Effect of disinfectants and manual wiping for processing the cell product changeover in a biosafety cabinet

Abstract

Introduction: The process of cell product changeover poses a high risk of cross-contamination. Hence, it is essential to minimize cross-contamination while processing cell products. Following its use, the surface of a biosafety cabinet is commonly disinfected by ethanol spray and manual wiping methods. However, the effectiveness of this protocol and the optimal disinfectant have not yet been evaluated. Here, we assessed the effect of various disinfectants and manual wiping methods on bacterial removal during cell processing.

Methods: The hard surface carrier test was performed to evaluate the disinfectant efficacy of benzalkonium chloride with a corrosion inhibitor (BKC + I), ethanol (ETH), peracetic acid (PAA), and wiping against Bacillus subtilis endospores. Distilled water (DW) was used as the control. A pressure sensor was employed to investigate the differences in loading under dry and wet conditions. The pre-spray for wiping was monitored by eight operators using a paper that turns black when wet. Chemical properties, including residual floating proteins, and mechanical properties, such as viscosity and coefficient of friction, were examined.

Results: In total, 2.02 ± 0.21-Log and 3.00 ± 0.46-Log reductions from 6-Log CFU of B. subtilis endospores were observed for BKC + I and PAA, respectively, following treatment for 5 min. Meanwhile, wiping resulted in a 0.70 ± 0.12-Log reduction under dry conditions. Under wet conditions, DW and BKC + I showed 3.20 ± 0.17-Log and 3.92 ± 0.46-Log reductions, whereas ETH caused a 1.59 ± 0.26-Log reduction. Analysis of the pressure sensor suggested that the force was not transmitted under dry conditions. Evaluation of the amount of spray by eight operators showed differences and bias in the spraying area. While ETH had the lowest ratio in the protein floating and collection assays, it exhibited the highest viscosity. BKC + I had the highest friction coefficient under 4.0-6.3 mm/s; however, that of BKC + I decreased and became similar to the friction coefficient of ETH under 39.8-63.1 mm/s.

Conclusions: DW and BKC + I are effective for inducing a 3-Log reduction in bacterial abundance. Moreover, the combination of optimal wet conditions and disinfectants is essential for effective wiping in specific environments containing high-protein human sera and tissues. Given that some raw materials processed in cell products contain high protein levels, our findings suggest that a complete changeover of biosafety cabinets is necessary in terms of both cleaning and disinfection.

Keywords: BKC + I, benzalkonium chloride with corrosion inhibitor; BSC, biosafety cabinet; Biosafety cabinet; CFU, colony forming unit; Cell-product processing; Changeover; Cross-contamination; DW, distilled water; ETH, ethanol; FBS, fetal bovine serum; PAA, peracetic acid; SUS, stainless steel; Wiping.

Fig. 1

Hard surface carrier test with wiping. (A) Experimental design of hard surface tests to evaluate the effects of disinfectants and wiping. CFU: colony forming unit. DW: distilled water. BKC + I: benzalkonium chloride with corrosion inhibitor. ETH: ethanol. PAA: peracetic acid. SUS: stainless steel. (B) Reduction effect of disinfectants. Log reduction of CFU from 1.0 × 10⁶ CFU B. subtilis in the collection solution after disinfectant treatment of SUS plates. ∗P < 0.05, and ∗∗P < 0.01. P values were calculated using Kruskal–Wallis test followed by Dunn's multiple comparison test. (C) Reduction effect of wiping. Log reduction of CFU from 1.0 × 10⁶ CFU B. subtilis in the collection solution immediately after wiping as the disinfectant treatment of SUS plates.

Fig. 2

Wiping pressure between dry and wet. (A) Experimental design of wiping between dry and wet using a pressure sensor. The pressure was evaluated independently six times. (B) The representative peak contact load distribution of each sensor cell recorded by an indenter moving in one direction. (C) Wiping contact load per sensor. P value was calculated using Mann–Whitney test. (D) The representative pressure of a section between dry and wet. (E) Contact area for the wiping to pressure sensor. (F) The representative quantified pressure of a section. (G) Average of maximum contact pressure per cell of the sensor. Data are presented as mean ± SD. ∗P < 0.05, and ∗∗P < 0.01. Data are presented as mean ± SD. P values were calculated using Mann–Whitney test.

Fig. 3

Survey on pre-spraying for wiping. (A) Experimental design of pre-spraying simulation for wiping. BSC: biosafety cabinet. (B) Results of a survey of eight operators. Scale bar: 10 cm. (C) Amount of spray used in the BSC by each operator. (D) Spray coverage in the BSC was evaluated for each operator. (E) Correlation analysis between usage volume and coverage area. R and P values were calculated using Spearman's correlation analysis. (F) Left-right difference of spray coverage area for each operator. The difference between the coverage areas were presented as an absolute value. Abs.: absolute.

Fig. 4

Wiping force, chemical, and mechanical properties of disinfectant. (A) Experimental design of wiping between force difference. FBS: fetal bovine serum. (B) Log reduction effect of wiping between force difference. (C) Experimental design of residual protein floating and collection assay. (D) Protein collection ratios were used to determine the chemical properties of the disinfectants. (E) Viscosity. (F) Friction coefficient by sliding velocity condition under 4.0–6.3 mm/s. (G) Friction coefficient by sliding velocity condition under 39.8–63.1 mm/s. (E) Viscosity and (F) Friction coefficient were used to determine the mechanical properties of the disinfectants. Data are presented as mean ± SD. ∗P < 0.05, and ∗∗P < 0.01. P values were calculated by Kruskal–Wallis test followed with Dunn's multiple comparison test.