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ORIGINAL RESEARCH article

Front. Bioeng. Biotechnol.

Sec. Industrial Biotechnology

Volume 13 - 2025 | doi: 10.3389/fbioe.2025.1567923

Enhancing therapeutic antibody production through amino acid-induced pH shift in Protein A affinity chromatography

Provisionally accepted
Lin Senzhu Lin Senzhu *Wangy Yue Wangy Yue
  • WuXi Biologics, Shanghai, China

The final, formatted version of the article will be published soon.

    Protein aggregation, denaturation, and loss of potency often occur during Protein A chromatography due to the harsh acidic conditions required for antibody elution. This study presents a pH shift-based elution strategy that effectively mitigates these issues by introducing amino acid-based elution buffers to create a milder elution environment and increase the final elution pH. By optimizing the combination of pre-elution and elution buffers, the elution pool pH was increased up to 7.2, significantly enhancing protein stability. Among various elution buffers tested, amino acids with non-polar or polar uncharged side chains-such as leucine, glycine, and serine-exhibited the most effective pH transition, resulting in 0.5-2.9 units pH shifts. Additionally, the use of 50 mM Bis-Tris, pH 7.2 as a pre-elution buffer demonstrated the highest capacity for stabilizing pH shifts. The scalability of this approach was validated using a 10 cm diameter column, where yields remained comparable to small-scale experiments, and elution pool stability was able to be maintained for 72 hours at 26°C. These findings establish pH-shifting elution as a scalable, cost-effective method for improving the recovery and stability of low pH-unstable therapeutic antibodies in Protein A chromatography.

    Keywords: pH Shift, low pH unstable protein, Protein A chromatography, amino acid-based elution, Downstream purification

    Received: 28 Jan 2025; Accepted: 17 Mar 2025.

    Copyright: © 2025 Senzhu and Yue. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Lin Senzhu, WuXi Biologics, Shanghai, China

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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