Comparing the relative robustness of anion exchange and the corresponding mixed-mode chromatography on removing a weakly-bound byproduct: A case study of bispecific antibody purification

Protein Expr Purif. 2021 Nov:187:105948. doi: 10.1016/j.pep.2021.105948. Epub 2021 Jul 22.


A certain impurity may bind weaker or tighter to a particular type of chromatography column (e.g., ion exchange, hydrophobic interaction or mixed-mode) than the target protein, and this forms the basis for separation. For impurities that bind weaker, they can be removed by an appropriate pre-elution wash. However, we previously showed that wash-enabled impurity clearance is usually sensitive to loading density, causing poor robustness of the process. In this work, with a bispecific antibody case study, we compared the relative robustness of anion exchange and the corresponding mixed-mode chromatography, which mediates both anion exchange and hydrophobic interactions, on removing a weakly-bound byproduct by wash. It was learned that under a fixed appropriate wash condition, the latter achieves consistent byproduct clearance and good yield over a much wider range of loading density than the former. As wide loading density range is highly desirable for large-scale manufacturing, the above finding suggests that for a chromatography step that employs stepwise elution and relies on pre-elution wash for removing weakly-bound impurities, mixed-mode chromatography could be a better choice than the corresponding monomode ion exchange chromatography.

Keywords: Anion exchange (AEX) chromatography; Bispecific antibody (bsAb); Column wash; Loading density; Mixed-mode chromatography; Weakly-bound byproduct.

Publication types

  • Comparative Study

MeSH terms

  • Antibodies, Bispecific / isolation & purification*
  • Chromatography, Ion Exchange / methods*
  • Hydrophobic and Hydrophilic Interactions
  • Reproducibility of Results


  • Antibodies, Bispecific