title: Ion-Exchange Chromatography to Analyze Components of a Clostridium difficile Vaccine.
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.
authors: Vaccine Analytical Department, Merck Research Laboratories, Merck & Co., Inc., 770 Sumneytown Pike, P.O. Box 4, WP42A-30, West Point, PA, 19486, USA. Richard_rustandi@merck.com.
authors: Richard R
authors: RR
authors: Rustandi
authors: Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, USA.
authors: Feng
authors: F
authors: Wang
authors: Vaccine Analytical Department, Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA.
authors: Catherine
authors: C
authors: Lancaster
authors: Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, USA.
authors: Adam
authors: A
authors: Kristopeit
authors: Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, USA.
authors: David S
authors: DS
authors: Thiriot
authors: Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, USA.|Sanofi Pasteur, Swiftwater, PA, USA.
authors: Jon H
authors: JH
authors: Heinrichs
mesh_terms: D036002:ADP Ribose Transferases / Q000302:isolation & purification / Q000633:toxicity; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000633:toxicity; D001427:Bacterial Toxins / Q000302:isolation & purification / Q000633:toxicity; D001428:Bacterial Vaccines / Q000096:biosynthesis / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D016360:Clostridioides difficile / Q000737:chemistry / Q000276:immunology*; D004761:Enterocolitis, Pseudomembranous / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*; D004768:Enterotoxins / Q000302:isolation & purification / Q000633:toxicity; D005557:Formaldehyde / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D012965:Sodium Chloride; D013696:Temperature; D014613:Vaccines, Attenuated
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004768:Enterotoxins; D014613:Vaccines, Attenuated; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D005557:Formaldehyde; D012965:Sodium Chloride; D036002:ADP Ribose Transferases; C410164:actin-specific ADP-ribosyltransferase, Clostridium
keywords: 5mTcdA; 5mTcdB; Binary toxins; C. difficile; HPLC; Ion-exchange chromatography; Vaccine