title: Characterization of a backbone cleavage product of BMS-196854 (Oncostatin M), a recombinant anti-inflammatory cytokine.
abstract: OBJECTIVE
  BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein.
  
  
  METHODS
  SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies.
  
  
  RESULTS
  Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction.
  
  
  CONCLUSIONS
  Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin inhibitor and/or controlling the solution pH and storage temperature.
authors: Pharmaceutics Research and Development, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, One Squibb Drive, P.O. Box 191, New Brunswick, NJ 08903, USA. fang.zhao@bms.com
authors: F
authors: F
authors: Zhao
authors: D J
authors: DJ
authors: Stein
authors: M
authors: M
authors: Paborji
authors: P W
authors: PW
authors: Cash
authors: B J
authors: BJ
authors: Root
authors: Z
authors: Z
authors: Wei
authors: C J
authors: CJ
authors: Knupp
mesh_terms: D000893:Anti-Inflammatory Agents / Q000737:chemistry*; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D053683:Oncostatin M; D010455:Peptides / Q000737:chemistry*; D014361:Trypsin Inhibitors / Q000494:pharmacology
chemical_list: D000893:Anti-Inflammatory Agents; D010455:Peptides; D014361:Trypsin Inhibitors; D053683:Oncostatin M