brazil - Master