title: Identifying and modulating disulfide formation in the biopharmaceutical production of a recombinant protein vaccine candidate.
abstract: Structural conversion of the serotype A recombinant botulinum neurotoxin heavy chain fragment (rBoNTA(Hc)) produced intracellularly in Pichia pastoris yeast was observed and characterized during purification development efforts. A pH screening study captured the transformation stages of the original recovered species into its derived counterpart and a number of analytical tools such as peptide mapping by LC/MS confirmed the formation of a disulfide bond, especially in samples of neutral to basic pH. A cation exchange chromatographic method proved useful in following the incidence of the reaction in various rBoNTA(Hc) samples. The disulfide formation kinetics were characterized using a one-quarter quadratic factorial design, following the investigation and development of controlled oxidation conditions using cysteine and cystamine as the redox pair. Temperature, pH and concentration of the redox pair had a significant effect on the yield and rate of the disulfide formation. This controlled reaction was eventually introduced as a functional unit operation in the purification process. The summation of preliminary scale-up and potency data showed scalability and robustness in the production of an active disulfide-bonded form of a recombinant botulism vaccine candidate. The presence of the disulfide bond did not effect the vaccine potency and it enhanced the molecule's thermal stability.
authors.affiliation: Diosynth RTP, Inc., 3000 Weston Parkway, Cary, NC 27513, USA. anne.bouvier@diosynth-rtp.com
authors.forename: Anne
authors.initials: A
authors.lastname: Bouvier
authors.forename: Jaymi
authors.initials: J
authors.lastname: Chapline
authors.forename: Renee
authors.initials: R
authors.lastname: Boerner
authors.forename: Shanthini
authors.initials: S
authors.lastname: Jeyarajah
authors.forename: Susan
authors.initials: S
authors.lastname: Cook
authors.forename: Prathima S
authors.initials: PS
authors.lastname: Acharya
authors.forename: Ian
authors.initials: I
authors.lastname: Henderson
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Schrimsher
authors.forename: Scot R
authors.initials: SR
authors.lastname: Shepard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001702:Biopharmaceutics / Q000379:methods*; D019274:Botulinum Toxins, Type A / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000627:therapeutic use; D001906:Botulism / Q000517:prevention & control; D004220:Disulfides / Q000737:chemistry*; D015195:Drug Design; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010843:Pichia / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use; D013329:Structure-Activity Relationship; D013696:Temperature; D014614:Vaccines, Synthetic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D019274:Botulinum Toxins, Type A