Ion-Exchange Chromatography to Analyze Components of a Clostridium difficile Vaccine

Methods Mol Biol. 2016:1476:269-77. doi: 10.1007/978-1-4939-6361-4_20.

Abstract

Ion-exchange (IEX) chromatography is one of many separation techniques that can be employed to analyze proteins. The separation mechanism is based on a reversible interaction between charged amino acids of a protein to the charged ligands attached to a column at a given pH. This interaction depends on both the pI and conformation of the protein being analyzed. The proteins are eluted by increasing the salt concentration or pH gradient. Here we describe the use of this technique to characterize the charge variant heterogeneities and to monitor stability of four protein antigen components of a Clostridium difficile vaccine. Furthermore, the IEX technique can be used to monitor reversion to toxicity for formaldehyde-treated Clostridium difficile toxins.

Keywords: 5mTcdA; 5mTcdB; Binary toxins; C. difficile; HPLC; Ion-exchange chromatography; Vaccine.

MeSH terms

  • ADP Ribose Transferases / isolation & purification
  • ADP Ribose Transferases / toxicity
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / toxicity
  • Bacterial Toxins / isolation & purification
  • Bacterial Toxins / toxicity
  • Bacterial Vaccines / biosynthesis
  • Bacterial Vaccines / isolation & purification*
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange / methods*
  • Clostridioides difficile / chemistry
  • Clostridioides difficile / immunology*
  • Enterocolitis, Pseudomembranous / immunology
  • Enterocolitis, Pseudomembranous / microbiology
  • Enterocolitis, Pseudomembranous / prevention & control*
  • Enterotoxins / isolation & purification
  • Enterotoxins / toxicity
  • Formaldehyde / chemistry
  • Hot Temperature
  • Humans
  • Hydrogen-Ion Concentration
  • Sodium Chloride
  • Temperature
  • Vaccines, Attenuated

Substances

  • Bacterial Proteins
  • Bacterial Toxins
  • Bacterial Vaccines
  • Enterotoxins
  • Vaccines, Attenuated
  • tcdA protein, Clostridium difficile
  • toxB protein, Clostridium difficile
  • Formaldehyde
  • Sodium Chloride
  • ADP Ribose Transferases
  • actin-specific ADP-ribosyltransferase, Clostridium