Two-step separation strategy with mixed-mode chromatography to purify recombinant human serum albumin from Pichia pastoris culture broth

Mixed-mode chromatography (MMC) with the specially-designed ligands shows good adsorption selectivity for protein separation. In this study MMC was explored to purify recombinant human serum albumin (rHSA) from Pichia pastoris culture broth, which combined bind-elute and flow-through modes. Four mixed-mode resins were evaluated for their adsorption capacities of HSA. It was found that two home-made MMC resins showed special performances. IC-B-6FF resin with indole-3-acetic acid-cysteine ligand had high adsorption and selectivity towards HSA, and SFA-B-6FF resin with sulfapyridine ligand showed minimal binding to HSA. Therefore, a two-step MMC separation strategy was designed that IC-B-6FF was used to capture rHSA directly from yeast culture broth and then SFA-B-6FF resin was used to polish the remaining impurities by flow-through mode. The results revealed that the purity of rHSA reached 95.1% with a yield of 81.2% after the two-step separation. The removal rate of host cell proteins (HCPs) was 99.8% with final HCP level as low as 50 ppm. The results demonstrate that MMC combining bind-elute and flow-through modes has a promising potential to separate protein with high selectivity and process efficiency.