WO2020263986A1 - System and method for sterilization - Google Patents

Abstract

A sterilization process uses a controlled amount of sterilant precursor gases in combination introduced into a sterilizing chamber containing a device to be sterilized. In one embodiment the sterilant precursor gases include NO, NO2 and H2O. The water gas in the chamber leads to the formation of water films on surfaces exposed to the water gas. The nitrogenous precursor gases can then produce a principle sterilizing molecular species (N2O3) in the water films.

Description

SYSTEM AND METHOD FOR STERILIZATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Application No. 62/865,366 filed June 24, 2019, the subject matter of which is incorporated herein by reference in entirety.

FIELD OF INVENTION

[002] This invention relates to sterilization devices and methods using precursor gases that react to form molecular microbicidal agents. Specifically, this invention relates to a device and method that uses nitric oxide, nitrogen dioxide and water gases to form a sterilizing molecule, N2O3, on surfaces and in microorganisms, which render these biological contaminants sterile.

BACKGROUND OF THE INVENTION

[003] Sterilization methods are known that rely on chemicals, pressure and temperature to eliminate biological contaminants such as bacteria, spores and fungi from a variety of substrates including medical devices, medical compounds and others. Alternately, radiation-based treatments may be used, avoiding some types of damage to the object to be sterilized that can result from heat and pressure. In another approach, gaseous sterilant chemicals have been used.

[004] Steam autoclaves have long been used for sterilizing most medical instruments. This method exposes materials to steam at 121° C. at a pressure for 15-30 minutes. The microbicidal process is mediated by heat denaturation of proteins, resulting in interruption of metabolic functions. The method requires cumbersome equipment, a power supply and plumbing, although benchtop models have Tillable water tables. Aside from these logistical problems, autoclaving is not suitable for many plastics and other heat labile materials.

[005] Sterilant gases can kill or control the growth of microorganisms. Some of these sterilant gases include chlorine dioxide, sulfur dioxide, hydrogen peroxide, carbon dioxide, hydrogen sulfide, ozone and ethylene oxide. One problem with many of the sterilant gases is that they are explosive in high concentrations (e.g. ethylene oxide, hydrogen peroxide, chlorine dioxide). Thus, storing, containing and using these gases in high concentrations represent a hazard to the user. For safety reasons, this limits the usable concentration of gas and creates an additional disadvantage. The concentration of the sterilant gas must be decreased due to safety concerns, while the exposure time must be increased to achieve effective sterilization.

[006] Although ethylene oxide is an effective sterilant, Ethylene oxide has been on the federal list of carcinogens since 1985. In December 2016, the U.S. EPA released a long-delayed reassessment that officially added the agency to a list of other national and international organizations declaring the chemical poses significant long-term cancer risks, in particular for breast cancer, leukemia and lymphomas. The risk of ethylene oxide residuals on medical devices and exhausted into the air near sterilization facilities has forced industry and regulators to attempt to reduce the amount of ethylene oxide used. This can be done by replacing ethylene oxide sterilization cycles with a sterilization method that is safer, scalable and can aerate effectively.

[007] Thus, there is a need for methods and devices that generate sterilant gases at the point of use in a safe and efficient manner. There is a further need for processes capable of producing significant concentrations of sterilant gas without the danger of explosion or oxidative fire. To minimize the risk of exposure to toxic, and possibly carcinogenic sterilant residual gases, the amount of sterilant gases used in the sterilization process needs to be minimized. Furthermore, to minimize the degradation of sterilized materials, the amount of the sterilizing chemicals used in the process should be reduced.

[008] Therefore, sterilization methods listed above have disadvantages and there is a need for a improved sterilization processes that reduce the amount of material degradation of the exposed devices, reduces the risks associated with sterilizing chemicals, and that improve the efficiency of the sterilization process. Furthermore, there is a need for controlling the reactions through the controlled delivery of sterilant precursors, where without such control the gases can either promote or inhibit the sterilization process.

[009] In view of the issues raised with ethylene oxide sterilization approaches, the inventor have determined that gas sterilization techniques based on molecular sterilizing precursors, including NO, NO2 and H2O, which, when applied together produce a sterilizing molecule and provide effective sterilization while mitigating damage to the sterilized substrate. In particular, the inventors have shown that using molecular sterilant precursor gases is an effective sterilization agent for a variety of medical. Furthermore, the risk of ethylene oxide release and human exposure is removed. DESCRIPTION OF THE INVENTION

[010] Nitrogen dioxide has been claimed as a sterilant, U.S. Pat. No. 9,180,217 B2 claims a method for sterilizing an object in a gas-tight sterilizing chamber using a sterilant gas consisting essentially of NO2. However, it is now known to the inventors that NO2 gas is not the sterilant, but a gas that contributes to the formation of the molecular sterilizing agent, N2O3. N2O3 reacts with DNA through the nitrosation of nucleosides cytosine and guanine. By applying NO2 alone, additional time and chemicals are needed to produce the sterilizing molecular agent. Given this, the present invention uses methods and devices to deliver the N2O3 sterilization method in a controlled manner.

[Oil] A microbicidal process that can be used for sterilization follows the same chemical reactions the lead to the immune response found in vivo. In the body, NO is produced by neutrophils and macrophages in response to inflammation and infection in vivo. The reactions that lead to the in vivo killing of infectious organism is summarized as follows: NO generated by a cell auto-oxidizes to form NO2; NO and NO2 combine to form N2O3; the N2O3 reacts with DNA through the nitrosation of nucleosides cytosine and guanine; the DNA is degraded and mitosis cannot occur rendering the microorganism non-viable. The reactions leading the formation of N2O3 can occur either in an aqueous environment (e.g., in the extracellular region, or in water films formed on inanimate surfaces), within the cell membrane, or within the microorganism. The sterilization methods and devices described herein are used in a chamber (in vitro) and follows this same chemical reaction cycle found in vivo, leading to the degradation of a microorganism’s DNA and microorganism death.

[012] One aspect of this invention relates to methods and devices that deliver precursor gases to a sterilization chamber to efficiently form a molecular sterilizing agent. In the case that the molecular sterilizing agent is a reactive molecular nitrogen species, the precursor gases that could be used include NO, NO2 and H2O. The molecular sterilizing agent formed from nitrogenous species is N2O3. A further aspect of the invention includes the methods and devices for using multiple chambers in preparing the measured delivery of the precursor gases to the sterilizing chamber.

[013] Aspects of embodiments relate to the methods and devices for using the ratio of precursor gases to facilitate and not hinder the formation of the molecular sterilizing agents. [014] Aspects of embodiments relate to systems and methods for delivering humidified air to a sterilization chamber, systems and methods for removing residual precursor gases an exhaust gas stream of a sterilization chamber, systems and methods for removing and replacing a source of sterilant gas, an exhaust gas scrubber and/or supplies for a humidification system.

[015] Aspects of embodiments relate to hardware and software for user interface in a sterilizing device and hardware and software for control of a sterilization cycle.

[016] Aspects of embodiments relate to chamber, chassis and door configurations of a sterilizer, including ornamental aspects thereof.

[017] Another aspect of the invention relates to a method for delivering gaseous precursor gases to a sterilization chamber containing one or more objects to be sterilized.

[018] Another aspect of the invention relates to a system configured to control the foregoing device or method including controlling one or more of precursor gas concentrations, temperature, gas circulation, total or partial gas pressure and/or duration of exposure of the object or objects to be sterilized.

[019] Aspects of embodiments of the invention relate to machine executable code embodied on a machine-readable medium which, when executed, performs methods as described herein.

[020] These and other objects, features, and characteristics of the present invention, as well as the methods of operation and functions of the related elements of structure and the combination of parts and economies of manufacture, will become more apparent upon consideration of the following description and the appended claims with reference to the accompanying drawings, all of which form a part of this specification, wherein like reference numerals designate corresponding parts in the various figures. It is to be expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the invention. As used in the specification and in the claims, the singular form of“a”,“an”, and“the” include plural referents unless the context clearly dictates otherwise.

DEFINITIONS

[021] As used herein, the term“molecular sterilizing agent” refers to the microbicidal chemical species.

[022] As used herein, the term“gas” or“gases” means any matter that is not in the solid state or liquid state, but rather, has relatively low density and viscosity, expands and contracts greatly with changes in pressure and temperature, diffuses readily and has the tendency to become distributed uniformly throughout any container.

[023] As used herein, the term“nitric oxide” or“nitrogen oxide” means the NO free radical or NO·. Similarly, as used herein, the term“nitrogen dioxide” means NO2, which is the free radical NO2·. As used herein,“dinitrogen trioxide” means N2O3 and“dinitrogen tetroxide” means N2O4.

[024] As used herein, the term“sterilant precursor” means a compound that is a chemical that contributes to producing a molecular sterilizing agent, including NO, NO2, H2O and N2O4.

[025] As used herein, the term“sterilization chamber” means any gas tight chamber of any size, whether comprised of hard or soft materials, where items to be sterilized or decontaminated can be contained. In an embodiment, the sterilization chamber is capable of; (i) receiving sterilant precursor gases; and (ii) receiving air. For vacuum processes, the sterilization chamber can maintaining a vacuum

[026] Sterilization is a high-level of decontamination that destroys all microbial life, including highly resistant bacterial endospores. Disinfection in an intermediate-level of decontamination, which eliminates virtually all pathogenic microorganisms, with the exception of bacterial spores. As used herein, the terms“sterilize”“sterilizing” and“sterilization” mean the killing or removal of all microorganisms in a material or on an object. When a material or object is“sterilized” or “sterile” there are no living organisms in or on a material or object. Since sterilization eliminates all microorganisms, including endospores, a method, system and/or device that sterilizes a material or object, therefore, also disinfects and decontaminates the material or object. As used herein, the term“object” refers not to a feature of the invention, but rather to the article or material being acted upon to be sterilized and/or decontaminated by the disclosed sterilizing methods, systems and devices. The term“object” can also include a material to be sterilized, no matter the physical form. An object may include, for example, without limitation, a medical device or medical instrument or any other article or combination of articles for which sterilization is desired. An object may have a wide variety of shapes and sizes and may be made from a variety of materials (e.g., without limitation, metal, plastic, glass).

[027] As used herein, the term“pre- chamber” means any container, of any size or composition, which may be used to contain a gas and/or a gas-generating compound. In an embodiment, the pre-chamber is made of a material that is impermeable to liquid, impermeable to gas, and can maintain a vacuum. [028] As used herein, the term “microbe” means any bacteria, virus, fungi, parasite, mycobacterium a or the like.

[029] As used herein, the term“extracellular” is used to describe the region external to a microorganism cellular membrane or spore coat.

[030] As used herein, the term“intracellular” is used to describe the region internal to a microorganism cellular membrane or spore coat.

[031] As used herein, the term“scrubbing” means the removal or conversion of toxic oxides of nitrogen from the exhaust stream of the sterilization device.

[032] As used herein, the term“medical device” means any instrument, apparatus, implement, machine, appliance, contrivance, implant, or other similar or related article, including any component, part, which is intended for use in the cure, mitigation, treatment, or prevention of disease, of a human or animal, or intended to affect the structure or any function of the body of a human or animal; and, which is intended to be inserted, in whole or in part, into intact tissues of a human or animal. As used herein, the term“implant” or“implantable” means any material or object inserted or grafted into intact tissues of a mammal.

[033] As used herein, the term flow measurement means the measurement of a gas stream using a mass flow meter, a rotometer, or other means of quantifying the flow of gas.

[034] As used herein, a humidity sensor means a means of quantifying humidity, which is a measure of the density of water gas, using sensors. Such sensors for quantifying water gas density include capacitive sensors, SAW sensors, spectrometric means, pressure difference methods, or other methods.

[035] As used herein, the term“impermeable” means a substance, material or object that prohibits over 95% of any liquid or gas from passing or diffusing through it, for at least one hour. As used herein, the term“permeable” means a substance, material or object that allows the passage of gases and/or liquid through it.

DETATEED DESCRIPTION OF THE INVENTION

[036] In one embodiment the sterilization process occurs in a chamber in which a controlled amount of sterilant precursor gases are combined. In one embodiment the sterilant precursor gases include NO, NO2 and H2O. Although there are chemical reactions that occur in the gas phase, the rate of these gas-phase reactions between NO, NO2 and water (humidity) is relatively slow and does not contribute to the generation of the principle sterilizing molecular species (N2O3). Furthermore, N2O3 is not stable as a gas and cannot be usefully delivered as a sterilant to a chamber in which sterilization processes are intended. However, the rate of the reactions on the surfaces exposed to the sterilant precursor gases (including water) is much faster. On surfaces exposed to the precursor gases, the rate of N2O3 generation is a function of the amount of water film created on surfaces to be sterilized, which in turn, is proportional to the amount of water gas in the sterilization chamber.

[037] Two scenarios where sterilant precursor gases can cause microorganism lethality include direct absorption of the sterilant precursors into the microorganism and microorganism, and these are shown in Figure 1. The first scenario is the direct interaction of microorganisms (eg, spores) with the precursor gases (Figure 1, Reaction A). The second scenario is the interaction of the microorganisms with the water film and chemical environment that develops on the surfaces ex posed to the precursor gases (see Figure 1, Reaction B).

[038] The first scenario (Reaction A) is more directly related to sterilization, when one considers the interaction of the humidified microorganisms with the gases in the sterilization chamber. The spore coat (or cell membrane) will become hydrated with the H2O gas. This humidity greatly increases the size of the spores, the water content of the spore coat, and the permeability of the spore to gases. The sterilant precursor gas constituents (NO2, N2O4, NO and additional H2O) enter the water-containing spore coat. Thereafter, N2O3 is formed and reacts with the internal nucleotides, leading to microbial lethality.

[039] The second scenario describes the reactions that occur in the water film that forms on all surfaces in the chamber exposed to the humidified air. These water film reactions result in new molecular nitrogen species. In the case that NO is not supplied to the chamber, the heterogeneous reactions will result in NO formation after some time. Some of these molecules remain in the water film; others leave the water film as gaseous products from the water film reactions. Gaseous reaction products like HONO and NO molecules supplement the NO2 monomer and dimer gases in the chamber, becoming sterilant precursor gases. In the case that a specified amount of NO is added to the chamber with NO2 and H2O, then the NO2, N2O4 and NO can enter the water film. The dissolved molecules in the water film can interact directly with the cell membrane or spore coat of the microorganisms in contact with the water film. Figure 1 shows this microbicidal scenario. [040] In the absence of adding NO directly with the NO2 and H2O gases, the reactions needed to form NO are outlined in Figure 2. These reactions have been found in the literature, determined by calculation, and directly measured in sterilization chambers.

[041] In Figure 2, step 1 consists of the dimerization of NO2 to form N2O4, as is described earlier in discussions of the equilibrium between the monomer NO2 and the dimer N2O4. This is a well- known behavior for NO2. The NO2 molecules that collide will momentarily bond and then return to the monomer state. The dissociation of the N2O4 is quite fast, with a half-life of less than 10 ps (at 25°C). Therefore, whenever NO2 gas is present, some fraction of the NO2 will be momentarily bound in the dimer form. Step 2 refers to the formation of a water film on surfaces in the sterilization chamber. The NO2 sterilization process occurs in a humidified environment. The microorganisms, spores, and all the surfaces (surfaces of the chamber, surfaces of the items to be sterilized, etc) will adsorb water in these conditions, forming a surface. In most applications of the NO2 sterilization process, a chamber will have roughly ambient temperature and relative humidity below 90%.

[042] The water gas in the chamber leads to the formation of water films on surfaces exposed to the water gas. The thickness of the water film on surface depends on the surface material and is measured in monolayers of water. In the literature, estimates of the thickness of a single monolayer of water range from 1.9 A (0.19 nm) to 2.7 A (0.27 nm). The thickness of the water film on a material surface will be some multiple of a water monolayer and is dependent on the surface material and the partial pressure of water at the surface (RH). The water film thickness will vary from 1 to 3 monolayers at 20% RH and increases to more than 60 monolayers of water at RH values above 75% RH. Figure 2, step 3 describes the interaction between the water film, the NO2 sterilant gas, and the other gases in the sterilization chamber. A gas will dissolve into water until the concentration in the water reaches equilibrium with the gas phase concentration. This phenomenon is called Henry’s law, which states that the equilibrium concentration of a gas dissolved into a liquid is Caq, and is given by

Caq = Pg HCP

[043] In this equation, P is the partial pressure of the gas at the water/gas interface and HCP is the Henry’s law constant. In thin films of water, this equilibrium is established very quickly. Table 1 lists the Henry’s law constants, at 25°C, for the prominent gases found in the NO2 sterilization process. Table 1. Henry’s law coefficients and enthalpy of dissolution for selected gases.

[044] The properties of this water film vary with the thickness of the water film. From the measurement of several physical and chemical properties, water films do not behave as bulk, aqueous water until the water film is several monolayers thick. Even with the thicker water film, surface reactions of NO2 are different from the bulk water reactions.

[045] The most reactive species on the water film surface is the asymmetric form of N2O4, nitrosonitrate (ONONO2), which results from the water-induced isomerization of symmetric N2O4. The asymmetric ONONO2 has two reaction pathways: it auto-ionizes to generate NCENCh or it has a back reaction with gas phase NO2 to form symmetric N2O4 (this reaction step is not shown in Figure 1). The NCriNCh complex reacts with water to generate HNO3 and HONO. Some of the HONO escapes from the water surface into the gas phase. The HNO3 on the surface generates N0₂ ⁺, a well-known reaction in concentrated solutions of HNO3. The NO is generated by the reaction of HONO with N02⁺.

[046] Given all these chemical reactions, the literature suggests that gaseous NO and HONO will be formed. The formation of the NO and HONO was confirmed in sterilization chambers using FT-IR spectroscopy. A measurement of the HONO and NO formed in the sterilization chamber. Data acquired during a cycle consisting of 10 mg/L NO2 dose added to a chamber that had been humidified to 75 % RH. After 6 minutes, the amount of HONO was found to be approximately 390 ppm and the amount of NO in gas was 502 ppm.

[047] Figure 2, step 5 is the reaction between the NO and NO2 to form the N2O3. The NO, NO2, and N2O3 can enter and cause critical damage to a microorganism. The N2O3 reacts with the cytosine and guanine to damage DNA and RNA. As previously described, DNA degradation within B subtilis spores was observed to proceed rapidly upon exposure to the NO2 process. Therefore, it may be concluded that the principle oxide of nitrogen associated with microorganism lethality is N2O3.

[048] Aspects of the present invention describe methods and devices for the addition of NO directly into the chamber. The rate of lethality in the sterilization chamber can be enhanced by the addition NO, as NO gas will drive the aqueous concentration of NO to the equilibrium level more quickly, due to Henry’s Law, than if the equilibrium level is achieved from water-generated NO alone.

[049] In another embodiment, the amount of NO is limited to a maximum level. When too much NO is added, the additional gaseous NO causes acid formation in the water film, decreasing the pH of the water films, thereby inhibiting the reactions that form N2O3. The inhibition stems from the relative weakness of the HNO2 acid (and Le Chatelier’s principle), and the subsequent increased solubility of the NO2 anion. These principles have been demonstrated by the measured results shown in Figure 2.

[050] In an embodiment, a low concentration (< 10 mg/L) of nitrogen dioxide gas and nitric oxide gas (< 2 mg/L) in the presence of air and water vapor is delivered to a sterilization chamber. In particular embodiments, concentrations of between about 4 and 10 mg/L of NO2 are used. One would expect that as lower concentrations are used, dwell time should be extended. But with the proportionate addition of NO, the dwell time can be further shortened, reducing overall process time and sterilant residuals on treated surfaces. As described in greater detail below, the process may be performed at or near room temperature and entails evacuating air from the chamber, introducing the sterilant precursor gases, each to a selected pressure. Depending on the physical characteristics and/or packaging of the item to be sterilized, the sequence of vacuum sterilant precursor gas injection dry air injection, may be repeated several times or the sequence changed. Furthermore, additional sequence steps of dry air injection and dwell may be included in one or all iterations of the sterilizing sequence. At the ordinary operating temperatures and pressures of the process, the sterilant precursor gases act as an ideal gases. The cycle may include a check for water or humidity levels, and should the levels be above a threshold, a drying sequence may be engaged. Drying may include heating of the chamber, evacuating the chamber to pressures below the vapor pressure of water at the temperature of the chamber, and/or passing dry gas such as dry air or a dry inert gas through the chamber. [051] The results of BI exposure cycles using sterilant precursor gases, according to this method and device, are shown in Table 2. This table shows the results from a series of tests where each exposure cycle had an amount of water gas in the chamber corresponded to 40 % RH, 10 Bis included in each exposure cycle, each BI had more than 10⁶ spores per BI carrier, and 4 mg/L of NO2 gas. The exposure time and amount of NO gas was varied to demonstrate the principles described herein. For each exposure time and NO gas concentration shown in Table 2, the number of negative Bis (Bis exhibiting no evidence of viable spores) is shown. From these test results the influence of NO is clear and as expected from the foregoing analysis. To achieve complete lethality without NO gas and with NO2 and H2O added to the sterilization chamber, partial lethality of the Bis is observed after 2 minutes, and complete lethality observed after 4 minutes. 4 mg/L of NO gas added to the 4 mg/L NO2 and H2O gas prevented the generation of the molecular sterilizing agent.

Table 2. The number of Bis that exhibit no viable spores.

[052] One observation from these results is that providing an amount of NO gas with the other precursor gases that agree with the amount of NO produced at equilibrium is the optimal cycle condition.

[053] Another observation is that acid formation is not a principle mechanism of spore lethality. With water films on surfaces, the NO and the NO2 both contribute to the formation of HNO3 and HNO2 acids the most amount of acid formed is with 4 mg/L of NO2 and 4 mg/L of NO in the chamber simultaneously. Because these exposure cycles resulted in the least amount of lethality, it is clear that the formation of acid is not a principle mechanism of spore lethality.

[054] An embodiment of a sterilizer that uses the sterilant precursor gases is shown in Figure 3, with the sterilizer 100 shown in accordance with the present invention. Note that while specific configurations are provided for certain flow paths, these should not be taken as limiting in any way, but are subject to variation in accordance with specific applications. A first portion of the sterilizer 100 is a source of diluent gas to be added to the sterilant precursor gases in the sterilization chamber 136. A diluent gas source may be either air or nitrogen (N2). Prior to addition to the sterilization chamber, the diluent gas can pass through a filter 106. The filter 106 reduces dust and other particulate impurities that are generally undesirable for the use of the diluent gas. Furthermore, the filter 106 may advantageously be designed to remove microbes from the diluent gas stream such that the gas delivered to the sterilizer, and in particular to the humidification system, is substantially pathogen free. As will be appreciated, sources of diluent gas may be provided by compressed air tanks, compressed N2 tanks, or a fixed air supply system that provides pressurized air to the room in which the sterilizer is housed.

[055] In an embodiment of the method and system, Nitrogen dioxide is provided to the system from a liquid supply tank 118. A valve controls flow from the supply tank 118. A pressure gage 124 allows monitoring of pressure in the lines and a valve or a pair of solenoid valves 126, 128 (shown as one valve in the Figures) control flow into a pre-chamber 130. Another valve 132 controls flow from the pre-chamber 130 to the sterilization chamber 136. Though illustrated in FIG. 3 as a simple pipe connection, the flow into the chamber 136 may alternately be provided using a manifold, allowing for improved distribution of the sterilant gas within the chamber. More detail of the operation of the NO2 delivery sub-system is discussed below. The amount of NO2 gas in the pre-chamber 130 is determined by pressure changes in the pre-chamber, as monitored by pressure sensor 180.

[056] In an embodiment, Nitric oxide is provided to the system from a source 119. This source of NO could be a compressed gas supply tank, or an NO gas generating chamber. A valve controls flow from the supply tank 119. A pressure gage 125 allows monitoring of pressure in the lines and a valve or a pair of solenoid valves 127, 129 (shown as one valve in the Figures) control flow into a pre-chamber 130. Another valve 132 controls flow from the pre-chamber 130 to the sterilization chamber 136. Though illustrated in Figure 3 as a simple pipe connection, the flow into the chamber 136 may alternately be provided using a manifold, allowing for improved distribution of the sterilant gas within the chamber. More detail of the operation of the NO delivery sub-system is discussed below. The amount of NO gas in the pre-chamber 130 is determined by pressure changes in the pre-chamber, as monitored by pressure sensor 180.

[057] An embodiment includes a sub-system for providing humidity to the sterilization chamber 136 begins with a water gas (humidity) source 138 that produces water gas. The water gas source can be a steam source, an ultrasonic humidifier, nebulizer or other water gas source. The water gas source 138 can use a diluent gas as a carrier for the water gas. While this is not necessary with a steam water gas source, other water gas sources can receive diluent gas from the diluent gas source 102. The flow of water gas is controlled by a valve 144.

[058] As illustrated in Figure 3, the sterilization chamber 136 includes access via a set of valves 150 so that samples of the chamber atmosphere may be taken and analyzed. Analysis may be, for example, by an FTIR, UV spectrophotometric, or other appropriate spectrometry system, not shown. Access for analysis has particular relevance to a test platform, and may be unnecessary in practice when the sterilizer is used in a production environment.

[059] The sterilization chamber 136 may include a fan or blower 152 that circulates gases in the chamber. Circulation helps to ensure both that the sterilant precursor gases and that objects to be sterilized are well exposed to the sterilant gas.

[060] The primary exhaust pathway proceeds through a solenoid valve 158 to a vacuum pump 164, and the through a scrubber 160, designed to eliminate and/or capture nitrogen dioxide before the exhaust reaches the environment. As will be appreciated, the order between the pump 164 and the scrubber 160 may be reversed, allowing for using a vacuum pump that is not compatible with the sterilant precursor gases and sterilant residual gases.

[061] A tank 118 containing liquid NCk acts as the source of NO2 sterilant precursor gas. A manual valve 120 provides a flow of gas from the tank 118 and may be integral with the tank. A pair of solenoid valves 126, 128 are actuatable to allow flow from the valve to the sterilizing system. As illustrated, there are four separate valves that ultimately control flow from the tank 118. As will be appreciated, other valve arrangements are possible, and redundancy may be reduced or eliminated, as desired.

[062] During use, NO2 sterilant precursor gas is allowed to flow from the final solenoid valve 126, 128 into a pre-chamber 130, where it expands and the dosage may be measured. As shown, the pre-chamber 130 includes a pressure transducer 180 that allows measurement of a total pressure which may be translated into dosage, given appropriate knowledge of the size of the chamber and optionally, temperature data derived from a temperature sensor, not shown. A solenoid valve 132 controls flow into the sterilizing chamber 136. An additional solenoid valve 182 controls flow of dry air into the pre-chamber.

[063] A NO sterilant precursor source 119 acts as the source of NO sterilant precursor gas. A manual valve 123 provides a flow of gas from the source 119 and may be integral with the tank. A pair of solenoid valves 127, 129 are actuatable to allow flow from the valve to the sterilizing system. As illustrated, there are four separate valves that ultimately control flow from the tank 118. As will be appreciated, other valve arrangements are possible, and redundancy may be reduced or eliminated, as desired.

[064] During use, NO sterilant precursor gas is allowed to flow from the final solenoid valve 127,

129 into a pre-chamber 130, where it expands and the dosage may be measured. As shown, the pre-chamber 130 includes a pressure transducer 180 that allows measurement of a total pressure which may be translated into dosage, given appropriate knowledge of the size of the chamber and optionally, temperature data derived from a temperature sensor, not shown. A solenoid valve 132 controls flow into the sterilizing chamber 136. An additional solenoid valve 182 controls flow of dry air into the pre-chamber.

[065] In one embodiment, NO precursor gas may be added to the pre-chamber 130 before NO2 precursor gas is added. In another method, NO2 precursor gas may be added to the pre-chamber

130 before NO precursor gas is added.

[066] In one method of operating the illustrated embodiment, the chamber 136 and pre chamber 130 are initially at low pressure, for example, they may be evacuated using appropriate vacuum pumps (for example, the pump 164 in the exhaust pathway illustrated in Figure 3). In an embodiment, an evacuation cycle is repeated prior to injection of the sterilant precursor gas. As an example, the chambers may be evacuated, re-filled with air, and then evacuated again prior to initiating the sterilant gas sequence.

[067] In order to begin delivery of sterilant gas, valves 126 and 128 are closed and 132 is opened, while valve 182 is held closed, equalizing the pressure in the sterilization chamber 136 and pre chamber 130 at a low pressure. Valve 132 is closed, isolating the pre-chamber 130 from the sterilizing chamber 136. Valves 126 and 128 are then opened (valve 122 and manual valve 120 having been already opened) and gas that has boiled-off of the liquid NO2 supply is allowed to enter the pre-chamber 130. The pressure transducer 180 may be used in a feedback arrangement to control solenoid valve 126 such that a selected total amount of NO2 is collected in the pre-chamber 130.

[068] In an embodiment, the pre-chamber 130 is directly connected to the vacuum pump and the pressure in the pre-chamber can be controlled with a valve 139.

[069] As will be appreciated, if the volume of the pre-chamber 130, pressure and temperature are known, for example via measurements using the pressure transducer 180 and a temperature gage (not shown), the total amount of NO2 in the pre-chamber 130 may be calculated. By way of example, an operating pressure of 10-20 inHg may be generated in order to provide an approximately 10 gm dose of sterilant to a sterilization chamber 136 having a volume of about 1200 liters. In this approach, a concentration of about 10 mg/L of sterilant gas is produced in the sterilization chamber 136.

[070] In an embodiment, the temperature of the sterilization chamber can be controlled and temperatures will not fluctuate significantly, the process of preparing a dose may be controlled with pressure measurements alone, without any calculating steps. Thus, when a pressure threshold is reached in the pre-chamber, the appropriate dose is present and the connection between the source and the pre-chamber may be closed. In an embodiment, there may be a table of pressure set points, for example, stored in memory accessible to the controller, that correspond to desired dosages. In another embodiment, there may be a curve stored in memory accessible to the controller that associates dose with pressure set points. In either approach, the measured pressure during a filling operation may be compared to the set point pressure derived from either the table or the curve. Where temperature is more variable, adjustments may be made based on a measured temperature, and those adjustments may be calculated, or derived from a table or curve stored in a memory.

[071] As will also be appreciated, NO2 and the dimer N2O4 exist in equilibrium. As a result, it may be useful to account for partial pressures of various species in calculating the total deliverable dose of sterilant gas. Because partial pressures of oxides of nitrogen in the (generally much larger) sterilization chamber 136 are substantially lower than in the pre-chamber 130, the dimerization of NO2 1S less prevalent and partial pressures remain at a low level and are not generally important. That is, the total mass of the NCh and N2O4 1S generally available as NC in the sterilization chamber 136.

[072] After the pre-chamber 130 is pressurized with sterilant precursor gases, the valves 126, 128, 127 and 129 are closed, isolating the pre-chamber 130 from the gas source. Valve 132 is opened, allowing the gas from the pre-chamber 130 to pass into the sterilizing chamber 136. Valve 182 is opened to allow dry air to enter into the sterilizing chamber 136, and to push any remaining sterilant gas out of the pre-chamber 130 and into the sterilizing chamber 136. Finally, valves 182 and 132 are closed, isolating the sterilizing chamber from the other portions of the system.

[073] As will be appreciated, other configurations and methods may be used to provide the sterilant gas to the sterilizing chamber 136. For example, the sterilant precursor gases NO2 and NO may have separate pre-chambers, as shown in figure 4. The NO pre-chamber 131 functions in a similar manner as the NO2 pre-chamber.

[074] A tank 119 containing liquid Mh acts as the source of NO2 sterilant precursor gas. A manual valve 123 provides a flow of gas from the tank 119 and may be integral with the tank. A pair of solenoid valves 127, 129 are actuatable to allow flow from the valve to the sterilizing system. As illustrated, there are four separate valves that ultimately control flow from the tank 119. As will be appreciated, other valve arrangements are possible, and redundancy may be reduced or eliminated, as desired.

[075] During use, NO2 sterilant precursor gas is allowed to flow from the final solenoid valve 127, 129 into a pre-chamber 131, where it expands and the dosage may be measured. As shown, the pre-chamber 131 includes a pressure transducer 126 that allows measurement of a total pressure which may be translated into dosage, given appropriate knowledge of the size of the chamber and optionally, temperature data derived from a temperature sensor, not shown. A solenoid valve 133 controls flow into the sterilizing chamber 136. An additional solenoid valve 183 controls flow of dry air into the pre-chamber.

[076] The NO pre-chamber can be evacuate through a valve 140 that is controlled to open when such evacuation is needed.

[077] In one embodiment, the NO pre-chamber can be supplied with NO2 and a catalyst can convert the NO2 to NO. [078] In another embodiment, the NO2 pre-chamber can be filled with NO gas and oxygen so that the oxidation of NO results in the NO2 precursor gas needed for the sterilization process. In a further embodiment, the source of oxygen could be air.

[079] In an embodiment, a diluent gas, being a non-reactive gas or gas mixture, rather than dry air is added to dilute the sterilant gas. Diluent gas addition to the sterilization chamber 136 is controlled by a valve 146. For example, N2 gas may be used in place of air. In this approach, the N2 gas may be used dry, humidified prior to adding to the sterilization chamber 136, or may alternately be humidified in the sterilization chamber 136, as with the embodiments using air.

[080] In an embodiment, the sterilization chamber 136 may include a track for loading objects to be processed and for unloading of processed objects. In the embodiment, the sterilization chamber may have a loading and an unloading door.

[081 ] In an alternate embodiment, water vapor may be directly added to the sterilization chamber as steam. In another approach, the nebulizer may be replaced with a humidifier of another design, such as evaporative, impeller or ultrasonic humidifiers.

[082] Because relative humidity is dependent in part on temperature, it may be useful to use temperature data to determine an amount of humidified air necessary to achieve a target humidity level in the chamber. In this regard, a temperature control means and a temperature sensor may be provided 147 in the sterilization chamber.

[083] As described above, the exhaust pathway includes a scrubber 160 for removing sterilant gas from the exhaust stream, prior to venting to the exterior of the system. The scrubber 160 may be, for example, embodied in a media-filled drum type of device not shown. A drum of this type may be placed in-line in the exhaust pathway, such that gases pulled out of the sterilization chamber 136 via the pump 164 will pass through the drum.

[084] In an embodiment, the drum is a cylinder having inlet connections and outlet connections and a volume where the gas to be treated enters. The inlet connections direct the gas flow to the treating medium, thence to an outlet to the exhaust pathway.

[085] The medium is selected to convert the reactive oxides of nitrogen into non-reactive products or otherwise neutralize any hazard associated with the sterilant gas. The scrubber material may be provided in granular form, smaller granules providing a greater surface area per unit volume of scrubbing material. In an embodiment, a sodium permanganate material has been found to be suitable for oxidation of NO to nitrate (i.e., NO to Mh and Mh to NO3). In particular, granular Purafil SP (14x28 mesh), a sodium permanganate including activated alumina has been found to be suitable. Once oxidized, the nitrate bonds with sodium to form a solid salt which is substantially non-toxic, non-corrosive, non-reactive and does not readily ignite. In embodiments, the exhaust gas may be considered to be remediated when NO2 levels are reduced below applicable safety standards, and particularly, when reduced below about 5 ppm and more particularly, when reduced below about 1 ppm. In embodiments where the gas is exhausted directly outdoors, a higher level of NCh may be acceptable, and levels of 10 ppm may be considered to be sufficiently remediated.

[086] In another embodiment, the scrubbing system may include multiple stages. In an embodiment, there may be an activated carbon stage after the permanganate stage.

[087] In an embodiment, the exhaust cycle may include a series of evacuations of the sterilization chamber. In this approach, the pressure in the sterilization chamber is reduced, for example, by 90%, exhausting all but 10% of the sterilant gas. Next, a charge of air or other diluent gas is added to the chamber, and the exhausting is repeated. As will be appreciated, each cycle reduces the amount of sterilant gas by 90%, so that after three cycles, only 0.1% remains in the chamber. This may be repeated as many times as necessary to render the interior of the chamber safe. For higher percentages of exhaust (e.g., 99%), fewer repetitions are needed while for lower percentages, more repetitions will be needed (e.g., 80%) to reach a selected threshold. Using this approach, the exhaust cycle can be controlled based on pressure measurements, and the interior toxic nitrogen gas levels may be known without requiring additional gas monitoring equipment in the chamber or at the outlet of the chamber.

[088] In an embodiment, replacement of the liquid NO2 bottle is controlled to improve safety. Interlocks may be provided to restrict removal until all supply valves are closed and a purge sequence is performed. Optionally, the exhaust pump may be held on during cylinder disconnect to ensure that any remaining sterilant gas is vented through the scrubber and exhaust pathway rather than backflowing through the input side of the system. Similar interlocking may be implemented to prevent removal/replacement of the scrubber when sterilant gas is present in the lines.

[089] In an embodiment, replacement of the NO source is controlled to improve safety. Interlocks may be provided to restrict removal until all supply valves are closed and a purge sequence is performed. Optionally, the exhaust pump may be held on during cylinder disconnect to ensure that any remaining sterilant gas is vented through the scrubber and exhaust pathway rather than backflowing through the input side of the system. Similar interlocking may be implemented to prevent removal/replacement of the scrubber when sterilant gas is present in the lines.

[090] In an embodiment, the surface reactions can be monitored by sensors designed for measuring molecular species dissolved into the water film. Methods for measuring the chemical reactions in the water film include spectroscopic methods, ATR FTIR, and others. Furthermore, in an embodiment of the method the addition of sterilant precursor gases is controlled by the monitoring of the water film chemistry.

[091] In an embodiment, the measurement of reaction by-products between the precursor gas molecules can be used to monitor and control the process. Molecules that can be monitored are gaseous HONO and NO in concentrations of NO that are more than originally added to the sterilization chamber.

[092] In an embodiment, and using the assumption that the very thin water films saturate very quickly, the amount of NO needed for dosing the sterilization chamber can be determined by adding only water gas (humidity) and NO2 and observing the equilibrium level of NO in the chamber in the sterilization chamber. If 500 ppm of gaseous NO is observed in the chamber, this can be a starting concentration for the NO sterilant precursor gas to add to the sterilization chamber.

[093] Control of the device may be implemented using a graphical user interface. The graphical user interface can include, for example, controls for run parameters (e.g., dosage/concentration of sterilant gas, pressure limits, cycles, dwell times, target pressures, etc.), readouts of metrology devices (e.g., timers, thermometers, pressure transducers, etc.) and controls or status outputs for various of the valves and compressors of the system. System warnings (e.g., interlock warnings) can also be displayed via the graphical user interface. Alternately, the entire process may be automated such that no user input is required and the user interface may simply be replaced by a display or other status indicators.

[094] In an embodiment, in addition to an automated sterilization cycle, the entire process including inserting and removing the object to be sterilized may be automated. In this approach, a robotic handler is provided that can convey the obj ect to be sterilized into the chamber, and remove it upon completion of the sterilization cycle. [095] Embodiments include the following examples:

[096] 1. A method of sterilizing a medical product, the method comprising adding sterilant precursor gases to the sterilization chamber wherein the sterilant precursor gases react to form the molecular sterilizing agent; adding a predetermined amount of NO2 as a precursor gas; adding a predetermined amount of NO as a precursor gas; adding a predetermined amount of H2O as a precursor gas; wherein the sterilant precursor gases are mixed in the sterilization chamber.

[097] 2. The method of example 1 wherein at least one pre-chamber is in fluid communication with the sterilization chamber.

[098] 3. The method of example 1 further comprising evacuating the sterilization chamber and at least one pre-chamber prior to the addition of the sterilant precursor gases.

[099] 4. The method of example 1 wherein the addition of sterilant precursor cases includes nitrogen oxide (NO), nitrogen dioxide (NO2) and H2O gas.

[0100] 5. The method of example 1 wherein sensors are connected to the chamber for measuring the precursor gases.

[0101] 6. The method of example 1 wherein H2O gas is provided by steam humidification in the sterilization chamber to create a principle molecular sterilizing agent.

[0102] 7. The method of example 1 wherein H2O gas is provided by ultrasonic humidification in the sterilization chamber.

[0103] 8. The method of example 6 wherein H2O gas is provided by steam humidification before the addition of NO and Mhto the sterilization and pre-chamber connection.

[0104] 9. The method of example 8 wherein H2O gas is provided by steam humidification after the addition of NO and M to the sterilization and pre-chamber connection.

[0105] 10. The method of example 6 wherein H2O gas is provided by ultrasonic humidification after the addition of NO and NO2 to the sterilization chamber and pre-chamber connection.

[0106] 11. The method of example 4 wherein NO, NO2, and H2O gas result in heterogeneous reactions that create a principle molecular sterilizing agent.

[0107] 12. The method of example 11 wherein the principle molecular sterilizing agent is dinitrogen tetroxide (N2O3)

[0108] 13. A method as in example 1, wherein a chemical scrubber comprises at least one container having media that capture the residual nitrogenous sterilant precursor molecules. [0109] 14. The method of example 4 wherein the predetermined amount of NO is added to sterilization chamber before the predetermined amount of NO2 is added to a pre-chamber and the amounts are determined by examining the stoichiometric ratios of the sterilant generating chemical reactions.

[0110] 15. The method of example 4 wherein the predetermined amount of NO2 is added to sterilization chamber before the predetermined amount of NO is added to a pre-chamber and the amounts are determined by examining the stoichiometric ratios of the sterilant generating chemical reactions.

[0111] 16. The method of example 14 wherein the predetermined amount of NO added to the sterilization chamber is a concentration less than 2.0 mg/L

[0112] 17. The method of example 15 wherein the predetermined amount of NO added to a pre-chamber chamber is a concentration less than 2.0 mg/L

[0113] 18. The method of example 4 wherein a vacuum pump connected to both the pre chamber and the sterilization chamber evacuates the sterilization chamber and pre-chamber before the addition of the precursor gases.

[0114] 19. The method of example 18 wherein the sterilization chamber and pre-chamber are evacuated at the same time.

[0115] 20. The method of example 4 wherein gas-phase reaction byproducts of generating the molecular sterilizing agent are measured for monitoring and controlling the sterilant exposure process;

[0116] 21. The method of example 4 wherein gas-phase reaction byproducts of generating the molecular sterilizing agent are measured to ensure sufficient conditions for effective sterilization have been achieved;

[Oil 7] 22. The method of example 4 wherein gas-phase reaction byproducts of generating the molecular sterilizing agent are measured by monitoring surface reaction or reactions using optical techniques;

[0118] 23. A method of example 22 wherein monitoring water film reactions with an FTIR- ATR prism surface mounted in the chamber;

[0119] 24. The method of example 4 wherein a vacuum pump is connected to the sterilization chamber and a separate vacuum pump is connected to the pre-chambers and the vacuum pumps can evacuate the sterilization chamber and pre-chamber separately before the addition of the precursor gases.

[0120] 25. A method of sterilizing a medical product, the method comprising: delivery of a predetermined amount of sterilant precursor gases and H2O gas to a sterilization chamber and pre chamber connection wherein the sterilant consists of the mixing of both nitrogen oxide (NO) and nitrogen dioxide (NO2) in the sterilization chamber; a humidifier that produces H2O gas; and a vacuum that reduces pressure in the sterilization chamber

[0121] 26. The method of example 25 wherein a spectrometric system measures the delivery concentrations of NO2 and NO.

[0122] 27. The method of example 25 wherein the first step of the method is for the vacuum to simultaneously reduce their atmospheric pressure in both the sterilization chamber and the pre chamber to a pressure of 0.3 inHg before the addition of NO and NO2

[0123] 28. The method of example 25 wherein a vacuum pump is connected to the sterilization chamber and a separate vacuum pump is connected to the pre-chambers and the vacuum pumps can evacuate the sterilization chamber and pre-chamber separately before the addition of the precursor gases.

[0124] 29. The method of example 25 wherein the last step of the method further comprises the chamber reaching ambient pressure (29.8 in Hg) and then reducing to a pressure of 0.3 in Hg at least 3 times.

[0125] 30. The method of example 27 wherein the sterilization chamber is dosed with NO directly from a compressed gas source before the pre-chamber doses NO2 to the sterilization chamber directly from a compressed gas source or liquid source.

[0126] 31. The method of example 30 wherein the NO is controllably dosed to the sterilization chamber based on a predetermined target pressure of the system, a mass flow meter, or a rotameter.

[0127] 32. The method of example 30 wherein a sensor the measures the NO concentration is used to control the valve that permits NO to flow into the sterilization chamber.

[0128] 33. The method of example 25, wherein the pre-chamber pressure increases above 0.3 in Hg until a target pressure is reached, allowing the NO2 in the pre-chamber to be flushed into the sterilization chamber. [0129] 34. The method of example 33 wherein reaching the target pressure involves having pressure transducers record the pressure rise associated with the addition of NO and NO2 to confirm the delivery of NO2 to the sterilization chamber.

[0130] 35. The method of example 34 wherein the H2O gas is generated until a target relative humidity of more than or equal to 25% RH is reached in the sterilization chamber and a predetermined set point below ambient pressure (29.8 inHg) is reached after the addition NO and NO2.

[0131] 36. The method of example 25 wherein a spectrometric system measures the relative humidity inside of the sterilization chamber due to the generation of H2O gas.

[0132] 37. The method of example 25 wherein the mixture of NO, NO2, and H2O gas dwells for a set period in the sterilization chamber and creates a water film.

[0133] 38. The method of example 25 wherein after the dwell period, the vacuum reduces pressure in the chamber to 3 inHg.

[0134] 39. The method of example 21 wherein the concentration of NO2 created in the water film after the addition of NO and NO2 is dependent upon the temperature of the chamber during the dwell.

[0135] 40. A sterilization system comprising: a sterilization chamber and a pre-chamber connection configured to allow for a fluid connection wherein the connection delivers a predetermined amount of sterilant precursor gases and compressed dry air to one or both chambers; the sterilant precursor gases consisting of both nitrogen oxide (NO) and nitrogen dioxide (NO2) mixing in the sterilization chamber; and a humidification means provides H2O gas in the sterilization chamber.

[0136] 41. The system of example 40 wherein the delivery of nitrogen dioxide (NO2), nitrogen oxide (NO), compressed dry air, and humidification of dry air into H2O gas create a water film in the sterilization chamber that generates a sterilizing environment and the additional sterilizing agent, dinitrogen tetroxide (N2O3).

[0137] 42. The system of example 40 wherein the addition of NO, NO2 and H2O gases is followed with the addition of compressed dry air and the conversion of compressed dry air to H2O gas in the sterilization chamber together generate the molecular sterilizing agents N2O3, nitrous acid (HNO2), and HN03 (nitric acid) in the water film of the sterilization chamber. [0138] 3. The system of claim 42 wherein the ratio of NO2 and NO in the sterilization chamber are determined by stoichiometric analysis of the chemical reactions that formN203, and additional chemical constituents generated in the chamber follows the determined ratio.

[0139] 44. The system of example 40 further comprising a mass flow meter to measure the delivery of gases.

[0140] 45. The system of example 40 further comprising a spectrometric system that measures the delivered concentrations any of the sterilant precursor gases.

[0141] 46. The system of example 40 wherein the sterilization chamber and pre-chamber simultaneously reduce their atmospheric pressure to a pressure of 0.3 inHg before the addition of NO, NO2, and dry air.

[0142] 47. The system of example 46 further comprising a compressed gas source that directly doses NO to the sterilization chamber before the pre-chamber doses NO2 to the sterilization chamber directly from a compressed gas source or liquid source following the reduction of pressure to 0.3 inHg.

[0143] 48. The system of example 40 wherein the NO is controllably dosed to the sterilization chamber based on a predetermined target pressure of the system.

[0144] 49. The system of example 40 further comprising a sterilization chamber valve that opens and waits after the dose of NO before flushing NO2 from the pre-chamber to the sterilization chamber.

[0145] 50. The system of example 40, wherein the pre-chamber flushes with the compressed dry air and allows for an increase in chamber pressure until a target pressure is reached, allowing the NO2 in the pre-chamber to be flushed into the sterilization chamber.

[0146] 51. The system of example 40 further comprising pressure transducers to record the pressure rise associated with the addition of NO and NO2 to confirm the delivery of NO2 to the sterilization chamber.

[0147] 52. The system of example 40 wherein the mixture of NO2, NO, H2O, and compressed dry air dwells for a set period in the sterilization chamber and creates a water film.

[0148] 53. The system of example 40 further comprising a vacuum source to evacuate the sterilization chamber pressure to 3 inHg following the dwell period.

[0149] 54. The method of example 40 wherein the diluent gas is dinitrogen. [0150] Although the invention has been described in detail for the purpose of illustration based on what is currently considered to be the most practical and preferred embodiments, it is to be understood that such detail is solely for that purpose and that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover modifications and equivalent arrangements that are within the spirit and scope of the appended claims. For example, it is to be understood that the present invention contemplates that, to the extent possible, one or more features of any embodiment can be combined with one or more features of any other embodiment.

REFERENCES

Won WD, Ross H. Reaction of Airborne Rhizobium Meliloti to Some Environmental Factors. Applied Micro 1969; 18:555-557.

Goersdorf S. et al, Nitrogen Dioxide induces DNA Single-strand Breaks in cultured Chinese Hamster Cells. Carcinogenesis 1990;11 :37-41.

Sander R. Compilation of Henry’s law constants for water as solvent. Atmos. Chem. Phys. 2015;15:4399-4981.

Lonkar P, Dedon PC. Reactive species and DNA damage in chronic inflammation: reconciling chemical mechanisms and biological fates. Int. J. Cancer 2010;128: 1999-2009.

Ngyuen T, et al. DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc. Natl. Acad. Sci. 1992;89:3030-3034.

Moeller MN, et al. Membrane“Lens” Effect: focusing the formation of reactive nitrogen Liebe HJ, Wolfe VL, Howe DA. Test of wall coatings for controlled moist air experiments. Rev. Sci. lustrum . 1984;55 : 1702- 1705.

oxides from the‘NO/Ck reaction Chem Res Toxicol 2007;20:709-714.

Leenson IA. Approaching Equilibrium in the N2O4-NO2 System: A common Mistake in Textbooks. Journal of Chemical Education. 2000;77: 1652-1659.

Westphal AJ, Price PB, Leighton TJ, Wheeler KE. Kinetics of size changes of individual Bacillus thuringiensis spores in response to changes in relative humidity. Proc Natl Acad Sci USA 2003;100:3461-6.

Finlayson-Pitts BJ, Wingen LM. Sumner AL, Syomin D, Ramazan KA. The heterogeneous hydrolysis of NO2 in laboratory systems and in outdoor and indoor atmospheres: An integrated mechanism. Phys. Chem. Chem. Phys 2003;5:223-242.

Caulfield JL, et al. Nitric Oxide-induced Deamination of Cytosine and Guanine in

Deoxynucleosides and Oligonucleotides /. Biol. Chem. 1998;273:12689-12695.

Knudsen SM, et al. Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores. J. Bacterial 2016; 198 : 168- 177.

Ewing GE. Ambient Thin Film Water on Insulator Surfaces 2006; Chem. Rev.;106(4):1511- 1526.

Liebe HJ, Wolfe VL, 1 lowe DA. Test of wall coatings for controlled moist air experiments. Rev. Sci. Instrum. 1984;55: 1702-1705.

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5UB5TITUTE SHEET (RULE 26) Svensson RE, et al. Kinetics of the Reaction between Nitrogen Dioxide and Water Vapor. Atmos. Environ. 1987;21 : 1529-1539.

Finlayson-Pitts BJ. Heterogeneous NOx chemistry in polluted urban atmospheres: implications for the formation of particles and ozone and control strategy development. Research Report, Accessed July 9, 2018 at https:/Av v.arb.ca.gov/research/apr/past/00-323.pdf.

Ramazan KA, et al. New experimental and theoretical approach to the heterogeneous hydrolysis of NO2: key role of molecular nitric acid and its complexes. J Phys Chem A. 2006;110:6886- 6897.

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5UB5TITUTE SHEET (RULE 26)

Claims

What is claimed is:

1. A method of sterilizing a medical product in a sterilization chamber the method comprising:

adding sterilant precursor gases to the sterilization chamber wherein the sterilant precursor gases react to form a primary molecular sterilizing agent, wherein the adding sterilant precursor gases comprises:

adding a predetermined amount of NO2 as a precursor gas;

adding a predetermined amount of NO as a precursor gas;

adding a predetermined amount of H2O as a precursor gas; and

mixing the sterilant precursor gases in the sterilization chamber to form the primary molecular sterilizing agent.

2. The method of claim 1 further comprising evacuating the sterilization chamber and at least one pre-chamber of the sterilization chamber prior to the addition of the sterilant precursor gases.

3. The method of claim 1 wherein the H2O gas is provided to the sterilization chamber by a method selected from the group consisting of: steam humidification and ultrasonic humidification, or a combination.

4. The method of any of claims 1-3 wherein the H2O gas is provided before the addition of NO and NO2 to the sterilization chamber.

5. The method of any of claims 1-3 wherein the H2O gas is provided after the addition of NO and NO2 to the sterilization chamber.

6. The method of any of claims 1-5 wherein the mixing of the NO, NO2, and H2O gases result in heterogeneous reactions to form the principle molecular sterilizing agent.

7. The method of any of claims 1-6 wherein the principle molecular sterilizing agent is dinitrogen tetroxide ( 2O3).

8. The method of claim 7, wherein the dinitrogen tetroxide forms in a water film on a surface of the medical product.

9. The method of any of claims 1-8, further comprising capturing residual nitrogenous sterilant precursor molecules using a chemical scrubber.

10. The method of any of claims 1-9, wherein the predetermined amount of NO is added to the sterilization chamber before the predetermined amount of NO2 is added to a pre-

28

5UB5TITUTE SHEET (RULE 26) chamber of the sterilization chamber, and the predetermined amounts are determined in accordance with stoichiometric ratios of sterilant generating chemical reactions that form the primary molecular sterilizing agent.

11. The method of any of claims 1-9, wherein the predetermined amount of NO2 is added to the sterilization chamber before the predetermined amount of NO is added to a pre chamber of the sterilization chamber, and the predetermined amounts are determined in accordance with stoichiometric ratios of sterilant generating chemical reactions that form the primary molecular sterilizing agent.

12. The method of any of claims 1-11 wherein the predetermined amount of NO added to the sterilization chamber is a concentration less than 2.0 mg/^'L

13. The method of any of claims 1-12, further comprising:

monitoring gas-phase reaction byproducts of generating the primary molecular sterilizing agent; and

controlling a process of exposing the medical product based on the monitoring.

14. The method of claim 13, wherein the monitoring gas-phase reaction byproducts comprises monitoring, using an optical technique, one or more surface reactions.

15. The method of claim 14, wherein the monitoring comprises using an FTIR-ATR prism surface mounted in the sterilization chamber.

16. A sterilization system comprising:

a sterilization chamber;

a pre-chamber;

a connection between the sterilization chamber and the pre-chamber, configured and arranged to provide a fluid connection therebetween, operable to deliver a predetermined amount of sterilant precursor gases and compressed dry air to one or both of the pre-chamber and the sterilization chamber;

one or more gas sources, configured and arranged to, in operation, provide the sterilant precursor gases comprising nitrogen oxide (NO) and nitrogen dioxide (NO2) such that they may mix in the sterilization chamber; and

a humidifier configured and arranged to provide H2O gas in the sterilization chamber

29

5UB5TITUTE SHEET (RULE 26)

17. The system of claim 16, wherein the system is controllable to deliver the nitrogen dioxide (NC ), the nitrogen oxide (NO), the compressed dry air, and H2O gas to create a water film in the sterilization chamber containing dinitrogen tetroxide (N2O3)

18. The system of any of claims 16-17, further comprising a mass flow meter to measure the delivery of gases.

19. The system of any of claims 16-18, further comprising a spectrometer configured and arranged to measure delivered concentrations of one or more of the sterilant precursor gases.

20. The system of any of claims 16-19, further comprising a vacuum source configured and arranged to reduce pressure of the sterilization chamber and pre-chamber simultaneously to a pressure of 0.3 inHg before the addition of NO, NO2, and dry air.

30

5UB5TITUTE SHEET (RULE 26)