Affinity-enhanced purification of human antibodies by aqueous two-phase extraction

The partition of human antibodies in polyethylene glycol (PEG)/dextran aqueous two-phase systems (ATPS) was investigated using charged, hydrophobic and affinity ligands, either in the form of free ligands or as phase forming components. Free ligands were based on triethylene glycol (TEG) molecules modified with glutaric acid, sulfonate, mercaptoethyl pyridine, aminoquinuclidine and pyrimidine groups. In addition, PEG molecules with a molecular weight of 1000 were modified with amino groups and also used as free ligands. Glutaric acid displayed a high affinity towards the antibodies with a 96% extraction yield and protein purity of 95%. PEG molecules with a molecular weight of 3350 were modified with glutaric acid, amino, mercaptoethyl pyridine, pyrimidine and benzyl groups, and used as phase forming components. Best results were obtained with glutaric acid, amino and benzyl groups with extraction yields higher than 75% and protein purities around 90%. The best performing system was composed of 5% dextran, 8% PEG diglutaric acid, 10 mM phosphate buffer pH 7, which allowed the extraction of 97% of IgG to the upper phase, with a purity of 94% in terms of proteins.