RESUMEN
Iminodiacetic acid (IDA) is one of the chelating ligands most frequently employed in immobilized metal-ion affinity chromatography (IMAC) due to its ability to act as electron-pair donor, forming stable complexes with intermediate and borderline Lewis metal ions (electron acceptor). Thus, IDA can also be employed in ion exchange chromatography to purify positively charged proteins at neutral pH values. This study aimed to evaluate IDA as an ionogenic group (ion exchanger) immobilized on poly (ethylene vinyl alcohol) (PEVA) hollow fiber membranes for immunoglobulin G1 (IgG1) monoclonal antibody (MAb) purification. IDA-PEVA membranes showed considerable promise for MAb purification, since IgG1 was recovered in eluted fractions with traces of contaminants as confirmed by Western blotting and ELISA analysis. Quantification of IgG1 showed that a purity of 94.2% was reached in the elution step. Breakthrough curve and batch adsorption experiments showed that the MAb dynamic binding capacity (DBC) of 3.10 mg g-1 and the maximum adsorption capacity of 70 mg g-1 were of the same order of magnitude as those found in the literature. The results obtained showed that the IDA-PEVA hollow fiber membrane could be a powerful adsorbent for integrating large-scale processes for purification of MAb from cell culture supernatant.
Asunto(s)
Anticuerpos Monoclonales/química , Cromatografía de Afinidad/métodos , Iminoácidos/química , Inmunoglobulina G/química , Membranas Artificiales , Adsorción , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Quelantes/química , Concentración de Iones de Hidrógeno , Inmunoglobulina G/aislamiento & purificación , Iones , Ligandos , Metales/química , RatonesRESUMEN
The aim of this work was to isolate and purify bromelain extracted from the pineapple peel by ammonium sulfate precipitation (40-80%), followed by desalting and freeze-drying with a 75% activity recovery and 2.2 fold increased specific activity. Ion exchange chromatography on DEAE-Sepharose was able to separate the polysaccharides from the enzyme, which was recovered in the elution step, maintaining its enzymatic activity. The batch adsorption of bromelain was evaluated in terms of total protein and enzymatic activity using Langmuir and Langmuir-Freundlich models. Results showed that the process could be suitable for the recovery and purification of the enzyme, maintaining its specific activity.