(438b) High-Throughput Process Development of Recombinant Human Serum Albumin Separation with Mixed-Mode Chromatography

The recombinant human serum albumin (rHSA) expressed by microorganisms, such as P. pastoris, is a potential pathogen-free and economic alternative for the plasma-derived HSA (pHSA), which could avoid virus infection and resolve blood shortage issues. However, high purity of rHSA is essential for therapeutic use because of high dosage requirement, which leads to a great challenge to purification processes.

In this work, four mixed-mode resins (Capto adhere, Capto MMC, MX-Trp-650M and Nuvia cPrime) were tested to capture rHSA from P. pastoris broth and compared with two ion-exchangers (Q Sepharose FF and SP Sepharose FF). The high-throughput screening method with microtiter filter plate was used to screen the optimal pH and dilution ratio for rHSA adsorption. The adsorption capacity and selectivity were investigated. It was found that the cation-exchange resins with the negatively-charged group (such as carboxyl or sulfonic group) were more suitable for the binding of rHSA, and mixed-mode resins showed high salt-tolerant property and the pretreatment of dilution was unnecessary. For MX-Trp-650M, 200 mM NaCl could even promote the adsorption of rHSA. In addition, four resins with cation-exchange group were used to screen the elution conditions for rHSA separation with microtiter filter plate. For Capto MMC and MX-Trp-650M, high recovery and purity could be obtained due to high adsorption selectivity. Finally, the separation conditions based on the high-throughput studies were verified by the column chromatography. The results indicated that high binding capacity and elution recovery could be obtained under the optimized loading and elution conditions. In general, Capto MMC and MX-Trp-650M showed an efficient practicability for rHSA separation with high capacity (~50 mg/ml dynamic binding capacity at 10% breakthrough), suitable salt-tolerance (about 20 mS/cm), high recovery (above 95%) and high purity (above 96%), as well as excellent removal of pigments and HCPs. The results indicated that high-throughput process development with microtiter filter plate is a powerful tool to determine a wide range of variables and then optimize the separation conditions. Mixed-mode chromatography showed high process efficiency for rHSA separation from cell broth with high capacity and purity, suitable salt-tolerance and high elution recovery.

This work was supported by the International Science & Technology Cooperation Program of China and National Natural Science Foundation of China.