Exploration of Recombinant Streptokinase Degradation Products Under Various pH Reduction Conditions in Downstream Processing.
2016
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.
Conclusion: 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.
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