Evaluation of preservative efficacy in pharmaceutical products: the use of psychrotolerant, low-nutrient preferring microbes in challenge tests

Abstract

Summary What is known and Objective: Preservative efficacy in medicines is typically investigated using challenge tests. In such tests, the product is artificially contaminated with a high concentration of standard bacterial and fungal test strains such as Pseudomonas aeruginosa and Candida albicans. The rate and extent of reductions in inoculum viability over a specified period forms the basis for acceptance/rejection of preservative efficacy. None of the strains named for inclusion in the challenge test outlined in the European Pharmacopoeia are associated with the contamination of high‐quality water used in pharmaceutical production. Alpha‐ and Betaproteobacteria are easily the most common microbes in waters intended for pharmaceutical production. In addition, none of the standard test strain panel prefer low‐nutrient, dilute conditions or grow at or around refrigeration temperatures. This is important because the water activity and nutrient content of medicines can vary greatly and medicines are often stored cold. We investigate the relevance of these factors when testing preservative efficacy by including other strains in challenge tests. Methods: Psychrotolerant, low‐nutrient preferring strains (Beta‐ and Alphaproteobacteria and a yeast) were isolated from pristine waters. These were compared in challenge tests with C. albicans and P. aeruginosa using different storage temperatures. Pharmaceutical products differing widely in water‐content, pH and preservative systems were included in the study. Results and Discussion: Regardless of the type of medicine tested C. albicans always showed superior survival characteristics to the yeast isolate (Cryptococcus terricola). One of the three screened bacterial strains (a Sphingomonas sp.) survived significantly better than P. aeruginosa in all but one product tested. However, the results for all products taken together cannot easily be explained by reference to this strain’s psychrotolerancy or its preference for dilute, low‐nutrient environments. This study supports previous work indicating that the inclusion of wild‐type test strains, in this instance strains that are suited to survival in high‐quality waters, improves preservative efficacy tests. What is new and Conclusion: Use of a Sphingomonas sp. isolated from a pristine water as a challenge test strain, gave a more stringent indication of preservative efficacy in a wide range of pharmaceuticals than did P. aeruginosa.