Tandem IMAC-HPLC purification of a cocaine-binding scFv antibody

Jason A. Moss, Avery R. Coyle, Jung Mo Ahn, Michael M. Meijler, John Offer, Kim D. Janda

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Immobilized metal affinity chromatography (IMAC) has rapidly become one of the most widespread affinity purification techniques employed in recombinant protein expression. However, the high purity demands of certain applications are occasionally unattainable through a single IMAC separation. GNC92H2scFv is a cocaine-binding single-chain antibody fragment that is unstable during long-term storage in aqueous solution. To circumvent this problem, a reversed-phase HPLC separation was performed following IMAC purification of GNC92H2scFv from Escherichia coli cell culture supernatant. The resulting HPLC effluent was then freeze-dried to afford a salt-free lyophilizate amenable to long-term storage with minimal loss in binding activity. HPLC purification also effectively removed an 80-kDa protein contaminant that co-eluted with the IMAC-purified protein. Of special importance for in vivo applications of recombinantly expressed protein therapeutics, an HPLC purification step afforded a 1000-fold reduction in lipopolysaccharide (LPS) endotoxin contamination in the final GNC92H2scFv product.

Original languageEnglish (US)
Pages (from-to)143-148
Number of pages6
JournalJournal of Immunological Methods
Volume281
Issue number1-2
DOIs
StatePublished - Oct 1 2003

Keywords

  • Antibody
  • IMAC
  • Protein
  • RP-HPLC
  • scFv

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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    Moss, J. A., Coyle, A. R., Ahn, J. M., Meijler, M. M., Offer, J., & Janda, K. D. (2003). Tandem IMAC-HPLC purification of a cocaine-binding scFv antibody. Journal of Immunological Methods, 281(1-2), 143-148. https://doi.org/10.1016/j.jim.2003.07.010