Separation of bispecific antibody related impurities with mixed-mode chromatography.

The complex structure of bispecific antibody molecules and the rapid increase in upstream expression pose a significant challenge for downstream purification. Compared to mAbs, bsAbs typically contain a higher proportion of aggregates and fragments that are difficult to remove. Extensive study and optimization are often required because the properties of impurities are close to the target molecules. In this investigation, ion exchange resins and mixed-mode resins were used to evaluate the effectiveness in removing byproducts. Mixed-mode anion chromatography was found to be effective in polishing step of bsAbs with strong hydrophobicity. Hydroxyapatite chromatography using arginine hydrochloride as an eluent provided excellent separation performance compared to that of cation exchange resins and conventional mixed-mode resins. The separation process was optimized and verified in a 200 L pilot batch. The final products were obtained with excellent purity (99.0 %) and yield (80.70 %). This work provides a new idea for high-yield polishing in downstream processing, which improves the economics of large-scale production. [Display omitted] • By-products can be removed through hydrophobic and ionic interactions. • Hydroxyapatite offers excellent resolution eluted with an Arg-HCl gradient. • Hydroxyapatite can effectively remove non-covalently bound aggregates. • Yield and purity show a high degree of consistency when scaled up to 200 L. [ABSTRACT FROM AUTHOR]