August 1, 2015 (Vol. 35, No. 14)
With Its Unique Advantages, H2O2 May Best High Heat, Outshine Other Chemical Sterilants
Biomanufacturing facilities and equipment provide nearly ideal environments for contaminating organisms to settle and grow. Extensively reported contamination events at major production facilities underscore the cliché that contamination is not an “if,” but a “when.”
Various decontamination methods are available for the sterilization of cell culture equipment, related equipment, and facilities. One of these methods, hydrogen peroxide vapor (HPV) sterilization, is garnering a reputation for safety, economy, and accessibility.
HPV has a well-known proprietary incarnation—VHP, for vaporized hydrogen peroxide. A registered trademark of Steris, VHP denotes a technology that encompasses dehumidification, conditioning, sterilization, and aeration.
VHP gas is generated in a Steris-manufacture unit that vaporizes a 35% liquid hydrogen peroxide product branded as Vaprox. In addition, VHP is maintained at a constant concentration while it is catalytically transforming to oxygen and water in the return air. According to Steris, the process is “dry” because it prevents condensation of the peroxide/water vapors.
HPV works with any equipment, enclosure, or room that may be enclosed or sealed, depending on material compatibility, efficacy requirements, and exposure limits. Rooms appropriate for HPV decontamination fall into three basic categories: filling areas for aseptic products, material pass-through rooms including those holding materials used in bioprocessing, and rooms specifically designated for decontaminating equipment.
Many devices used in bioprocessing and biotech R&D are equipped with sanitization utilities. Stainless steel tanks have SIP/CIP capabilities, and all incubators today come with some type of self-cleaning capability.
For incubators, high heat disinfection is completely labor-free and therefore close to error-proof. The downside is long cycle time. It can last up to 14 hours. HPV sterilization, by contrast, takes three hours. Gas introduced into the chamber rapidly penetrates nooks and crannies to provide high-log clearance of bacteria and spores.
Panasonic has published an extensive comparison of HPV versus heat for sterilizing CO2 incubators. To summarize:
- The sterilization cycle for HPV lasts just 3 hours, whereas that for heat lasts 8–14 hours.
- No special materials or construction issues arise for peroxide, whereas incubators designed for heat sterilization components must withstand high-temperature conditions.
- Incubator components, including the CO2 sensor and HEPA components, can remain inside during sterilization with HPV—not so for heat.
ENV Services not only certifies controlled-environment equipment (such as biological safety cabinets, isolators, laminar flow hoods, HEPA filters, and cleanrooms), it also provides decontamination services for that equipment. Although the company still performs formaldehyde-based disinfection, many customers now request HPV sterilization. “Formaldehyde exposure has been linked with cancer,” says Trevor Lewis, a technical support specialist at ENV Services.
Exposure limits for HPV are 10 times higher than for gaseous forms of formaldehyde, ethylene oxide, chlorine dioxide, and other decontaminating agents. A concentration as high as 1 ppm of HPV is still acceptable, compared to just 0.1 ppm for the others.
Lewis admits that not all materials are compatible with HPV sterilization: “Peroxide is a strong, corrosive oxidizer. Nylon and other plastics that come into contact with the gas will become brittle over time. Some surface coatings, paints, and anodizations don’t hold up well, either.”
Top disinfection services companies will perform materials compatibility testing before exposing rooms or equipment to HPV. ENV Services, which primarily works with incubators, refrigerators, and biosafety cabinets, maintains a list of specific products that may present issues.
“The insides of these devices are usually made of stainless steel, which easily stands up to hydrogen peroxide gas,” Lewis notes. “It’s the external structures and surfaces that raise issues, particularly when we’re decontaminating an entire room.”
Companies that provide decontamination services are well versed in disinfection protocols that produce 6-log pathogen removal. If they adhere to established criteria for the number of grams of peroxide per cubic meter of room space, things go well. But problems arise, Lewis warns, when customers suggest pushing those limits or trying something new.
Not everyone is ready to jump on the HPV bandwagon. At Genzyme, director Mark Yang, Ph.D., and colleagues recently completed a study that cautions against incomplete removal of peroxide from isolator systems. The Genzyme team demonstrated that in situations where lyophilized protein is exposed to the isolator environment for significant periods, residual peroxide caused protein oxidation in a dose-dependent manner. Oxidation, in turn, caused a slight increase in protein aggregation.
The effects were noticeable even at peroxide concentrations of 0.04 ppm, which is well below what is considered a safe concentration. The take-home lesson here is to have the isolator undergo more-extensive purging before introducing lyophilized products in open vials.
Similarly, literature from Thermo Fisher Scientific warns that residual peroxide could negatively affect cell culture processes when HPV is used to sterilize materials entering clean areas, particularly through pass-through isolators: “Peroxide residues possibly left behind … could affect sensitive systems that the materials are used in. For example, peroxide residue remaining on materials used in cell culture would adversely affect the growth of cells grown on those materials.”
And according to ChlorDiSys, a company specializing in decontamination and sterilization equipment and services, effective decontamination can be ensured only if a sterilant reaches all surfaces, penetrates into all areas, and remains in place long enough to kill all contaminants. Since HPV is a vapor and not a gas, and since the vapor is considered “lazy” with respect to penetration, ChlorDiSys recommends chlorine dioxide—a true gas—over HPV. However, various health organizations have noted the low permissible exposure levels of chlorine dioxide.
There is a misconception that the final step in HPV decontamination involves the application of ultraviolet light to convert peroxide to water and oxygen. Commercial HPV generators do not require this step.
Units from Bioquell and Steris, for example, employ drying systems to eliminate traces of water. Both companies sell a range of standalone HPV generators for disinfecting equipment, enclosures, or entire rooms. Steris also manufactures an HPV sterilization chamber, the VHP LTS-V, for terminal, low-temperature sterilization of packaged single-dose biopharmaceuticals, as well as medical devices and small equipment that is unsuitable for harsh chemical or high-heat sterilization.