The Validation of Vapor Phase Hydrogen Peroxide Microbial Reduction for Planetary Protection and a Proposed Vacuum Process Specification

The Jet Propulsion Laboratory, in conjunction with the NASA Planetary Protection Officer, has selected the vapor phase hydrogen peroxide sterilization process for continued development as a NASA approved sterilization technique for spacecraft subsystems and systems. The goal is to include this technique, with an appropriate specification, in NPR 8020.12C as a low temperature complementary technique to the dry heat sterilization process. To meet microbial reduction requirements for all Mars in-situ life detection and sample return missions, various planetary spacecraft subsystems will have to be exposed to a qualified sterilization process. This process could be the elevated temperature dry heat sterilization process (~115oC for 40 hours) which was used to sterilize the Viking lander spacecraft. However, with utilization of such elements as highly sophisticated electronics and sensors in modern spacecraft, this process presents significant materials challenges and is thus an undesirable bioburden reduction method to design engineers. The objective of this work is to introduce vapor hydrogen peroxide (VHP) as an alternative to dry heat microbial reduction to meet planetary protection requirements. The VHP process is widely used by the medical industry to sterilize surgical instruments and biomedical devices, but high doses of VHP may degrade the performance of flight hardware, or compromise material properties. Our goal for this study was to determine the minimum VHP process conditions to achieve microbial reduction levels acceptable for planetary protection. In order to evaluate the effectiveness of VHP for the inactivation of the standard spore challenge organism, Geobacillus stearothermophilus, the STERIS Corporation, under contract to the Jet Propulsion Laboratory (JPL), conducted several series of experiments. The experiments were conducted to determine VHP process parameters that provided significant reductions in spore viability while allowing survival of sufficient spores for statistically significant enumeration. In addition to the obvious process parameters – hydrogen peroxide concentration, number of pulses, and exposure duration – the investigation also considered the possible effect of environmental parameters. Temperature, relative humidity, and material substrate effects on lethality were also studied. Finally, a comparison of assays performed by STERIS and JPL of sporeinoculated coupons exposed to VHP under the same test conditions was included. Biological indicators were inoculated with more than 1 million Geobacillus stearothermophilus (ATCC 7953) spores on stainless steel coupons and packaged in Tyvek/Mylar pouches. For the tests on the effect of material substrates, the same inoculation procedure was employed on the selected material substrates. All exposures were conducted in a STERIS VHP MD2000 Series Sterilization System. The process involves a conditioning phase, injection of liquid hydrogen peroxide, a sterilization phase (in vacuum), and an aeration phase with high-efficiency particulate air filter (HEPA)-filtered air. The derivation of D-values from the statistically significant (i.e., non-zero) lethality data permitted conservative recommendations for a planetary protection specification. The outcome of this study provided an optimization of test sterilizer process conditions: VHP concentration, process duration, a process temperature range for which the worst case D-value may be imposed, a process humidity range for which the worst case D-value may be imposed, and robustness to selected spacecraft material substrates.