Evicting hitchhiker antigens from purified antibodies

J Chromatogr B Analyt Technol Biomed Life Sci. 2009 May 15;877(14-15):1543-52. doi: 10.1016/j.jchromb.2009.03.042. Epub 2009 Apr 1.


Antibodies that target common cellular structures may have a propensity to bind those very same antigens as they become exposed in dead or dying cells during production in a bioreactor. Those tendencies can be accentuated if the targeted epitope is highly conserved across species. While attention to contaminants such as endotoxin, viral particles, cellular DNA and even prions has grown coincident with the emergence of the monoclonal antibody industry, it is surprising how little attention has been focused on hitchhiker antigens that may co-elute while bound to the supposedly pure antibody. In this case study, we will focus on anti-histone antibodies and the measures we have taken to eliminate stowaways, such as histone-DNA complexes. These simple measures include the addition of a quartenary amine guard column to the protein A, adjusting the ionic strength of the cell culture supernatant to 400 mM sodium chloride, and establishing a mobile phase gradient from 400 mM to 2M during protein A chromatography. Initially adjusting the cell culture to 600 mM can compromise the quartenary amine guard column. Also, we demonstrate the applicability of these techniques in both the R&D lab and the manufacturing plant, particularly in improving the apparent potency of antibodies destined for the clinic. Given the prominence of anti-histone antibodies in chromatin immunoprecipitation (ChIP), the implications of hitchhiker antigens interferring with the results of an experiment are far-reaching, indeed, we detect them in some popularly used antibodies. Moreover, a wide variety of monoclonals that may target antigens expressed by the producer cell line may face similar problems, resulting in a decreased production yield, as well as a diminished apparent binding potency.

Publication types

  • Evaluation Study

MeSH terms

  • Antibodies / immunology
  • Antibodies / isolation & purification*
  • Antigens / immunology*
  • Chromatography, Affinity / methods*
  • DNA / immunology
  • Histones / immunology*
  • Humans


  • Antibodies
  • Antigens
  • Histones
  • DNA