Sterilization method

Abstract

A method of sterilizing the interior of a sealed container containing a disinfecting solution and a disinfecting composition therein. The method includes adding a disinfecting solution to the container and then sealing the container to form a sealed container having an inner wall defining an interior volume. The method further includes wetting the inner wall of the container with a disinfecting solution. The method further includes subjecting the sealed container to heating at a predetermined sterilization temperature of 50 DEG C to 80 DEG C for a predetermined sterilization time of 50 minutes to 500 minutes.

Description

Background Technology

[0001] Methods for sterilizing disinfectant compositions have been described, for example, in U.S. Patents 9,724,437 and 9,895,455. Attached Figure Description

[0002] Figure 1 A side view of a liquid applicator according to some embodiments of the present disclosure is shown.

[0003] Figure 2 Examples Figure 1 The cross-section of the liquid applicator.

[0004] Figure 3 It is used to determine Bacillus subtilis ( Bacillus subtilis The logarithm of the D value of spores in 2% CHG / 70% IPA at 65°C 10 A curve comparing the number of surviving spores with the exposure time. Detailed Implementation

[0005] In the industrialized world, it is standard practice to disinfect the skin before any invasive procedure, such as surgery, catheter insertion, or acupuncture, to reduce the risk of infection. These products are often referred to as skin preparations or simply "preparations." The routine preparation of a patient's skin for such procedures involves washing the affected area with disinfectant soap for 30 seconds to 10 minutes. These solutions are typically applied using a foam sponge tip applicator. The foam sponges are usually saturated by soaking them in an open solution dish or with a solution from a container of disinfectant solution held in a hollow handle connected to the foam sponge.

[0006] As mentioned, in many cases, the disinfecting solution is held in a hollow cavity within an applicator in a container (e.g., a frangible ampoule). In the manufacture and use of disinfectant formulations and associated containers, there are various challenges associated with providing and maintaining sterility. For example, certain high-temperature sterilization processes (e.g., pressurized steam at temperatures greater than 120 °C for a period of time sufficient for the steam to cause sterilization of the object (e.g., 3 minutes to 15 minutes)), while effective to sterilize the disinfecting solution and the interior walls of the container, can inactivate the sterilizing solution or compromise the integrity of the primary packaging. As another example, certain low-temperature sterilization processes (e.g., dry heat at temperatures less than 80 °C for 60 minutes to 600 minutes), while effective to sterilize the solution without significant inactivation or packaging issues, have been found to be insufficient to sterilize the interior walls of the container. For example, it has been found that naturally occurring biological loads present on the interior surfaces of the container, within the headspace, or introduced during the manufacturing process can be of a high enough quantity and resistant to the sterilization process to not be adequately sterilized without additional processes, which can present an undue risk to the patient.

[0007] Accordingly, there is a need for sterilization techniques that fully sterilize the disinfecting solution and the interior walls of the disinfecting solution container without significantly inactivating the disinfecting solution.

[0008] When these terms appear in the specification and claims, the terms "comprising," "including," and "having," and variations thereof, are not intended to exclude any element unless otherwise indicated.

[0009] As used herein, "one," "a," "an," "at least one," and "one or more" are used interchangeably and are meant to encompass one or more than one unless otherwise indicated.

[0010] In some embodiments, the present disclosure relates to methods for sterilizing disinfecting solutions (e.g., chlorhexidine-based disinfecting solutions) and containers (e.g., bottles or ampoules) for holding such solutions Figures 1-2 A liquid applicator 100 according to some embodiments of the present disclosure is illustrated. As shown, the liquid applicator 100 can include an elongated hollow body 110 (which can serve as a handle for the user) that includes a wall 160. The wall 160 can define a chamber 170 having a closed end 118 and an open end 174. A container 175 or ampoule containing a disinfecting solution can be disposed within the chamber 170.

[0011] The closed end 172 can be sealed in any of a variety of known ways to inhibit or prevent fluid contained within the chamber from escaping through the closed end. For example, a cap that can be press fit, threaded, or otherwise attached can seal the chamber forming the closed end 172. The open end 174 can include one or more holes that allow fluid to flow out of the chamber 170. A pad 178 (e.g., an absorbent foam pad) can be fluidly coupled with the open end 174 such that fluid flowing through the holes directly or indirectly contacts the pad 178. The pad 178 can be coupled to the hollow body (directly or indirectly) by conventional fastening mechanisms (e.g., adhesive or ultrasonic welding).

[0012] In some embodiments, the pad 178 can be made from a variety of commercially available materials having a wide range of compressive modulus of ratio (i.e., density) and porosity. In some embodiments, the pad 178 can include (or be formed from) a polyurethane foam (e.g., an open cell polyester or polyether polyurethane foam). In some embodiments, the foam can have a material type described in U.S. Patents 6,841,586 or 8,247,466, which are incorporated by reference herein in their entirety. In some embodiments, the porosity of the pad 178 can be selected such that the pad will release a uniform amount of liquid when pressed against a surface onto which the liquid is to be dispensed. A wide variety of pad shapes are known. Generally, the shape of the pad 178 can be oval, square, or rectangular, and the thickness can be uniform (or substantially uniform).

[0013] It should be appreciated that while the present disclosure has been described with respect to a particular applicator 100, the articles and methods of the present disclosure can be used with any applicator in which a container holding a disinfecting solution is present in conjunction with the applicator.

[0014] In some embodiments, the disinfecting solution container can be a self-contained structure formed from a material suitable for containing a disinfecting solution. In some embodiments, the container can be made from a frangible material such that, upon application of sufficient force, the container breaks. For example, the material can include plastic or glass. For purposes of the present disclosure, the terms "container" and "ampoule" are used interchangeably. Generally, one or more walls of the container can have a thickness sufficient to withstand sterilization processes, shipping, and storage. In embodiments in which the container is frangible, the material and thickness can also be sufficient to allow the container to break upon application of localized pressure. The range of thicknesses can vary depending on the size of the container. In some embodiments, the wall thickness of a glass or plastic container can be 0.15 mm to 0.45 mm. In some embodiments, the container can be formed from a non-frangible material, such as a metal (steel, aluminum, etc.).

[0015] In some embodiments, the container can hold a sufficient volume of the disinfecting solution to be applied to a desired surface and have an antimicrobial effect on the desired surface. In some embodiments, the desired surface is the skin of a patient. In this regard, in some embodiments, the container can have an internal volume of between 0.5 mL and 3 mL, or between 4 mL and 7 mL, or between 10 mL and 26 mL.

[0016] In some embodiments, the disinfecting solution of the present disclosure can include water, an alcohol, and a disinfectant.

[0017] In some embodiments, suitable disinfectants include bis-(dihydropyridinyl)-decane (or derivatives thereof) (e.g., salts of octenidine) and / or biguanides (or derivatives thereof) (e.g., salts of chlorhexidine). Examples of biguanides / biguanide derivatives other than salts of chlorhexidine include alexidine, salts of alexidine, polyhexamide, salts of polyhexamide, polyaminopropyl biguanide, salts of polyaminopropyl biguanide, and other alkyl biguanides. In some embodiments, the disinfectant includes a salt of octenidine, such as octenidine dihydrochloride, or a salt of chlorhexidine, such as chlorhexidine gluconate (CHG). In some embodiments, the disinfectant includes CHG.

[0018] In some embodiments, the disinfecting solution can include one or more alcohols. In some embodiments, the one or more alcohols can include a secondary alcohol having a single hydroxyl group. In some embodiments, the one or more alcohols can include isopropyl alcohol (or consist essentially of isopropyl alcohol).

[0019] In some embodiments, the disinfectant (e.g., CHG) can be present in the disinfecting solution in an amount of between 0.5% w / v and 5% w / v, between 1% w / v and 3.5% w / v, or between 1.5% w / v and about 2.5% w / v. As used herein, % w / v is a measure of weight in grams of the (disinfectant) per 100 mL of the disinfecting solution.

[0020] In some embodiments, the one or more alcohols (e.g., isopropyl alcohol) can be present in the disinfecting solution in an amount of between 50% v / v and 80% v / v, between 55% v / v and 80% v / v, or between 65% v / v and 75% v / v. As used herein, % v / v is a measure of volume in mL of a particular component per 100 mL of the disinfecting solution

[0021] In some embodiments, water can be present in the disinfecting solution in an amount of between 15% v / v and 45% v / v, or between 20% v / v and 30% v / v.

[0022] In some embodiments, the formulation can contain various concentrations of other additives, e.g., one or more dyes for coloring the formulation in an amount sufficient to provide a desired color (so that when present on human skin, the color is readily perceived by the human eye). Alternatively or additionally, the formulation can include one or more polymers (e.g., to facilitate film formation upon drying).

[0023] In some embodiments, the present disclosure further relates to methods of sterilizing the above-described disinfectant solution container (including the inner and outer walls of the container) and the disinfectant solution contained within the disinfectant solution container.

[0024] In some embodiments, the method can include adding the disinfectant solution to the container, and then sealing the container to form a sealed container. For example, in some embodiments, the container can be hermetically sealed. In some embodiments, adding the disinfectant solution can include adding the disinfectant solution such that there is a first region within the interior volume of the container that holds the disinfectant solution, and a second region within the interior volume of the container that can be used to contain air and / or disinfectant solution vapor. For example, the disinfectant solution can be added to the container such that the first region is between 30% and 99% by volume, between 30% and 95% by volume, between 30% and 90% by volume, or between 50% and 70% by volume, based on the total interior volume of the container, and the second region makes up the remainder of the total interior volume of the container.

[0025] In some embodiments, the methods of the present disclosure can further include subjecting the sealed container to heat at a temperature and for a heating time sufficient to sterilize the disinfectant solution as well as the outer and inner walls of the sealed container. In this regard, as noted above, it was discovered that for certain low temperature sterilization techniques, while sufficient to sterilize the disinfectant solution and the outer walls of the container, the inner walls of the sealed container were not sufficiently sterilized. More specifically, it was discovered that the adsorption of disinfectant molecules onto the surface of the indicator bacterial spores was necessary for the disinfectant to exert a sporicidal effect at these relatively low temperatures, and that the vapor generated from the disinfectant (e.g., CHG / IPA vapor) did not have a sufficient sporicidal effect at such temperatures.

[0026] However, it was further discovered that if the inner walls of the sealed container are immediately wetted with the disinfectant solution prior to (e.g., not more than 120 minutes prior, not more than 60 minutes prior, not more than 30 minutes prior, or not more than 10 minutes prior) and / or during the heating of the sealed container, sufficient sterilization of the inner walls of the sealed container (along with the disinfectant solution and outer walls) can be achieved even using such low temperature sterilization techniques. As noted, it is believed that this approach ensures that disinfectant molecules are adsorbed onto the surface of the microbial / biological load within the container in order to exert a sterilizing effect of the disinfectant.

[0027] In some embodiments, wetting of the interior walls of the sealed container, and particularly the interior walls of the second region of the interior of the container, can be performed using any conventional mechanism, including, for example, agitation of the container, inversion of the container, or a combination thereof. In some embodiments, the method can include wetting all or substantially all of the walls of the interior of the sealed container immediately prior to and / or during heating of the sealed container.

[0028] In some embodiments, subjecting the sealed container to heat can further include bringing the sealed container to a particular temperature (or temperature range) and maintaining that temperature (or temperature range) for a particular amount of time (such that the solution is sufficiently sterilized, while at the same time maintaining sufficient antimicrobial efficacy as a disinfectant). In some embodiments, the disinfecting solution within the sealed container can be brought to a temperature of 50 °C to 80 °C, 55 °C to 75 °C, or 60 °C to 70 °C (referred to herein as a "predetermined sterilization temperature").

[0029] As used herein, the term "predetermined sterilization time" refers to the duration of time that the solution is at the predetermined sterilization temperature. In this manner, the "sterilization time" does not include the time taken for the solution to reach the sterilization temperature (i.e., does not include the "warm-up" time), and also does not include the time taken for the solution to return to the temperature at which the solution was prior to heating (i.e., does not include the "cool-down" time). In some embodiments, the predetermined sterilization time can be 50 minutes to 500 minutes, 100 minutes to 400 minutes, or 150 minutes to 320 minutes.

[0030] In some embodiments, after the predetermined sterilization time has ended, the disinfecting solution can be cooled. For example, about 10 minutes to about 40 minutes can be required to cool the disinfecting solution after the predetermined sterilization time.

[0031] In some embodiments, subjecting the sealed container to heat can be performed using conventional heating equipment, such as an oven (e.g., dry heat with or without convection), a water bath, an oil bath, an autoclave, etc. As noted above, it has been found that the sterilization method of the present disclosure produces a sterilized solution and a container (including the interior walls of the container) that contains the sterilized solution with at least a 10 -6 log SAL (for a given test microorganism spore, when subjected to sterilization conditions sufficient to reduce a population of 1 x 10 6 test microorganisms by about 6 logs (i.e., to about zero, as measured by lack of outgrowth of the test microorganism). The spores that are resistant to the sterilization process can include, for example, spores from the genera Bacillus ( Bacillus ), Geobacillus ( Geobacillus ), Clostridium ( Clostridium ), Neurospora ( Neurospora ), and Candida (Candida ) or a combination thereof. For purposes of the present disclosure, SAL can be determined according to ANSI / AAMI / ISO 11138-1: Sterilization of healthcare products - Biological indicators - First Section: General Requirements Sterilization of health care products - Biological indicators - Part 1: General requirements ) to determine.

[0032] In some embodiments, following the above-described sterilization process, the sterilized container and the sterilized solution contained therein can be implemented in various applications. In some embodiments, the sterilized container and the sterilized solution contained therein can be placed into a liquid applicator of the type described above.

[0033] In some embodiments, the present disclosure can further relate to methods of preparing a site (e.g., a surgical site) on the skin of a mammal using the above-described liquid applicator. The method can include introducing a sterilizing solution into a foam pad (e.g., such that the foam pad is saturated or nearly saturated with the solution), and then contacting (e.g., scrubbing) the site with the foam pad for a period of at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, or at least 10 minutes.

[0034] The following examples further illustrate purposes and advantages of the present disclosure, but the particular materials and amounts thereof, as well as other conditions and details, recited in these examples should not be construed to unduly limit this disclosure.

[0035] Examples

[0036] Sporicidal activity of 2% w / v chlorhexidine gluconate / 70% v / v isopropyl alcohol solution: This example determines whether alcohol vapor generated within an ampule during the heating process is active against a selected biological indicator (BI). A mock-up device designed to mimic an ampule is used for this purpose. The mock-up device consists of a top, a bottom, and a clamp that holds the top and bottom together during the experiment. The bottom is a 10 mL short necked round bottom borosilicate ampule with notches to enhance mixing. The top is a hollow glass stopper sized to fit the 24 / 40 joint of the ampule. The clamp is a standard plastic Keck clamp. The lower half of the mock-up ampule is filled with the formulation of interest, while the upper half of the ampule is inoculated with the BI. This is done by pipetting an alcohol solution of spores onto the inner surface of the upper half of the ampule and allowing the alcohol solution to dry for at least 24 hours prior to the experiment. The top and bottom are fastened together, sealed with vacuum grease and a paraffin film, and fastened with the clamp. The assembled device is then exposed to heat for a period of time. Spores are recovered from the solution as well as the upper half of the mock-up ampule. Spores are recovered from the top of the ampule by adding a volume of neutralizer and glass beads, followed by vigorous vortexing.

[0037] Prior to determining process parameters or BIs, a 2% w / v chlorhexidine gluconate / 70% v / v isopropyl alcohol solution (2% CHG / 70% IPA) and Bacillus subtilis were used to investigate headspace sporicidal activity. Approximately 10 7 spores were placed in the upper half of the ampule and allowed to dry for 24 hours. The bottom of the ampule was filled with a 2% CHG / 70% IPA solution, 70% IPA only, or left empty, and the ampule was assembled and sealed. One set of ampules was briefly inverted, exposing the spores directly to the representative formulation, and placed upright back before heating. The ampules were then placed in a 65°C water bath for 24 hours.

[0038] Table 2. Top space sporicidal activity of 2% CHG / 70% IPA solution at 65°C .

[0039]

[0040] The results show that the vapor from both 2% CHG / 70% IPA and 70% IPA (2% CHG / 70% IPA HS and 70% IPA HS, respectively) did not exhibit sporicidal activity (Table 2). In both 70% IPA and 2% CHG / 70% IPA ampoules, the headspace (HS) spore population was slightly reduced after 24 hours; however, this can be explained as spore loss into the solution during heating. Condensation formed inside the ampoule and dripped into the solution below, most likely carrying spores with it. These spores were observed in the IPA solution after 24 hours, but not in the 2% CHG / 70% IPA solution, as the heated 2% CHG / 70% IPA solution had sporicidal activity. The presence of spores in the 70% IPA solution at 0 hours most likely resulted from spores falling into the solution during ampoule assembly, as the ampoule containing only 70% IPA was not inverted.

[0041] When the ampoule was briefly inverted prior to heating, this significantly increased the sporicidal activity of the top of the ampoule. Some of the headspace spores were lost into the solution due to the inversion (2% CHG / 70% IPA inverted solution, 0 hours), but were not immediately killed due to the lack of heating. Any spores lost into the solution during inversion were killed during heating, as no spores were recovered from the 2% CHG / 70% IPA solution after 24 hours (2% CHG / 70% IPA inverted solution, 24 hours). When the spores in the headspace of the ampoule were briefly exposed to the solution, a 5.8 log reduction was observed. This indicates that brief exposure to a CHG / IPA solution prior to heating is sufficient to kill spores that can be on the inner surface of the ampoule, above the liquid level. Therefore, it is important to incorporate an ampoule inversion step prior to heating the ampoule. 10 of the ampoule. Therefore, it is important to incorporate an ampoule inversion step prior to heating the ampoule.

[0042] After the process parameters and BI were determined, additional experiments were performed to evaluate a representative 2% CHG / 70% IPA formulation. Approximately 10 6 spores of Bacillus subtilis were placed in the upper half of a mock ampoule and allowed to dry for 24 hours. The bottom of the ampoule was filled with the formulation, and the ampoule was assembled and sealed. In this case, no IPA controls or non-inverted samples were evaluated. The ampoule was briefly inverted and then placed in a 65 °C water bath for 2 hours.

[0043] Table 3. Sporicidal activity of representative 2% CHG / 70% IPA formulations against B. subtilis top space spores D-value determination for B. subtilis spores .

[0044]

[0045] The results show that heating alone did not have sporicidal activity, and brief exposure to the formulation followed by heating resulted in a significant decrease in viable spores (Table 3). As observed in previous experiments, inversion of the ampoule resulted in loss of spores into solution. This alone resulted in a 1.4 log reduction of spores in the headspace relative to the control. Some of these spores were recovered from the solution at 0 hours. Brief exposure of the spores to the solution followed by heating resulted in a 5.3 log reduction relative to the control, and a 4.2 log reduction relative to the headspace recovery at 0 hours. The estimated bioburden of the ampoule was less than 3 colony forming units. The inversion step prior to heating was sufficient to eliminate this bioburden.

[0046] Figure 3 : The decimal reduction time (D-value) of B. subtilis spores in 2% w / v chlorhexidine gluconate / 70% v / v isopropyl alcohol (2% CHG / 70% IPA) at 65°C was determined by inoculating 11 mL of 65°C 2% CHG / 70% IPA with 0.11 mL of a spore suspension (1.81 x 10 10 CFU / mL) to form a spore-CHG mixture. At regular intervals (every 45 minutes), a 0.1 mL aliquot of the spore-CHG mixture was transferred to 9.9 mL of Dey-Engley neutralizing broth (D / E broth) to form a neutralized spore suspension, while the remaining portion of the spore-CHG mixture was maintained at 65°C. Ten-fold serial dilutions of the neutralized spore suspension were used to plate on tryptic soy agar (TSA) plates, which were then incubated at 35°C for 48 hours. Bacterial colonies on the TSA plates were counted to determine the number of surviving spores at each sampling time point (Table 4). A log 10 (number of surviving spores) - (exposure time) curve was made Table 4. Experiment to determine D-value for B. subtilis spores in 2% CHG / 70% IPA at 65°C , and the inverse of the slope of the regression line in this curve was determined to be 49.5 minutes, which is the D-value of B. subtilis spores in 2% CHG / 70% IPA at 65°C.

[0047] Low heat sterilization process .

[0048]

[0049] ND: not determined.

[0050] Sterility validation : A forced air oven was used as a low heat batch sterilizer (LHBS). The LHBS was used for sterilization of the interior surfaces of the hermetically sealed glass ampoules held in trays with temperature monitor assemblies (TMAs) (for monitoring the temperature of the 2% CHG / 70% IPA solution throughout the process) and process challenge devices (PCDs) (for sterility validation). Each TMA utilized a wireless temperature data logger sensor with the probe end immersed in the 2% CHG / 70% IPA content (26 mL) of the glass ampoule, the cap opening of which was sealed with silicone sealant to minimize the amount of 2% CHG / 70% IPA lost to evaporation during processing. Each PCD consisted of a spore disk (stainless steel disk, 6 mm in diameter, with >1.0 x 10 6 CFU of Bacillus subtilis spores) in a 26 mL 2% CHG / 70% IPA immersed in a threaded clear glass vial (28 mm x 108 mm) that was capped with a PTFE lined cap. The thickness of the PCD vial was greater than the thickness of the glass ampoule and the capacity of the PCD vial was similar to the capacity of the glass vial; therefore, the use of the PCD was valid. The TMAs and PCDs were assigned to appropriate locations within the trays holding the glass ampoules. After inverting the samples once to allow the 2% CHG / 70% IPA to wet the interior surfaces of the glass ampoules and PCD vials, the trays were loaded into the LHBS. The sterilization process began with a preheat phase in which the temperature of the 2% CHG / 70% IPA in the TMAs was raised from room temperature to 65 °C, then the temperature of the chamber of the LHBS was maintained at 65 °C for a 300 minute hold time (which is 6 times the D-value of the Bacillus subtilis spores in 2% CHG / 70% IPA at 65 °C; 6 x 49.5 minutes = 297 minutes).

[0051] ​ : After the glass ampoules and PCDs were cooled to room temperature, the PCDs were removed for analysis.

[0052] For each PCD, the 2% CHG / 70% IPA contents of the glass vial were transferred to be filtered through a PVDF membrane filter (0.45 mm pore size; 47 mm diameter) to recover dislodged spores or microorganisms, if any, from the inside surface of the Petri dish while the Petri dish remained in the glass vial. Two wash procedures were performed with 70% IPA on the Petri dish, the interior of the glass vial, and the PVDF membrane filter, each by washing the Petri dish and the interior of the glass vial with 26 mL of 70% IPA and then transferring the 70% IPA wash from the glass vial to be filtered through the PVDF membrane filter while the Petri dish remained in the glass vial. Then, two procedures were performed to neutralize residual CHG on the Petri dish, the interior of the glass vial, and the PVDF membrane filter with D / E broth in the same manner as the wash procedures with 70% IPA. Subsequently, the Petri dish, the interior of the glass vial, and the PVDF membrane filter were washed twice with tryptone water in the same manner as the wash procedures with 70% IPA. The PVDF membrane filter was aseptically transferred to a TSA plate, and 35 mL of Letheen broth (LB) was added to the clear glass vial with the Petri dish. The PVDF filter-TSA plate assembly and the glass vial with the Petri dish in LB were incubated at 35 °C for 7 days.

[0053] All removed PCDs were processed for analysis. No bacterial growth halo was observed on the PVDF filter-TSA plate assembly or from the Petri dish in LB after incubation at 35 °C for 7 days, indicating that the low heat sterilization process was effective for sterilization of the 2% CHG / 70% IPA contents and the interior surface of the hermetically sealed glass ampule and achieved a 10 -6 log sterilization assurance level. Example 2: Sterilization of PCDs with 2% CHG / 70% IPA Contents

Claims

1. A method for sterilizing a disinfectant solution and the interior of a sealed container therein containing a disinfectant composition, the method comprising: A disinfectant solution is added to the container, and then the container is sealed to form a sealed container with an inner wall that defines the internal volume. Wet the inner wall of the container with the disinfectant solution; and The sealed container is subjected to a predetermined sterilization time of 50 to 80°C for 50 to 500 minutes.

2. The method of claim 1, wherein adding the disinfectant solution comprises adding the disinfectant solution such that there exists a first region within the internal volume for retaining the disinfectant solution, and a second region within the internal volume for containing air and / or disinfectant solution vapor.

3. The method of claim 2, wherein the first region is between 30% and 99% of the total internal volume of the container, and the second region constitutes the remainder of the total internal volume, and wherein wetting the inner wall includes wetting the inner wall of the second region.

4. The method according to any one of claims 1 to 3, wherein wetting the inner wall comprises agitating or inverting the sealed container.

5. The method of claim 4, wherein the sealed container is agitated or inverted for no more than 60 minutes prior to the step of subjecting the sealed container to heating.

6. The method according to any one of claims 1 to 5, wherein the sealed container is a glass ampoule.

7. The method according to any one of claims 1 to 6, wherein the disinfectant composition comprises a disinfectant, an alcohol, and water.

8. The method of claim 7, wherein the disinfectant is present in the disinfectant solution in an amount between 0.5% w / v and 5% w / v.

9. The method according to any one of claims 7 to 8, wherein the disinfectant comprises chlorhexidine gluconate or ostinidine dihydrochloride.

10. The method according to any one of claims 1 to 8, wherein the disinfectant comprises chlorhexidine gluconate.

11. The method according to any one of claims 7 to 10, wherein the alcohol is present in the disinfectant solution in an amount between 50% v / v and 80% v / v.

12. The method according to any one of claims 7 to 11, wherein the alcohol comprises ethanol, isopropanol, or n-propanol.

13. The method according to any one of claims 7 to 12, wherein the alcohol comprises isopropanol.

14. The method according to any one of claims 7 to 13, wherein the water is present in the disinfectant solution in an amount between 15% v / v and 45% v / v.

15. A method for manufacturing a liquid applicator, the method comprising: Provide an applicator, the applicator comprising: A hollow body, the hollow body including a wall defining an internal cavity having a closed end and an open end; and Absorbent pad, the absorbent pad being connected to the open end of the hollow body; and The method according to any one of claims 1 to 14 sterilizes the disinfectant solution and the interior of the sealed container therein containing the disinfectant composition; The sealed container is inserted into the internal chamber to form the liquid applicator.

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