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Abstract
Purpose: β-D(+) glucose–glucose oxidase (E.C.# 1.1.3.4)–catalase (E.C.# 1.11.1.6) (GO–CAT) is being investigated as a new antioxidant system for use in pharmaceutical solutions. This study reports the results of tests for pyrogenicity and antigenicity of GO–CAT derived from Aspergillus niger when used parenterally in autoclaved preparations. Methods: The Limulus amebocyte lysate (LAL) method was used to test the pyrogenicity of native GO–CAT. Pyrogenicity/antigenicity was evaluated in vivo by injecting autoclaved GO–CAT into New Zealand white rabbits. Antigenicity was also evaluated by Ouchterlony and Western blotting. Results: None of the native GO–CAT concentrations tested (up to 30.83 u/ml) produced a positive gel clot in the LAL test, thereby suggesting its non-pyrogenicity. The rabbits, which received seven injections of autoclaved GO–CAT over a period of eleven weeks, remained healthy during and after the GO–CAT injections. All Ouchterlony and Western blot assays using sera from rabbits injected with autoclaved GO–CAT were negative. Furthermore, autoclaved GO–CAT could not be detected in Ouchterlony assays using a mouse monoclonal antibody (GO40 mAb) to native A. niger glucose oxidase. Control samples containing native GO–CAT produced an antigen–antibody complex reaction in Ouchterlony assays against the GO40 mAb. Antigen-antibody complexes could be detected by non-denaturing PAGE in samples containing native GO–CAT/GO40 and boiled GO–CAT/GO40, but not in samples containing autoclaved GO–CAT/GO40. These results indicate autoclaved GO–CAT is neither pyrogenic nor antigenic. Conclusions: Based on these results, there is potential for the use of β-D(+) glucose–glucose oxidase–catalase as an antioxidant system in pharmaceutical solutions, particularly in terminally autoclaved aqueous formulations for parenteral use.