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2000
Volume 32, Issue 10
  • ISSN: 0929-8665
  • E-ISSN: 1875-5305

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.

© 2025 Bentham Science Publishers
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2025-10-03
2026-02-20

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/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
PPL 32, 711 (2025); 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
Keyword(s): acid-base pair; buffer strength; Cation exchange chromatography (CEX); pH transition; shoulder-peak

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