Performance of phospho-L-tyrosine immobilized onto alginate/polyacrylamide-based cryogels: Effect of ligand coupling on human IgG adsorption and Fab fragments separation

Autor(es)

Marcuz, Camila ; Mourao, Cecilia Alves ; Haupt, Karsten ; Alves Bueno, Sonia Maria

• Tipo de documento: Artigo Científico
• Fonte: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES; v. 1165, p. 12-pg., 2021-01-21.

Resumo

The ortho-phospho-tyrosine (P-Tyr) pseudoaffinity ligand was immobilized via ether linkage onto polyacrylamide-alginate (PAAm-Alg)-epoxy cryogels prepared according to two different approaches in order to explore their performance in the immunoglobulin G (IgG) purification from human serum. In the first approach, the P-Tyr was attached to cryogel prepared by cryocopolymerization of acrylamide and alginate with allyl glycidyl ether (AGE) as functional comonomer, and methylenebisacrylamide and Ca(II) as crosslinkers, obtaining the PAAm-Alg-AGE-P-Tyr. In the second approach, the PAAm-Alg was synthesized under the same conditions, but without AGE, and the P-Tyr was coupled to epichlorohydrin (ECH)-activated cryogel, obtaining the PAAmAlg-ECH-P-Tyr. Both pseudoaffinity cryogels were characterized by scanning electron microscopy, swelling tests, porosity, ligand density, and flow dynamics. The human IgG differently interacted with the PAAm-Alg-ECH-PTyr and PAAm-Alg-AGE-P-Tyr cryogels, depending on the pH and adsorption buffer system used. The selectivity analyzed by electrophoretic profiles was similar for both cryogels, but PAAm-Alg-ECH-P-Tyr achieved higher IgG adsorption capacity (dynamic capacity of 12.62 mg of IgG/mL of cryogel). The IgG purity assayed by ELISA was 95%. The maximum IgG adsorption capacity and dissociation constant of the PAAm-Alg-ECH-P-Tyr, determined by Langmuir isotherm, were found to be 91.75 mg IgG/g of dry cryogel and 4.60 x 10-6 mol/L at pH 6.0 from aqueous solutions. The PAAm-Alg-AGE-P-Tyr showed potential to purify the Fab fragments from papain-digested human IgG solution at pH 7.0. Fab fragments were separated from Fc fragments (but with uncleaved IgG) in eluted fractions (analyzed by the Western blot technique), with yield of 82% and purity of 95% (determined by radial immunodiffusion). (AU)

• Processo FAPESP: 17/21751-5 - Adsorção de IgG humana em criogel monolítico de acrilamida/alginato com o-fosfotirosina (OPT) imobilizado
• Beneficiário: Camila Marcuz
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 16/10855-1 - Criogel Monolítico de Acrilamida/Alginato com o-Fosfotirosina (OPT) Imobilizado como Fase Estacionária para Purificação de IgG Humana
• Beneficiário: Sonia Maria Alves Bueno
• Modalidade de apoio: Auxílio à Pesquisa - Regular