Group seeks to create a high-level, pure-aqueous ozone disinfectant system that leaves no chemical residue
By Durand M. Smith, Xuan Xiao Wang and Vicki Ellen Wile
Present methods for high-level disinfectants in the pharmaceutical industry leave a chemical residue that can alter the effectiveness of the drugs being produced.
For example, one disinfectant that uses a combination of hydrogen peroxide and peracetic acid is known to leave residual amounts of hydrogen peroxide for hours after application. The presence of hydrogen peroxide interferes with the efficacy of certain drugs produced on a line disinfected by this method.
Ozone is a well-known, broad-spectrum biocide that is unstable, will breakdown to oxygen and reacts with other molecules through an oxidation process in a relatively short time. It is generated by ultraviolet (UV) or corona discharge breakdown of molecular oxygen or by electrolysis in water.
For general disinfecting applications where users can be exposed to ozone, the safest method of application is done by dissolving the ozone in water and applying the aqueous ozone directly to the surfaces to be disinfected. While it is common to produce ozone gas and then contact the ozone gas with water, it is much safer and more efficient to use the electrolysis method because the electrolysis method creates ozone directly in water through electrochemical oxidation of water.1
Electrolytic ozone generators have been developed by several suppliers, however, the generator selected for this project is unique in the materials of construction. It is common for the electrolytic cell to use lead dioxide because it provides very high dissolved ozone concentrations.
We have chosen to use a Kobelco D-Ozone system because no lead dioxide is used that may contaminate the aqueous ozone. A high-purity, noble metal catalyst is used in the Kobelco electrolytic cell that assures no harmful metals are present in the ozonated water.
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For some drug production applications, the water purity is also extremely important. For this application, pharmaceutical water for injection is used. This assures there are no harmful residuals from the water itself. However, because the electrolysis process depends on the conductivity of the water, high-purity water is expected to yield much lower concentrations of dissolved ozone—this was not found in our development work.
The objectives of this study were to develop a high-level disinfectant that can be applied safely in a restricted area barrier filling line and inactivate 1,000 spores/ml in 10 minutes exposure time according to a defined protocol. Hydrogen peroxide levels had to be less than 0.1 parts per million (ppm) at the time of visible dryness. Ozone levels had to be less than 0.2 ppm for two hours during and immediately after application and less than 0.1 for an 8-hour total weighted average. The disinfectant must be applied in a similar manner to the current practice of applying a chemical disinfectant.
Pure disinfection system described
The pure disinfection system consists of three major components, various plumbing components, dissolved ozone and ambient-air ozone sensors as shown in Figure 1.2
The major components are a stainless-steel tank or reservoir for the system, a Kobelco Do-030 electrolytic ozone generator and an integration cabinet. The integration cabinet houses the water pump, pressure gauge, flow meter and a redundant dissolved ozone sensor.
A separate ambient ozone sensor is provided to monitor the air while the system is operating and in the area where the aqueous ozone is applied. The reservoir must be at least ten liters and could be the disinfection spray tank with the appropriate modifications. This would eliminate the need to transfer the aqueous ozone at the completion of the ozonation process.
The Do-030 creates aqueous ozone directly in the water through an electrolytic process. Water is pumped to the Do-030 and thereby through its major component, an electrolytic cell. The cell voltage (nominally 7.5 VDC) and current (up to 64 Amps) are controlled by the programmable logic controller, while monitoring its own dissolved ozone sensor in a feedback control circuit. The dissolved ozone sensor is based on ultraviolet absorption of the ozone dissolved in the water.
A macromolecular membrane with a platinum coating divides the electrolytic cell. Ozonated water passes on the anode side. Water that serves to cool the cell and carry away positively charged ions passes on the cathode side. As the ozonated water passes through the cell, molecules of water and oxygen are ionized and ozone is created.
Positively charged ions migrate through a membrane to the cooling water stream. Because of the purity of the water—water for injection (WFI)—used in the system, the positively charged ions consist primarily of hydrogen. The cooling water is separated from the aqueous ozone and collected in a separate tank of at least three liters volume.
The water pump, plumbing components and the redundant dissolved ozone sensor are contained in the integration cabinet. The pump draws water from the reservoir tank and delivers it to the generator. A three-way valve also permits the ozonated water to be diverted to fill a spray tank, while a pressure-relief valve between the pump and generator assures that the pump will not go over pressure and damage the pump.
With the redundant dissolved ozone sensor (Model 3660.300; Orbisphere), approximately 300-milliliters/minute out of the 1.3-liter/minute total flow is sampled for dissolved ozone. A flow control valve and calibrated flow gauge is provided in the integration cabinet. An aqueous ozone sample flows through a sensor cell and returns to the system in the line to the Do-030. This sensor is redundant with the dissolved ozone sensor in the Do-030 and provides a second phenomenology method for measuring the dissolved ozone. The redundant sensor is based on current flow through the sensor due to ozone reduction at the cathode.
 The disinfection system is manually shut down when the set ozone level is achieved. |
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In operation, the Do-030 is set to the desired dissolved ozone level and the system is run until a steady level of ozone is achieved. The Do-030 ozone sensor and the optional dissolved ozone sensor in the integration cabinet will read the same value at steady state. Once the set value is achieved, the system is manually shut down. A typical ozone production curve is shown in Figure 2.
An ambient air ozone gas monitor (Model 450; Advanced Pollution Instrumentation Inc.) ensures that the ozone gas levels released by the system do not exceed Occupational Safety and Health Administration (OSHA) permissible exposure levels. The monitor is separate from the three major pure disinfection system components so that it can also be used in the area where the aqueous ozone is to be applied.
Constructing the spray booth
To ensure ambient ozone level is always below OSHA limit of 0.1 ppm in a typical pharmaceutical cleanroom environment, a spray booth was constructed with a fan and a box filter to smooth out the airflow in the test volume.
The fan blows air downward from the top of the booth, passing through the air filter, into the test volume. Airflow was adjusted to simulate standard pharmaceutical cleanroom airflow of about 124 feet per minute (fpm) at the center of the test volume. Airflow in the test volume was not uniform because of the small flow area and distances from the fan to the filter. Measurement of nine positions across the filter resulted in a mean flow of 108.4 fpm with a standard deviation of 28.7 fpm.
Spray application characterization
A series of tests were conducted for various conditions of dissolved ozone and spray nozzles in the spray booth. The results are:
- Ambient ozone level in the spray booth did not exceed the OSHA safety limit of 0.1 ppm, with as high as 15 ppm of dissolved ozone in the spray tank and with the spray nozzles in this study. In fact, the ozone levels were <3 parts per billion (ppb), the lowest detectable level of the ozone sensor used.
- In long-term evaporation tests of the ozonated water, there were no detectable levels of hydrogen peroxide.
- A nozzle was found that closely matched the spray angle and flow rate of an air-atomizing nozzle currently used by pharmaceutical companies. Tests were run to characterize ozone and hydrogen peroxide off gassing with that nozzle.
- Dissolved ozone concentrations in the selected nozzle showed that 35 percent remained in the spray water. In practice with the pure disinfection system, we expect to be able to deliver >1.5 ppm dissolved ozone to the surface to be disinfected.
- Ozone decay rate in the spray tank is shown in Figure 3. WFI water is used for the study.
The nozzle selected was based on spray angle, followed by the ozone in the spray, and then the flow rate. Table 1. records data from six of the 15 nozzles tested. Observe that the percent of dissolved ozone (%dO3) is inversely proportional to pressure. The nozzle selected is highlighted.
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Microbial testing
The microbiological studies for the efficacy of aqueous ozone as sporicidal agent were conducted.
The efficacy challenge to kill 1,000 spores/ml within 10 minutes of exposure to ozonated water was accomplished with a dissolved ozone concentration of approximately 1.00 ppm.
Earlier work on B. subtilis spore reported a CT of 10 mg-min/l for one log reduction and for B. cereus and B. megaterium spores a threshold of 2.29 mg/l ozone concentration.3,4,5 Our work results indicate that at a CT of 10mg-min/l an average spore reduction for Bacillus subtilis var. niger—American Type Culture Collection (ATCC) 9372—was 1.4 log (12,187/ml) and for Bacillus stearothermophilus (ATCC 7953) was 0.14 log (3,300/ml).
Test results indicate B. subtilis var. niger is approximately four times more susceptible to a dissolved ozone concentration of ~1.00 ppm than B. stearothermophilus. Dissolved ozone has proven to be an efficient sporicidal agent. The beneficial effects are expected to be even greater at higher concentrations and longer exposure times. Table 2. and Table 3. summarize the study results.
The concentration of dissolved ozone of 1.00 ppm was chosen for the study based on the worst-case scenario for the potential loss of ozone in the spray, the half-life, and the maximum time delay of ozone generation to application.
Durand M. Smith Ph.D., MBA, is president of Cyclopss Corp. (Albuquerque, N.M.) and has more than 25 years experience in research and development in commercial, governmental and scientific projects. Smith can be reached at [email protected].
(Jim) X. Wang Ph.D., MSEE, senior scientist for Cyclopss Corp. is responsible for ozone research and the design and manufacture of control systems for ozone products. He has more than 15 years experience with instrumentation and control systems used in commercial and scientific projects.
Vicki Ellen Wile, BS, microbiologist for Cyclopss Corp., specializes in research with common foodborne and bloodborne pathogens and medical biological indicators. She has 20 years experience in microbiology and chemistry in the education, medical, environmental and food industry.
References
1. Rice, R. G. and A. Netzer, Handbook of Ozone Technology and Applications, Ann Arbor, Mich., Ann Arbor Science Publishers, 1982, pp. 85-102.
2. Cyclopss Corporation patent pending
3. Owens, James H. et. al., “Pilot-scale ozone inactivation of cryptosporidium and other microorganisms in natural water”, Ozone Sci. & Engrg, 22:501-517 (2000).
4. Kim, J.G., and A.E. Yousef, “Inactivation of Bacillus subtilis spores by ozone in combination with heat or pulsed electric field,” Inst. Food Tech. Annual Meeting, June 10-14, 2000, 78F-3.
5. Broadwater, W.T., R.C. Hoehn, and P.H. King, “Sensitivity of three selected bacterial species to ozone,” Appl. Micro., 26:391-393 (1973).