Purificação da imunoglobulina M a partir da utilização de frações do plasma humano empregando técnicas de cromatografia líquida

Abstract

Immunoglobulin M (IgM) is a pentamer of approximately 950kDa, consisting of two heavy chains of 75kDa each, two light chains of 25kDa and a J chain of 15kDa. IgM is a promising therapeutic candidate due to increasing evidence suggesting its potential as a tumor marker, anti-inflammatory and immunomodulatory activities. In order to exploit its full potential, it is important that large amounts of IgM are available at low cost. In this work, two purification steps that were already being developed by the laboratory were explored. Purification by metal affinity chromatography (IMAC) and cation exchange of the pool from plasma purification on Sepharose 4FF followed by ANX Sepharose FF was evaluated. In IMAC, the purification was evaluated at pHs 5.0, 6.0 and 7.0 and the results indicate that with pH 6.0 in IMAC-Co2+ we obtained IgM with better recovery and greater purity. By varying the NaCl concentration in the equilibrium buffer between 250mM and 500mM, we found that IgM was obtained with greater purity in the purification in which 250mM NaCl was used in the equilibrium buffer. Using the cation exchange column, the results obtained were not satisfactory. The purity of IgM, calculated by the ImageJ program, was approximately 75%. To increase the purity of IgM, purification on the Superdex 200 gel filtration column will be evaluated.