SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield

Protein Expr Purif. 2024 Apr:216:106418. doi: 10.1016/j.pep.2023.106418. Epub 2023 Dec 21.


For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield.

Keywords: Aggregate; Culture harvest; Fc-fusion; Flow-through; Protein A chromatography; SEC-HPLC; bsAb; mAb.

MeSH terms

  • Antibodies, Bispecific* / chemistry
  • Antibodies, Monoclonal / chemistry
  • Cell Culture Techniques*
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Staphylococcal Protein A / chemistry


  • Antibodies, Monoclonal
  • Antibodies, Bispecific
  • Staphylococcal Protein A