Microesferas de quitosana sulfatadas com potencial aplicação em purificação de IgG: estudo das condições operacionais

Abstract

Immunoglobulins of G isotype (IgG) are blood proteins of great interest for therapeutic applications and diagnosis of some diseases. To make these applications viable, it is necessary that the IgG obtained has a high degree of purity, which has motivated the use of several purification techniques. This work aimed to find better adsorption conditions for human IgG purification, using sulfated chitosan microspheres (SCM). The adsorbent assays were performed in batch with high purity human IgG to analyze the influence of the buffer’s type, the pH range, the adsorbent mass, the initial concentration og IgG solution and the contact time in the process. According to the obtained results, it was observed a greater adsorptive capacity in the assays using 5 mg of the adsorbent kept in contact with 0.75 mg/mL IgG solution in HEPES buffer at pH 6.8, in which, under these conditions, the adsorbed amount was about 260 mg/g. The kinetics and isotherms were obtained in the best adsorption condition. The kinetic adsorption profile shows that the equilibrium time was 10 hours and the isotherm adjustments were satisfactorily represented by the Langmuir and Langmuir- Freundlich models, providing maximum adsorption capacity values of 306 and 284 mg/g, respectively. Comparatively, the kinetic profiles and the isotherms were obtained for assays using as adsorbent non-sulfated chitosan microspheres (CM). To these, the adsorption of IgG reached the equilibrium in 6 hours, with adsorbed amount of 160 mg/g and the isothermal adjustments made with the Langmuir and Langmuir-Freundlich models resulted in adsorptive capacities of 405 and 466 mg/g respectively. Comparing the isothermal curves obtained for the two adsorbents, it was found that, in relation to the adsorbed amounts of immunoglobulin G, the SCM surpasses the CM only in cases where the IgG concentration in solution, at equilibrium, is up to 0.8 mg/mL. From the assays with IgG/BSA binary mixtures, in different mass proportions, it was possible to verify that the presence of BSA in solution interferes in the adsorption of IgG and, by SDS-PAGE electrophoresis, it can be observed that the SCM are more selective to immunoglobulin G than to albumin. Thus, it is possible to conclude that SCM have potential for application in IgG purification processes, being more appropriate for immunoglobulin concentration processes.