Application of aqueous two-phase systems to antibody purification: a multi-stage approach.
The single-stage equilibrium aqueous two-phase extraction (ATPE) of human antibodies from a Chinese hamster ovary (CHO) cells supernatant was investigated. The performance of polyethylene glycol 3350 (PEG 3350)/phosphate aqueous two-phase systems (ATPS) was evaluated by studying several experimental conditions, such as pH, ionic strength, volume ratio and initial antibody concentration in the feed stock. The conditions that favoured the extraction of human immunoglobulin G (IgG) were low pH values, high NaCl concentration and high volume ratios. The percentage of contaminants removal did not depend on the pH value and NaCl concentration, increasing, however, as the volume ratio decreased. About 66% of total contaminants were removed when a volume ratio of 0.4 was used. The multi-stage equilibrium ATPE was also investigated by simulating a four stages cross-current operation in test tubes. According to the IgG equilibrium curves and respective McCabe Thiele diagrams, a predicted optimised scheme of a counter-current multi-stage ATPE was described. An IgG recovery yield of 89% and a protein purity of 75% can be achieved using a PEG/phosphate ATPS containing 10% (w/w) NaCl, five stages and a volume ratio of 0.4. This represents significant improvements in the recovery yield and purity when compared to a single-stage trial performed at the same experimental conditions, where a 61% recovery yield and 55% protein purity were attained. Based on the CHO cells supernatant components equilibrium curves, it was observed that IgG can be completely purified from the higher molecular weight (MW) components and partially purified from the lower MW components.