title: Exploration of Recombinant Streptokinase Degradation Products Under Various pH Reduction Conditions in Downstream Processing.
abstract: BACKGROUND
  Methods of producing streptokinase, which can be used in the treatment of myocardial infarction, by hemolytic streptococci and recombinant E. coli have been described in patents since 1955. Degradation products in active pharmaceutical ingredients (APIs) and finished pharmaceutical products are considered as impurities and it is required that these degradation impurities are minimized or rather avoided throughout manufacturing process.
  
  
  OBJECTIVE
  The aim of this study was to explore the occurrence of rSK degradation during acidification step in downstream processing.
  
  
  METHODS
  The polyclonal antibody was produced by immunization of New Zealand white (NZW) rabbit with pure rSK (purity>98%). The solubilized inclusion bodies with various pH values (4.2, 5.0 and 6.0) were analyzed by Western blotting using rSK polyclonal antibody.
  
  
  RESULTS
  Western blot analysis demonstrated the generation of rSK degradation products (with the molecular weight of about 27, 20 and 17 kDa) when the pH value of the solubilized inclusion bodies was reduced to 5.0 and 4.2, while no degradation of rSK observed at pH 6.0.
  
  
  CONCLUSIONS
  This study demonstrates that the level of pH reduction in the solubilized inclusion bodies during downstream processing plays an important role in generating rSK degradation products, and substantial post-solubilization degradation of rSK occurs at pH lower than 6.0. Development of these degradation impurities, which cannot be eliminated by subsequent chromatographic purifications, can be exclusively avoided during acidification procedure by appropriate pH adjustment approach in downstream processing.
authors.affiliation: Production & Research Complex, Pasteur Institute of Iran, Tehran-Karaj Highway, Postal Code: 31599, Karaj, Iran. rjalalirad@gmail.com.
authors.forename: Reza
authors.initials: R
authors.lastname: Jalalirad
authors.forename: Alireza
authors.initials: A
authors.lastname: Kavianpour
authors.forename: Ahmad
authors.initials: A
authors.lastname: Beiroti
authors.forename: Mina
authors.initials: M
authors.lastname: Sepahi
authors.forename: Parinaz
authors.initials: P
authors.lastname: Tavakoli Zaniani
authors.forename: Firooz
authors.initials: F
authors.lastname: Arsalani
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010330:Patents as Topic; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013291:Streptococcus / Q000235:genetics; D013300:Streptokinase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D013300:Streptokinase