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image of Improving Acid-Base Pair Concentration in Wash/Elution Buffer Eliminates Elution Peak-Shouldering in Cation Exchange Chromatography

Abstract

Introduction

Peak-shouldering elution behavior was a common and unexpected result in bind-and-elute mode Cation Exchange Chromatography (CEX), which may be due to the pH transition during the elution step and the aggregation tendency of target proteins.

Methods

Improving the concentration of acid-base pairs in the wash buffers or elution buffers without changing pH or conductivity effectively resolved the peak-shouldering issue in CEX.

Results

In the case of molecule A, the shoulder peak was eliminated in the CEX run by increasing the NaAc-HAc concentration from 50 mM to 100 mM in the elution buffer or from 50 mM to 75 mM in the wash buffer. Higher NaAc-HAc concentrations affect the pH transition in the early stages of the elution step, which may explain the elimination of the shoulder peak. A similar result was observed for molecule B, where increasing the Tris-HCl concentration in the elution buffer from 50 mM to 80 mM also removed the shoulder peak during elution.

Discussion

The successful elimination of peak-shouldering behavior by increasing acid-base pair concentrations highlights the critical role of buffer capacity in modulating pH transitions during CEX. While this strategy offers a simple and effective solution, further investigation is needed to assess its applicability across diverse protein types and buffer systems.

Conclusion

These results demonstrate that increasing the concentration of acid-base pairs in the elution buffer or wash buffer of CEX using NaAc-HAc or Tris-HCl buffers is an effective strategy for eliminating the shoulder-peak.

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2025-10-03
2025-11-08

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References

  1. Yu L. Zhang L. Sun Y. Protein behavior at surfaces: Orientation, conformational transitions and transport. J. Chromatogr. A 2015 1382 118 134 10.1016/j.chroma.2014.12.087 25601319
    [Google Scholar]
  2. Shukla A.A. Thömmes J. Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends Biotechnol. 2010 28 5 253 261 10.1016/j.tibtech.2010.02.001 20304511
    [Google Scholar]
  3. Luo H. Cao M. Newell K. Afdahl C. Wang J. Wang W.K. Li Y. Afdahl, C.; Wang, J.; Wang, W.K.Double-peak elution profile of a monoclonal antibody in cation exchange chromatography is caused by histidine-protonation-based charge variants. J. Chromatogr. A 2015 1424 92 101 10.1016/j.chroma.2015.11.008 26596869
    [Google Scholar]
  4. Guo J. Creasy A.D. Barker G. Carta G. Surface induced three-peak elution behavior of a monoclonal antibody during cation exchange chromatography. J. Chromatogr. A 2016 1474 85 94 10.1016/j.chroma.2016.10.061 27802880
    [Google Scholar]
  5. Huang C. Wang Y. Xu X. Mills J. Jin W. Ghose S. Li Z.J. Mills, J.; Jin, W.; Ghose, S.; Li, Z.J, Hydrophobic property of cation-exchange resins affects monoclonal antibody aggregation. J. Chromatogr. A 2020 1631 461573 10.1016/j.chroma.2020.461573 33010710
    [Google Scholar]
  6. Guo J. Carta G. Unfolding and aggregation of monoclonal antibodies on cation exchange columns: Effects of resin type, load buffer, and protein stability. J. Chromatogr. A 2015 1388 184 194 10.1016/j.chroma.2015.02.047 25739785
    [Google Scholar]
  7. Guo J. Zhang S. Carta G. Unfolding and aggregation of a glycosylated monoclonal antibody on a cation exchange column. Part I. Chromatographic elution and batch adsorption behavior. J. Chromatogr. A 2014 1356 117 128 10.1016/j.chroma.2014.06.037 25015241
    [Google Scholar]
  8. Guo J. Carta G. Unfolding and aggregation of a glycosylated monoclonal antibody on a cation exchange column. Part II. Protein structure effects by hydrogen deuterium exchange mass spectrometry. J. Chromatogr. A 2014 1356 129 137 10.1016/j.chroma.2014.06.038 25011681
    [Google Scholar]
  9. Guo J. Xu X. Conformational changes of biomolecules in ion-exchange chromatography. In: Ion-Exchange Chromatography and Related Techniques, Elsevier Amsterdam, Netherlands 2024 521 534 10.1016/B978‑0‑443‑15369‑3.00020‑1
    [Google Scholar]
  10. Kimerer L.K. Pabst T.M. Hunter A.K. Carta G. Chromatographic behavior of bivalent bispecific antibodies on cation exchange columns. I. Experimental observations and phenomenological model. J. Chromatogr. A 2019 1601 121 132 10.1016/j.chroma.2019.04.012 31056270
    [Google Scholar]
  11. Luo H. Macapagal N. Newell K. Man A. Parupudi A. Li Y. Li Y. Effects of salt-induced reversible self-association on the elution behavior of a monoclonal antibody in cation exchange chromatography. J. Chromatogr. A 2014 1362 186 193 10.1016/j.chroma.2014.08.048 25182858
    [Google Scholar]
  12. Esfandiary R. Hayes D.B. Parupudi A. Casas-finet J. Bai S. Samra H.S. Shah A.U. Sathish H.A. A systematic multitechnique approach for detection and characterization of reversible self-association during formulation development of therapeutic antibodies. J. Pharm. Sci. 2013 102 1 62 72 10.1002/jps.23369 23150484
    [Google Scholar]
  13. Poplewska I. Piątkowski W. Antos D. A case study of the mechanism of unfolding and aggregation of a monoclonal antibody in ion exchange chromatography. J. Chromatogr. A 2021 1636 461687 10.1016/j.chroma.2020.461687 33246679
    [Google Scholar]
  14. Pabst T.M. Carta G. pH transitions in cation exchange chromatographic columns containing weak acid groups. J. Chromatogr. A 2007 1142 1 19 31 10.1016/j.chroma.2006.08.066 16978635
    [Google Scholar]
  15. Pabst T.M. Antos D. Carta G. Ramasubramanyan N. Hunter A.K. Protein separations with induced pH gradients using cation-exchange chromatographic columns containing weak acid groups. J. Chromatogr. A 2008 1181 1-2 83 94 10.1016/j.chroma.2007.12.054 18194806
    [Google Scholar]
  16. Ghose S. McNerney T.M. Hubbard B. pH transitions in ion-exchange systems: Role in the development of a cation-exchange process for a recombinant protein. Biotechnol. Prog. 2002 18 3 530 537 10.1021/bp020002i
    [Google Scholar]
  17. Bankston T.E. Dattolo L. Carta G. pH Transients in hydroxyapatite chromatography columns—Experimental evidence and phenomenological modeling. J. Chromatogr. A 2010 1217 14 2123 2131 10.1016/j.chroma.2010.02.004 20193952
    [Google Scholar]
  18. Soto Pérez J. Frey D.D. Behavior of the inadvertent pH transient formed by a salt gradient in the ion-exchange chromatography of proteins. Biotechnol. Prog. 2005 21 3 902 910 10.1021/bp040025s 15932272
    [Google Scholar]
  19. Gillespie R. Nguyen T. Macneil S. Jones L. Crampton S. Vunnum S. Cation exchange surface-mediated denaturation of an aglycosylated immunoglobulin (IgG1). J. Chromatogr. A 2012 1251 101 110 10.1016/j.chroma.2012.06.037 22771262
    [Google Scholar]
  20. Chen Z. Huang C. Chennamsetty N. Xu X. Li Z.J. Insights in understanding aggregate formation and dissociation in cation exchange chromatography for a structurally unstable Fc-fusion protein. J. Chromatogr. A 2016 1460 110 122 10.1016/j.chroma.2016.07.023 27452990
    [Google Scholar]
  21. Hirano A. Shiraki K. Kameda T. Effects of arginine on multimodal chromatography: Experiments and simulations. Curr. Protein Pept. Sci. 2018 20 1 40 48 10.2174/1389203718666171024115407 29065827
    [Google Scholar]
  22. Lange C. Rudolph R. Suppression of protein aggregation by L-arginine. Curr. Pharm. Biotechnol. 2009 10 4 408 414 10.2174/138920109788488851 19519416
    [Google Scholar]
  23. Fu Y. Xu Y. Zhang M. Lv F. Removal of signal peptide variants by cation exchange chromatography: A case study. Protein Expr. Purif. 2025 225 106581 10.1016/j.pep.2024.106581 39168393
    [Google Scholar]
  24. Hu L. Tang J. Zhang X. Li Y. Sodium caprylate wash during Protein A chromatography as an effective means for removing protease(s) responsible for target antibody fragmentation. Protein Expr. Purif. 2021 186 105907 10.1016/j.pep.2021.105907 34022391
    [Google Scholar]
  25. Liu C. Tian M. Dong W. Lu W. Zhang T. Wan Y. Zhang X. Li Y. SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield. Protein Expr. Purif. 2024 216 106418 10.1016/j.pep.2023.106418 38141898
    [Google Scholar]
  26. Hardin A.M. Ivory C.F. Buffer salt effect on pH in the interior of an anion exchange resin. J. Colloid Interface Sci. 2006 302 2 560 567 10.1016/j.jcis.2006.06.055 16870202
    [Google Scholar]
  27. Tsumoto K. Ejima D. Senczuk A.M. Kita Y. Arakawa T. Effects of salts on protein–surface interactions: Applications for column chromatography. J. Pharm. Sci. 2007 96 7 1677 1690 10.1002/jps.20821 17221853
    [Google Scholar]
  28. Cummins P.M. Rochfort K.D. O’Connor B.F. Ion-exchange chromatography: Basic principles and application. Methods Mol. Biol. 2017 1485 209 223 10.1007/978‑1‑4939‑6412‑3_11 27730555
    [Google Scholar]
/content/journals/ppl/10.2174/0109298665394172250926073117
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Improving Acid-Base Pair Concentration in Wash/Elution Buffer Eliminates Elution Peak-Shouldering in Cation Exchange Chromatography
Bentham Science Publishers ; https://doi.org/10.2174/0109298665394172250926073117
/content/journals/ppl/10.2174/0109298665394172250926073117
/content/journals/ppl/10.2174/0109298665394172250926073117
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  • Article Type:     Research Article
Keywords: shoulder-peak ; acid-base pair ; buffer strength ; Cation exchange chromatography (CEX) ; pH transition

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