High-throughput screening and optimization of mixed-mode resins for human serum albumin separation with microtiter filter plate

High-throughput approaches become increasingly important on process development for protein separation. In this work, four mixed-mode resins (Capto adhere, Capto MMC, MX-Trp-650 M and Nuvia cPrime) were investigated for human serum albumin (HSA) separation and compared with two ion-exchangers (Q Sepharose FF and SP Sepharose FF). High-throughput method based on microtiter filter plate was used to determine adsorption properties and optimize separation conditions. For resins with anion-exchange groups alkaline condition would be favorable for HSA adsorption, while acidic condition for resins with cation-exchange groups. Mixed-mode resins showed obviously salt-tolerant properties. Salt addition had little effect on Nuvia cPrime, and could promote HSA adsorption for Capto MMC and MX-Trp-650 M. The elution conditions were also optimized and high recovery could be obtained. In addition, the optimized conditions obtained by high-throughput approaches were verified by lab-scale column chromatography. It was found that Capto MMC and MX-Trp-650 M would be more suitable for HSA separation and DBC10% could reach more than 40 mg/ml under 200 mM NaCl addition. Finally, rHSA separation from P. pastoris culture broth was tested with MX-Trp-650 M, and high purity and recovery could be obtained. This study demonstrated that high-throughput screening method is a powerful tool to optimize the chromatographic separation. Mixed-mode cation-exchange resins showed an efficient practicability for HSA separation with high capacity, suitable salt-tolerance and high elution recovery.