Salt tolerant chromatography provides salt tolerance and a better selectivity for protein monomer separations

Salt tolerant chromatography (STC) is an attractive method as buffer exchange during protein purification processes can be skipped; however, the retention and separation mechanism of such STC are still not fully understood. We carried out linear gradient elution (LGE) experiments of bovine serum albumin (BSA) including its dimer form by using poly-amine ligand STC. The peak salt concentration IR was measured as a function of normalized gradient slope GH, and the number of binding sites B was determined. The separation performance of monomer and dimer was much higher for STC. The IR values of BSA monomer and dimer for STC were much higher (IR > 0.5M) than those for conventional IEC. The IR values of arginine-Cl gradient decreased markedly compared to those of NaCl gradient whereas they did not change for conventional IEC. This might be due to combined effects of electrostatic and hydrophobic interaction to the retention of proteins in STC. Adding polyethylene glycol (PEG) into the mobile phase of IEC also increased the retention (salt tolerance) and the resolution of BSA monomer and dimer. Higher viscosity and low solubility of proteins due to PEG were disadvantages of this method. STC with poly-amine ligand might be also suited for the continuous flow-through separation of monomer.