Cleaning methods for biosafety cabinet to eliminate residual mycoplasmas, viruses, and endotoxins after changeover

Introduction

Cell-processing operations can potentially contaminate biosafety cabinets, which should be maintained sterile. However, unintended contamination can occur owing to the presence of viruses, mycoplasmas, and bacteria in the raw materials. Moreover, although several methods for expunging these contaminants have been proposed, an optimal method has not yet been determined. Additionally, the effectiveness of conventional methods for eliminating these contaminants remains unclear owing to their unique characteristics and potential resistances to cleaning. Therefore, this paper proposes a risk-based approach to identify appropriate cleaning methods and reduce the likelihood of cross-contamination in biosafety cabinets by these contaminants.

Methods

Various cleaning methods for eliminating mycoplasmas, viruses, and endotoxins from biosafety cabinets were evaluated, including ultraviolet (UV) irradiation at 200 mJ/cm² for 20 min and wiping with disinfectants such as distilled water, benzalkonium chloride (BKC), and 70 % ethanol (ETH). The effectiveness of each method was evaluated by applying the contaminants on stainless steel plates and cleaning them using each method. Mycoplasma orale was cultured for 2 weeks in a liquid medium after cleaning. Feline calicivirus (FCV) was used for evaluating the virus-cleaning effectiveness and its presence was tested using the TCID50 test, whereas endotoxins obtained from the dried extract of Escherichia coli were measured via endotoxin testing.

Results

UV irradiation and wiping with BKC inhibited the growth of mycoplasma and significant decreased their presence compared with the other cleaning methods. Notably, mycoplasma were detected after wiping all SUS304 plates with ETH, which is a widely used cleaning method. Additionally, the cleaning efficacy for virus showed that the TCID₅₀ of the wet group was 132,000 TCID₅₀/plate, whereas those after UV irradiation or cleaning with BKC or DW were below the detection limit. Finally, UV irradiation did not significantly reduce the endotoxin production compared with that in the dry group. Additionally, wiping with ETH did not significantly reduce endotoxins compared with the dry group and their residues were higher than those detected after wiping with BKC or DW.

Conclusions

The changeover protocols currently employed in most cell-processing facilities may be ineffective as pathogenic or nonpathogenic materials may remain even after ETH wiping, leading to unintended cross-contamination. To the best of our knowledge, this is the first study to provide reference data of different cleaning methods for mycoplasmas, viruses, and endotoxins in cell-product manufacturing facilities, and can potentially support the development of evidence-based management strategies for ensuring safe cell-product processing.