Processing of high-salt-containing protein A eluate using mixed-mode chromatography in purifying an aggregation-prone antibody

Protein Expr Purif. 2019 Dec:164:105458. doi: 10.1016/j.pep.2019.105458. Epub 2019 Jul 22.


We previously developed a method that can significantly improve Protein A chromatography's capability of removing antibody aggregates. That particular method requires polyethylene glycol (PEG) and 400 mM or more of calcium chloride/sodium chloride to be added to wash and elution buffers. Consequently, Protein A chromatography performed using this method has relatively high concentration of salt in its eluate. The high salt content prevents the neutralized eluate from binding to ion exchange columns without conductivity adjustment. In the current study we demonstrated that mixed-mode chromatography can be used as a subsequent step to Protein A chromatography with high-salt-containing eluate. As mixed-mode ligand mediates salt-tolerant adsorption, it allows the neutralized Protein A eluate to be directly loaded without the need of conductivity adjustment, and thus enables a smooth and convenient connection between capture and polishing steps. In this work we also showed that the mixed-mode chromatography, performed in bind-elute mode, removed most of PEG in the Protein A eluate.

Keywords: Antibody aggregate; Capto MMC; Ceramic hydroxyapatite (CHT); Mixed-mode chromatography; Polyethylene glycol (PEG); Protein A chromatography.

MeSH terms

  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal / isolation & purification*
  • Chlorides / chemistry*
  • Chromatography, Ion Exchange / methods*
  • Durapatite / chemistry
  • Polyethylene Glycols / chemistry
  • Protein Aggregates
  • Staphylococcal Protein A / chemistry*


  • Antibodies, Monoclonal
  • Chlorides
  • Protein Aggregates
  • Staphylococcal Protein A
  • Polyethylene Glycols
  • Durapatite