The antioxidant system beta-D(+) glucose-glucose oxidase-catalase: tests for pyrogenicity and antigenicity.
OBJECTIVE
beta-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 beta-D(+) glucose-glucose oxidase-catalase as an antioxidant system in pharmaceutical solutions, particularly in terminally autoclaved aqueous formulations for parenteral use.
Uppoor
R
R
Department of Ophthalmology, Medical University of South Carolina, 167 Ashley Avenue, Charleston, SC 29403, USA.
Niebergall
P J
PJ
James
E R
ER
D000818:Animals; D000917:Antibody Formation; D000941:Antigens; D000975:Antioxidants; D015153:Blotting, Western; D002374:Catalase / Q000276:immunology / Q000494:pharmacology*; D005260:Female; D005334:Fever / Q000139:chemically induced*; D005949:Glucose Oxidase / Q000276:immunology / Q000494:pharmacology*; D006737:Horseshoe Crabs; D011817:Rabbits
D000941:Antigens; D000975:Antioxidants; D005949:Glucose Oxidase; D002374:Catalase