Understanding the role of arginine and citrate as eluents in affinity chromatography

Diwakar Shukla, Bernhardt L. Trout

Research output: Chapter in Book/Report/Conference proceedingChapter


Industrial scale purification of therapeutically useful antibodies (immunoglobulin, IgGs) typically rely on Protein A affinity chromatography, as a first step. Eluents i.e aqueous solution of small organic molecules, are typically used to elute the antibodies from the affinity column. The molecular interactions between the antibody and the Protein A affinity resin are exceedingly dependent on pH and eluent concentration. This complex mechanism is not well understood, beyond the application of general trends, for eluents such as arginine and citrate. We have performed molecular mechanics simulations of antibody and Protein A to rationalize the experimental observations of the effects of arginine and citrate on purification yield. It was found that the highly positively charged arginine reduced the binding free energy (ΔΔGB) between the Protein A affinity resin and the antibody. While the negatively charged citrate increases the ΔΔGB for Protein A affinity resin and the antibody. The results provide a rationale for the observed fluctuations in the recovery of antibodies using citrate and arginine, and also provided insight into of the nature of the molecular interactions, which can be exploited for eluent design on Protein A affinity chromatography.

Original languageEnglish (US)
Title of host publicationDevelopments in Biotechnology and Bioprocessing
PublisherAmerican Chemical Society
Number of pages20
ISBN (Print)9780841229105
StatePublished - Mar 8 2013
Externally publishedYes

Publication series

NameACS Symposium Series
ISSN (Print)0097-6156
ISSN (Electronic)1947-5918

ASJC Scopus subject areas

  • General Chemistry
  • General Chemical Engineering


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