Optimization of the polishing process by integrating experimental design and high-throughput screening

Abstract

Complex bispecific antibody formats tend to form more product-related impurities than monoclonal antibodies. The primary constraints are yield and purity in the polishing stage. The purpose of this study was to enhance the understanding of the optimal working window for four mixed-mode resins and to reduce the burden of resin screening and parameter optimization during process development. This study optimized the loading and elution conditions of four different mixed-mode cationic resins to enhance the yield and purity by integrating Design of Experiments with High-Throughput Screening. It was observed that despite being weakly acidic mixed-mode cationic resins, these four resins exhibited significant differences in their adsorption and elution performances and varied tolerances to salt concentrations. Capto MMC demonstrated strong hydrophobicity, while the performance profiles of MX-Trp-650 M and Nuvia cPrime were similar, with Nuvia cPrime showing superior purification effects. Eshmuno CMX, by extending its side chain ligand, achieved higher binding efficiency and capacity. Through the optimization of salt concentrations or the application of dual-gradient elution strategies, the target protein with high yield (77 %) and purity over 99 % was successfully obtained. This research not only provides in-depth insights into the application of mixed-mode chromatography in the biopharmaceutical field but also offers practical optimization strategies for the industrial-scale purification of bispecific antibodies.