Recent studies were performed using different additives to mediate the residual effects of VHP on microbial growth
By Damien Tuleu
Microbiological monitoring of air is a critical component of any pharmaceutical or biopharmaceutical manufacturer's environmental program. It provides information on the processing environment's quality during manufacturing and enables the study of microbiological air quality trends—and, of course, is a Good Manufacturing Practice (GMP) requirement for both the United States and Europe.
Choosing and validating an air sampler is a critical factor in air monitoring, while selecting a sampler typically entails comparison to an “industry standard.” This can be challenging due to particle capture and cutoff size that vary among air samplers.
 Choosing and validating an air sampler is a critical factor in air monitoring, and can be challenging due to samplers’ varying particle capture and cutoff size. |
Additionally, the devices must be oriented to minimize positional bias and air turbulence due to sampler exhaust. One method of microbial air sampling is agar impaction, which collects and concentrates microbial organisms by impaction on pre-filled agar cassettes, where microbes are then grown and counted.
Typically, hydrogen peroxide has been used as a sanitant in isolators and other barrier systems. But once the hydrogen peroxide is introduced into a closed environment, such as an isolator, removing it completely presents difficulties. Due to production cost and time constraints, aeration purge cycle times are often set to reach 1 part per million (ppm)—and, in some cases, even higher concentrations-of residual vaporous hydrogen peroxide (VHP).
Unfortunately, even 1 ppm of residual hydrogen peroxide can inhibit the growth of microorganisms when impacted and concentrated on agar media, making accurate air monitoring difficult and causing false-negative test results.
We recently conducted studies to improve isolator air monitoring post-VHP sterilization. Experiments were performed using different additives to mediate the residual effects of VHP on microbial growth.
The studies utilized the Millipore M Air T Isolator air sampling system, consisting of a programmable pump (which remained outside the controlled area) and an independent sampling head that used pre-poured, easy-to-handle M Air T agar cassettes. Using this system, we conducted a study to investigate the effect of Trypticase Soy Agar (TSA) medium supplemented with pyruvate on the recovery of selected USP ATCC strains after sampling air in an isolator containing from 0.3 ppm sprayed hydrogen peroxide (SHP) up to 300 ppm VHP.
Impact on bacterial recovery
To demonstrate the effect that low hydrogen peroxide concentrations may have on bacterial recovery, air sampling was performed in the presence of 0.3 ppm sprayed hydrogen peroxide. The results showed that the recovery dramatically decreased to a zero value, showing that this level of peroxide is sufficient to inhibit subsequent growth of microorganisms when applied on the media (see Figure 1).
 Air sampling results showing microbial recovery inhibited by sprayed hydrogen peroxide (SHP). |
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Knowing that many isolators deliver hydrogen peroxide in a vaporized form, we investigated the difference between the effect of SHP and VHP on bacterial recovery. The inhibitory effect of VHP on bacterial recovery was significantly reduced.
The observed difference between SHP and VHP could be explained partially. Actually, due to differences in their physical properties, desorption of SHP is likely to be slower than VHP. The effective residual peroxide remaining on the agar when bacteria are spread on the media may be reduced with VHP, explaining the higher recovery rates obtained with VHP.
Catalase is known to degrade peroxide through an enzymatic reaction, leading to H2O and O2 as end products. Increasing quantities of enzyme were incorporated in the media, giving rise to increasing recovery. Nevertheless, the highest quantity of enzyme tested proved that catalase is not as good as pyruvate.
Colony-forming units were difficult to detect due to the presence of bubbles in the agar media, rendering visualization of colonies very tricky. These bubbles originated from the production of gaseous oxygen during the enzymatic reaction.
The optimal pyruvate concentration of 1 percent, was able to tolerate as much as 15 ppm of vaporous hydrogen peroxide. Our team investigated the effect of this concentration on all selected test strains. Sampling was performed in an isolator delivering VHP. Automatic VHP injections were done to stay at 5 to 15 ppm VHP during the entire sampling.
As this media is finally sterilized by gamma-irradiation after aseptic filling and packaging, we did a comparison study between TSA supplemented with 1 percent pyruvate gamma-sterilized and non-gamma-sterilized to ensure that irradiation would not negatively affect the pyruvate neutralizing activity.
Both gamma-sterilized and non-gamma-sterilized media were equivalent, allowing us to use gamma-irradiation as final sterilization without any impact on pyruvate activity.
Before investigating the activity of 1 percent pyruvate and higher concentrations during air monitoring, we tested increasing concentrations of pyruvate for their non-toxicity on bacteria. Various results occurred with different microorganisms. But the bacterial recovery dramatically decreased at 20 percent pyruvate—all strains were completely inhibited at this concentration.
A media supplemented with either 1 percent or 5 percent pyruvate was able to trigger bacterial growth after air sampling containing 100 ppm VHP-98 percent and 105 percent recovery, respectively. A concentration of 1 percent pyruvate was not able to neutralize 300 ppm VHP (no recovery). But a concentration of 5 percent pyruvate was still efficient with 300 ppm VHP, as we obtained a recovery of 69 percent (see Figure 2).
 In tests, adding one percent pyruvate reduced the effect of hydrogen peroxide. |
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Critical process
Microbiological air monitoring in isolators is critical and must be done after sufficient aeration to remove as much of the VHP used during the sterilization process as possible. Due to production cost and time constraints, however, the time of aeration is often set to reach 1 ppm of residual VHP, and in some cases, even higher concentrations. These higher concentrations have been shown in this paper to have a negative effect on bacterial recovery.
In our recent studies, a 1 percent pyruvate supplement to TSA agar proved to be optimal for efficient recovery on a range of organisms. Data shows that this formulation will tolerate as much as 100 ppm residual VHP, ensuring microbial growth and minimizing false-negative results when air monitoring.
DAMIEN TULEU is QA/QC group product manager, Millipore Corp. He can be reached at: [email protected]