Methods of sterilization

Abstract

A method of sterilizing an antiseptic solution and an interior of a sealed container in which the antiseptic composition is contained. The method includes adding an antiseptic solution into the container and then sealing the container to form a sealed container having interior walls that define an interior volume. The method further includes wetting the interior walls of the container with the antiseptic solution. The method further includes subjecting the sealed container to heat at a predetermined sterilization temperature of from 50 to 80 degrees Celsius for a predetermined sterilization time of from 50 to 500 minutes.

Description

[0001] METHODS OF STERILIZATION

[0002] BACKGROUND

[0003] Methods of sterilizing antiseptic compositions have been described, for example, in U.S. Patent 9,724,437 and U.S. Patent 9,895,455.

[0004] BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1 illustrates a side view of a liquid applicator according to some embodiments of the present disclosure.

[0006] FIG. 2 illustrates a cross-section of the liquid applicator of FIG. 1.

[0007] FIG. 3 is a plot of logio(number of surviving spores) vs. (exposure time) used to determine the D-value of Bacillus subtilis spores in 2%CHG / 70%IPA at 65°C.

[0008] DETAILED DESCRIPTION

[0009] It is a standard practice in the industrialized world to disinfect the skin prior to any invasive procedure such as surgery, catheterization, or needle puncture to reduce the risk of infection. These products are often referred to as skin preps or simply “preps.” Antiseptic preparation of patient's skin for such procedures conventionally includes a 30 second- 10 minute scrubbing of the affected area with an antiseptic soap solution. These solutions are often applied with foam sponge tipped applicators. The foam sponges are often saturated by soaking them in open pans of solution or with solution from an antiseptic solution container housed in a hollow handle fluidically coupled to the foam sponge.

[0010] As mentioned, in many cases, the antiseptic solution is held in a hollow cavity within an applicator in a container (e.g., frangible ampoule). In the manufacturing and use of antiseptic formulations and associated containers, there are various challenges related to providing and maintaining sterility. For example, certain high temperature sterilization processes (e.g., pressurized steam at a temperature of greater than 120°C, for a period of time sufficient for the steam to cause sterilization of the object (e.g., 3-15 minutes)), while effective in sterilizing the antiseptic solution and interior walls of the container, can deactivate the sterilizing solution or compromise the integrity of the primary packaging. As another example, certain low temperature sterilization processes (e.g., dry heat at temperature less than 80°C for 60 to 600 minutes), while effective in sterilizing the solution without appreciable deactivation or packaging issues, have been discovered to not adequately sterilize the interior walls of the container. For example, it was discovered that natural bioburdens present on the internal surfaces of the containers, within the headspace or introduced during the manufacturing process, may be high in number and resistant to the sterilization process. Without additional processes, these native microorganisms are not adequately sterilized and can present undue risk to patients.

[0011] Consequently, sterilization techniques that fully sterilize the antiseptic solution and interior walls of the antiseptic solution container, and do not appreciably deactivate the antiseptic solution, are desirable.

[0012] The term "comprising" and variations thereof (e.g., comprises, includes, etc.) do not have a limiting meaning where these terms appear in the description and claims.

[0013] As used herein, "a," "an," "the," "at least one," and "one or more" are used interchangeably, unless the context clearly dictates otherwise.

[0014] In some embodiments, the present disclosure is directed to methods for sterilizing antiseptic solutions (e.g., chlorhexidine based antiseptic solutions) and containers (e.g., bottles or ampoules) used to hold such solution FIGS. 1-2 illustrate a liquid applicator 100 in accordance with some embodiments of the present disclosure. As shown, liquid applicator 100 may include an elongate hollow body 110 (which may serve as a handle for the user) that includes a wall 160. Wall 160 may define chamber 170 having a closed end 118 and an open end 174. An antiseptic solution containing container 175, or ampoule, may be disposed within the chamber 170.

[0015] Closed end 172 may 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, which may be press-fit, screwed, or otherwise attached, may seal the chamber forming closed end 172. The open end 174 may include one or more orifices that permit fluid to flow out of the chamber 170. A pad 178 (e.g., an absorbent foam pad) may be fluidically coupled to the open end 174 such that fluid flowing through the orifice directly or indirectly contacts the pad 178. The pad 178 may be coupled to the hollow body (directly or indirectly) by a conventional fastening mechanism (e.g., adhesives or ultrasonic welding).

[0016] In some embodiments, the pad 178 can be prepared from a variety of commercially available materials having a wide range of compression set ratios (i.e., density) and porosities. In some embodiments, the pad 178 may include (or be formed of) a polyurethane foam (e.g., an open- celled polyester or polyether polyurethane foam). In some embodiments, the foam may be of the material type described in U.S. Pats. 6,841,586 or 8,247,466, which are herein incorporated by reference in their entirety. In some embodiments, the porosity of the pad 178 may be selected such that the pad will release a uniform amount of liquid when it pressed against the surface upon which the liquid is to be dispensed. A wide variety of pad shapes are known. Generally, pad 178 may be oval, square, or rectangular in shape and may be uniform (or substantially uniform) in thickness.

[0017] It is to 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 may be employed with any applicator in which an antiseptic solution containing container is present in connection with the applicator.

[0018] In some embodiments, the antiseptic solution container may be a self-contained structure, formed of a material suitable for containing the antiseptic solution. In some embodiments, the container may be made of a frangible material such that upon application of sufficient force, the container fractures. For example, the material may comprise plastic or glass. For purposes of the present disclosure, the terms “container” and “ampoule” are used interchangeably. Generally, the wall or walls of the container may have a thickness sufficient to withstand the sterilization process, transport, and storage. In embodiments in which the container is frangible, the material and thickness may also be sufficient to allow the container to be fractured upon the application of localized pressure. The thickness range may vary depending on the container size. In some embodiments the wall thickness of the glass or plastic containers may be from 0.15 mm to 0.45 mm. In some embodiments, the container may be formed of a non-frangible material, such as a metal (steel, aluminum, etc.).

[0019] In some embodiments, the container may contain antiseptic solution of a sufficient volume to be applied to a desired surface and have an antimicrobial effect on the desired surface. In some embodiments, the desired surface is a patient's skin. In this regard, in some embodiments, the container may have an inner volume of between 0.5 and 3 mL or between 4 and 7 mL or between 10 and 26 mL.

[0020] In some embodiments, the antiseptic solutions of the present disclosure may include water, an alcohol, and an antiseptic.

[0021] In some embodiments, suitable antiseptics include bis-(dihydropyridinyl)-decane (or derivatives thereof) (e.g. octenidine salts) and / or biguanides (or derivatives thereof) (e.g.. chlorhexidine salts). Examples of bsguanides / bigua de derivatives other than ehlorhexidine salts include alexidine, alexidine salts, polyhexamide, polyhexamide salts, polyaminopropyl biguanide, poly aminopropyl biguanide salts, and other alkyl biguanides. In some embodiments, the antiseptic includes an octenidine salts, such as octenidine dihydrochloride or a ehlorhexidine salts, such as ehlorhexidine gluconate (CHG). In some embodiments, the antiseptic includes CHG.

[0022] In some implementations, the antiseptic solutions may include one or more alcohols. In some embodiments, the one or more alcohols may include secondary alcohols having a single hydroxyl group. In some embodiments, the one or more alcohols may include (or consist essentially of) isopropyl alcohol.

[0023] In some embodiments, antiseptic (e.g., CHG) may be present in the antiseptic 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 to about 2.5% w / v. As used herein, % w / v is the measure of weight in grams (of antiseptic) per 100 mL of the antiseptic solution.

[0024] In some embodiments, the one or more alcohols (e.g., isopropyl alcohol) may be present in the antiseptic 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 the measure of volume in mL of a particular component per lOOmL of the antiseptic solution

[0025] In some embodiments, water may be present in the antiseptic solution in an amount of between 15% v / v and 45% v / v or between 20% v / v and 30 % v / v.

[0026] In some embodiments, the formulation may contain other additives in various concentrations, for example, one or more dyes for tinting the formulation in an amount sufficient to provide a desired color (such that it is readily perceptible by the human eye when present on human skin). Alternatively, or additionally, the formulation may include one or more polymers (e.g., to facilitate thin film formation upon drying).

[0027] In some embodiments, the present disclosure is further directed to methods of sterilizing the antiseptic solution containers (including interior and exterior walls of the container) and antiseptic solutions contained therein discussed above.

[0028] In some embodiments, the methods may include adding the antiseptic solution into the container and then sealing the container to form a sealed container. For example, in some embodiments, the container may be hermetically sealed. In some embodiments, adding the antiseptic solution may including adding the antiseptic solution such that there is a first region within the interior volume of the container holding the antiseptic solution and a second region within the interior volume of the container that is available to contain air and / or antiseptic solution vapor. For example, the antiseptic solution may be added to the container such that the first region is between 30 and 99 vol. %, between 30 and 95 vol. %, between 30 and 90 vol. %, or between 50 and 70 vol. %, based on the total interior volume of the container, and the second region makes up the balance of the total interior volume of the container.

[0029] In some embodiments, the methods of the present disclosure may further include subjecting the sealed container to heat at a temperature and heating time sufficient to sterilize the antiseptic solution as well as the exterior and interior walls of the sealed container. In this regard, as mentioned above, it was discovered that for certain low temperature sterilization techniques, while adequate in sterilizing the antiseptic solution and exterior walls of the container, the interior walls of the sealed container are not adequately sterilized. More specifically, it was discovered that adsorption of antiseptic molecules onto the surfaces of indicator bacterial spores is a requisite for the antiseptic to exert sporicidal effect at these relatively low temperatures, and that the vapors generated from that antiseptic (e.g., CHG / IPA vapors) do not have sufficient sporicidal effect at such temperatures.

[0030] It was further discovered, however, that adequate sterilization of the interior walls of the sealed container may be achieved (along with the antiseptic solution and exterior walls), even with such low temperature sterilization techniques, if the interior walls of the sealed container are wetted with the antiseptic solution immediately prior to (for example, no more than 120 minutes before, no more than 60 minutes before, no more than 30 minutes before, or no more than 10 minutes before) and / or during heating of the sealed container. As mentioned, it is believed that this approach ensures that antiseptic molecules adsorb on the surfaces of microorganisms / bioburdens inside the containers in order to exert a sterilization effect of the antiseptic.

[0031] 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, may be carried out using any conventional mechanism including, for example, agitation of the container, inversion of the container, or a combination thereof. In some embodiments, the methods may 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.

[0032] In some embodiments, subjecting the sealed containers to heat may 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 maintaining sufficient antimicrobial efficacy as an antiseptic). In some embodiments, the antiseptic solution within the sealed containers may be brought to a temperature (referred to herein as the “predetermined sterilization temperature”) from 50° C to 80° C., from 55° C to 75° C, or from 60 C to 70° C.

[0033] As used herein, the term “predetermined sterilization time” refers to the duration of time at which the solution is at the predetermined sterilization temperature. In this manner, the “sterilization time” does not include the time it takes for a solution to reach the sterilization temperature (i.e., does not include “ramp up” time) and also does not include the time it takes for the solution to return to the temperature the solution was at prior to the heating (i.e., does not include “cool down” time). In some embodiments, the predetermined sterilization time may be from 50 to 500 minutes, from 100 to 400 minutes, or from 150 to 320 minutes.

[0034] In some embodiments, after the predetermined sterilization time ends, the antiseptic solution may be cooled. For example, it may take about 10 to about 40 minutes to cool the antiseptic solution following the predetermined sterilization time. In some embodiments, subjecting the sealed containers to heat may be carried out using conventional heating apparatus such as an oven (e.g., dry heat with or without convection), water bath, oil bath, autoclave, or the like. As mentioned, the sterilization methods of the present disclosure have been found to produce sterilized solutions and containers that contain the sterilized solutions (including the interior walls of the containers). In this regard, the sterilization methods of the present disclosure may have a sterility assurance level (SAL) of at least 10-6 (for a given test microorganism spore, when subjected to sterilization conditions which would be sufficient to decrease a population of 1x10° test microorganisms by about 6 logs (i.e,, to a population of about zero as measured by lack of outgrowth of the test microorganisms)), Sterilization process resistant spores may include at least one of species of microorganisms from the genera Bacillus, Geobacillus, Clostridium, Neurospora, and Candida, for example, or combinations thereof. For purposes of the present disclosure, SAL may be determined in accordance with ANSI / AAMI / ISO 11138-1: Sterilization of health care products - Biological indicators - Part 1: General requirements.

[0035] In some embodiment, the sterilized containers and antiseptic solutions contained therein, after undertaking the above-described sterilization process, may be implemented in a variety of applications. In some, embodiments, the sterilized containers and antiseptic solutions contained therein may be placed into a liquid applicator of the type described above.

[0036] In some embodiments, the present disclosure may be further directed to methods of using the above-described liquid applicators to prepare a site on the skin of a mammal (e.g., a surgical site). The method may include introducing the antiseptic solution into the 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.

[0037] Objects and advantages of this disclosure are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this disclosure. EXAMPLES

[0038] Sporicidal activity of a 2% w / v chlorhexidine gluconate / 70% v / v isopropyl alcohol solution:

[0039] This Example determined whether the alcohol vapor generated within the ampule during the heating process is active against the selected Biological Indicator (BI). A design device designed to simulate an ampule was used for this purpose. This design device consisted of a top, a bottom, and a clamp to hold the top and bottom together during experimentation. The bottom was a 10 mL short-necked round-bottom borosilicate ampule with indentations to enhance mixing. The top was a hollow glass stopper sized to fit the 24 / 40 joint of the ampule. The clamp was a standard plastic Keck clamp. The bottom half of the simulated ampule was filled with the formulation of interest, while the top half of the ampule was inoculated with the BI. This was done by pipetting an alcoholic solution of spores onto the inner surface of the top half of the ampoule, and allowing it to dry for at least 24 hours prior to experimentation. The top and bottom were secured together, sealed with vacuum grease and parafilm, and secured with the clamp. The assembled device was then exposed to heat for a period of time. Spores were recovered from the solution, as well as the top half of the simulated ampule. Spores were recovered from the top of the ampule by adding a volume of neutralizer and glass beads, followed by vigorous vortexing.

[0040] Prior to identification of process parameters or a BI, head space sporicidal activity was studied using a 2% w / v chlorhexidine gluconate / 70% v / v isopropyl alcohol solution (2%CHG / 70%IPA) and B. subtilis. Approximately 107spores were placed into the top half of the ampule and allowed to dry for 24 hours. The bottoms of the ampules were filled with 2%CHG / 70%IPA solution, 70% IPA alone, or nothing, and the ampules were assembled and sealed. One set of ampules was briefly inverted, exposing the spores directly to the representative formulation, and set back upright before heating. The ampules were then placed into a 65 °C water bath for 24 hours.

[0041] Table 2. Head Space Sporicidal Activity of 2% CHG / 70%IPA Solution at 65°C.

[0042] The results show that the vapor from 2% CHG / 70%IPA and 70%IPA (2%CHG / 70% IPA HS and 70%IPA HS, respectively) exhibited no sporicidal activity (Table 2). There was a slight decrease in the head space (HS) spore populations after 24 hours in both the 70% IPA and 2% CHG / 70%IPA ampules; however, this can be accounted for as loss of spores into the solution during heating. Condensation formed on the inside of the ampule and dripped into the solution below, most likely carrying spores with it. These spores are observed in the IPA solution after 24 hours, but not in the 2% CHG / 70%IPA solution, because the heated 2% CHG / 70%IPA solution has sporicidal activity. The presence of spores in the 70%IPA solution at 0 hr is most likely the result of spores shedding into the solution during ampule assembly, as the ampules containing 70%IPA alone were not inverted.

[0043] When the ampules were inverted briefly prior to heating, this significantly increased sporicidal activity in the top of the ampule. Some of the head space spores were lost to the solution as a result of inversion (2% CHG / 70%IPA Inverted Soln, 0 hr), but were not killed immediately because of the absence of heat. Any spores lost to 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 Soln, 24 hr). A 5.8 logic reduction was observed when spores in the head space of the ampule were briefly exposed to solution. This indicates that a brief exposure to CHG / IPA solution prior to heating is sufficient to kill spores that may be on the interior surface of the ampule, above the level of liquid. As a result, it is important to incorporate an ampule inversion step prior to heating the ampules.

[0044] After identification of the process parameters and BI, an additional experiment was performed to evaluate a representative 2% CHG / 70%IPA formulation. Approximately 106B. subtilis spores were placed into the top half of the simulated ampule and allowed to dry for 24 hours. The bottom of the ampule was filled with the formulation, and the ampule was assembled and sealed. In this case, no IPA control or non-inverted samples were evaluated. The ampules were briefly inverted and then placed into a 65°C water bath for 2 hours.

[0045] Table 3. Sporicidal Activity of representative 2% CHG / 70%IPA formulation against B. subtilis Head Space Spores.

[0046] The results show that heat alone does not have sporicidal activity, and that brief exposure to the formulation followed by heating results in a significant reduction in viable spores (Table 3). Inversion of the ampules results in a loss of spores into the solution, as was observed in the previous experiment. This alone resulted in a 1.4 log reduction of spores in the head space, relative to the control. Some of these spores were recovered from the solution at 0 hr. Brief exposure of 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 head space recovery at 0 hours. The estimated bioburden of the ampules is less than 3 colony forming units. An inversion step prior to heating was sufficient to eliminate this bioburden.

[0047] D-value determination for Bacillus subtilis spores:

[0048] The decimal reduction time (D-value) of Bacillus 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 IO10CFU / mL) to form a spore-CHG mixture. At regular intervals (every 45 minutes), 0.1 mL aliquots of the spore - CHG mixture were transferred to 9.9 mL of Dey-Engley Neutralizing Broth (D / E Broth) to form neutralized spore suspensions while the temperature of the rest of the spore-CHG mixture was maintained at 65°C. Ten-fold serial dilutions of the neutralized spore suspensions were used for plating on Tryptic Soy Agar (TSA) plates which were then incubated for 48 hours at 35°C. The bacterial colonies on the TSA plates were counted to determine the number of surviving spores at each sampling time point (Table 4). The logio(number of surviving spores)-(exposure time) plot (Fig. 3) was prepared and the reciprocal of the slope of the regression line in the plot was determined to be 49.5 minutes which is the D-value of Bacillus subtilis spores in 2%CHG / 70%IPA at 65 °C.

[0049] Table 4. Experiments to determine the D-value of B. subtilis spores in 65°C 2%CHG / 70%IPA.

[0050] ND: not determined.

[0051] Low heat sterilization process:

[0052] A forced-air oven was used as a Low Heat Batch Sterilizer (LHBS). The LHBS was used for sterilization of 2%CHG / 70%IPA contents (26 mL / ampoule) and the inner surfaces of hermetically sealed glass ampoules held in pallets along with Temperature Monitor Assembles (TMAs) (to monitor the temperature of the 2%CHG / 70%IPA solution throughout processing) and Process Challenge Devices (PCDs) (for sterility validation). Each TMA utilized a wireless temperature data logger sensor with its probe end submerged in the 2%CHG / 70%IPA content (26 mL) of a glass ampoule of which the cap opening 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 disc (stainless steel disc, 6 mm in diameter, with > 1.0 x 106CFU of Bacillus subtilis spores) submerged in 26 mL of 2%CHG / 70%IPA in a threaded clear glass vial (28 x 108 mm) that was enclosed with a PTFE lined cap. The thickness of the PCD vial was greater than that of the glass ampoule and the capacity of the PCD vial was similar to that of the glass vial; therefore, the use of the PCDs was valid. The TMAs and PCDs were distributed to appropriate locations within the pallets that held the glass ampoules. After inverting the samples once to allow 2%CHG / 70%IPA to wet the inner surfaces of the glass ampoules and PCD vials, the pallets were loaded into the LHBS. The sterilization process was started with a preheat stage in which the temperature of the 2%CHG / 70%IPA in the TMAs increased from room temperature to 65°C, and then the temperature of the chamber of the LHBS was maintained at 65 °C for a dwell time of 300 minutes (which is greater than 6 times of the D-value of Bacillus subtilis spores in 2%CHG / 70%IPA at 65°C; 6 x 49.5 minutes = 297 minutes).

[0053] Sterility validation:

[0054] After the glass ampoules and the PCDs were cooled to room temperature, the PCDs were retrieved for analyses.

[0055] For each PCD, the 2%CHG / 70%IPA content in the glass vial was transferred for filtration through a PVDF membrane filter (0.45 mm pore size; 47 mm in diameter) to recover dislodged spores or microorganisms (if there were any; from the spore disc or the inner surface of the PCD vial) while the spore disc was left in the glass vial. A washing procedure with 70% IPA for the spore disc, the inside of the glass vial and the PVDF membrane filter was conducted twice, each being conducted through washing the spore disc and the inside of the glass vial with 26 mL of 70% IPA and then transferring the 70% IPA wash from the glass vial for filtration through the PVDF membrane while the spore disc was left in the glass vial. Then, the procedure to neutralize residual CHG on the spore disc, the inside of the glass vial and the PVDF membrane filter was conducted twice with D / E Broth in the same manner as of the washing procedure with 70% IPA. Subsequently, the spore disc, the inside of the glass vial and the PVDF membrane filter were washed twice with Peptone water in the same manner as of the washing procedure with 70% IPA. The PVDF membrane filter was aseptically transferred onto a TSA plate and 35 mL of Letheen Broth (LB) was added to the clear glass vial with the spore disc. The PVDF filter-TSA plate assemble and the glass vial with the spore disc in LB were incubated at 35°C for 7 days.

[0056] All the retrieved PCDs were processed for analysis. No bacterial outgrowth was observed on the PVDF filter-TSA plate assembles or from the spore discs in LB after incubation at 35°C for 7 days, indicating that the low heat sterilization process was efficacious for sterilization of 2%CHG / 70%IPA contents and inner surfaces of the hermetically sealed glass ampoules, and a sterilization assurance level of 106was achieved.

Claims

WHAT IS CLAIMED IS:

1. A method of sterilizing an antiseptic solution and an interior of a sealed container in which the antiseptic composition is contained, the method comprising: adding an antiseptic solution into the container and then sealing the container to form a sealed container having interior walls that define an interior volume; wetting the interior walls of the container with the antiseptic solution; and subjecting the sealed container to heat at a predetermined sterilization temperature of from 50 to 80 degrees Celsius for a predetermined sterilization time of from 50 to 500 minutes.

2. The method of claim 1, wherein adding the adding antiseptic solution comprises adding the antiseptic solution such that there is a first region within the interior volume holding the antiseptic solution and a second region within the interior volume that is available to contain air and / or antiseptic solution vapor.

3. The method of claim 2, wherein the first region is between 30 and 99 vol. %, based on the total interior volume of the container, and the second region makes up the balance of the total interior volume, and wherein wetting the interior walls comprises wetting the interior walls of the second region.

4. The method of any one of claims 1-3, wherein wetting the interior walls comprises agitating or inverting of the sealed container.

5. The method of claim 4, wherein the agitating or the inverting of the sealed container occurs no more than 60 minutes before the step of subjecting the sealed container to heat.

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

7. The method of any one of claims 1-6, wherein the antiseptic composition comprises an antiseptic, an alcohol, and water.

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

9. The method of any one of claims 7-8, wherein the antiseptic comprises chlorhexidine gluconate or octenidine dihydrochloride10. The method of any one of claims 1-8, wherein the antiseptic comprises chlorhexidine gluconate.

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

12. The method of any one of claims 7-11, wherein the alcohol comprises ethanol, isopropanol, or n-propanol13. The method of any one of claims 7-12, wherein the alcohol comprises isopropanol.

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

15. A method of making a liquid applicator, the method comprising: providing an applicator comprising: a hollow body comprising a wall defining an internal chamber having a closed end and an open end; and an absorbent pad coupled to the open end of the hollow body; and sterilizing an antiseptic solution and an interior of a sealed container in which the antiseptic composition is contained in accordance with the methods of any one of claims 1-14; inserting the sealed container into the internal chamber to form the liquid applicator.

Images