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

Academic Article
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Overview

related to degree

  • Moss, Jason, Ph.D. in Chemistry, Scripps Research 2000 - 2005

authors

  • Moss, Jason
  • Coyle, A. R.
  • Ahn, J. M.
  • Meijler, M. M.
  • Offer, J.
  • Janda, Kim

publication date

  • October 2003

journal

  • Journal of Immunological Methods  Journal

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.

subject areas

  • Chromatography, Affinity
  • Chromatography, High Pressure Liquid
  • Cocaine
  • Enzyme-Linked Immunosorbent Assay
  • Immunoglobulin Fragments
  • Lipopolysaccharides
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Research

keywords

  • IMAC
  • RP-HPLC
  • antibody
  • protein
  • scFv
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Identity

International Standard Serial Number (ISSN)

  • 0022-1759

Digital Object Identifier (DOI)

  • 10.1016/j.jim.2003.07.010

PubMed ID

  • 14580888
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Additional Document Info

start page

  • 143

end page

  • 148

volume

  • 281

issue

  • 1-2

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