Purificação de imunoglobulina G a partir do plasma ou soro humano utilizando cromatografia de afinidade com ions metalicos imobilizados

AUTOR(ES)
DATA DE PUBLICAÇÃO

1999

RESUMO

Purification of plasma proteins through selective methods, such as cromatography, has been recognised in nowadays as an important operation by pharmaceutical industry as the highly purified proteins reduce side effects. Therefore, the development of purificaton selective methods for large scale operations are extremely important to broad diversity, improve the yield and purity of therapeutic proteins extracted from plasma. In this work a flexibility study on the separation of immunoglobulin G (IgG) from human plasma through immobilized metal ion affinity chromatography (IMAC) is carried out. This technique exploit the protein affinity for immobilized metal ion and has been used to purity several proteins and peptides. Chromatography were carried out with pure human IgG in order to select the metal ion amongst Cu2+, Ni2+, Zn2+ e Co2", to be immobilized on IDA. Several buffers were tested in the adsorption stage as well. The proteins adsorbed on the support were eluted by the protonation of the electron-donating species on the protein using a decreasing pH step gradient or increasing imidazole concentration. The metal ion bonding chelating agents (IDA) showed distinct interactions with protein (pure human IgG). Since Ni2+ was selected, the support selectivity to adsorb IgG was determined with human plasma from healthy people. As IgG has not been completely purified on IDA-Ni2^, a second column (containing immobilized Ni2") has been used in series with the first one (containing immobilized Zn2") in order to achieve a ligher purity degree of IgG. Nephelometric analysis of the retained fraction on ID A-Zn2" columns in series with IDA-Ni2+, and eluted at pH 5.0 determinated 41.90% of IgG, 1.34% of IgM, 2.10% of IgA and 54.69% of transferrin. The contaminant proteins (transferrin and IgM) could be removed using gel filtration. The analysis carried out by isoelectric focusing of wash e elution fractions using human IgG (Sigma), showed that the isoelectric point distribution in the wash and elution fractions were not dependent neither on the immobilized metal ion or the buffer used. It could be observed that the non-retained fractions (wash) contained IgG with low pi (between 5.0 e 6.0). Selectivity in IgG elution by decreasing pH step gradient (immobilized Ni2+) or increasing imidazole concentration (immobilized Ni2r and Zn2+) was not reached, all retained fractions contained IgG with pi between 6.0 and 8.65, and separation was not observed

ASSUNTO(S)

cromatografia de afinidade imunoglobulinas imunoglobulinas - purificação

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