DETAILED DESCRIPTION OF THE INVENTION

[0048] It has been discovered herein that despite the complete inactivation of certain bacterial proteases with high concentrations of per-compound oxidants such as peracetic acid (CH₃COOOH, hereafter abbreviated as PAA) and mixtures of hydrogen peroxide and PAA required to achieve decontamination, represented herein as high- or intermediate-level disinfection and/or sterilization, one can nonetheless combine selected cleaning enzymes with per-compound solutions to achieve single-use simultaneous cleaning and decontamination (high- or intermediate-level disinfection or sterilization) of a device such as an endoscope. Moreover, it has been discovered herein that through the proper selection of an amount of chelating agent and pH buffer of appropriate hydrophobicity, along with the application of medical device reprocessing steps involving a first bulk soil removal step followed by contacting the device with a combined solution of enzyme and per-compound, then rinsing the solution from the device and thereafter repeating the reprocessing steps for each device, one can achieve a simultaneous cleaning and decontaminating solution and method which is reusable for processing a plurality of medical devices such as endoscopes. Moreover, longer solution reuse times can be achieved with a method step wherein the enzyme is added to the solution in intervals so that enzyme activity is replenished. Additionally, it has been discovered that metal corrosion inhibitors can be combined with cleaning enzymes and per-compounds without inactivating the cleaning activity of the enzymes. The compositions of the present invention can also be prepared as a concentrate designed to be diluted with water to a final use dilution just prior to use. The compositions and methods of the present invention can be used through either manual or automated instrument applications, at various temperatures and with ultrasonic or other energy input.

[0049] It has also been discovered herein that the compositions and methods of the present invention can be utilized for safely and efficiently simultaneously cleaning and decontaminating an artificial kidney dialyzer, such that dialyzer reuse life can be significantly extended.

[0050] Advantages of the Present Invention

[0051] One advantage of the compositions and methods of the present invention is the reduced number of device processing steps. Prior art methods employ an initial separate precleaning treatment with an enzymatic detergent, followed by a rinsing step, followed by a disinfecting step, and thereafter a last rinsing step. The method of the present invention includes an optional first bulk soil removal rinsing step, followed by a simultaneous cleaning and decontaminating step employing a per-compound oxidant and an enzyme, and a last rinsing step to remove the oxidant and enzyme solution from the device. Thus, the invention combines the former separate enzymatic detergent precleaning treatment with the decontaminating step so that cleaning and decontaminating are performed simultaneously. There are several advantages of this combination, such as (1) the reduced number of device processing steps making processing easier, faster and hence less costly for the user; (2) the enhancement of enzymatic cleaning, which takes place with the combination of enzyme and decontaminant, that results in better cleaning and hence longer device lifetime; and (3) regimen compliance is imposed, with the result that proper cleaning is carried out so proper decontamination is ensured. The latter advantage is perhaps the most significant, as non-compliance with proper device cleaning can result in insufficient decontamination/sterilization and infection.

[0052] A second advantage is that the combined cleaning and decontaminating solution is reusable. This is due to the incorporation of a combination of enzyme plus decontaminant plus a unique chelating agent and/or buffer, the latter which provides adequate buffering capacity for a large volume of decontaminant during multiple device processing cycles. The chelating agent in the proper concentration prevents destabilization of the decontaminant due to contact with blood and trace metals. The reusability of the compositions of the present invention is also made possible by the unique device reprocessing steps employing a bulk soil removal rinsing step, followed by a simultaneous cleaning and decontaminating step employing a per-compound oxidant and an enzyme, and a last rinsing step to remove the oxidant and enzyme solution from the device. The foregoing steps are then repeated for a plurality of devices such as a medical device wherein each device is contacted with the same per-compound oxidant and enzyme composition. Either of the aforementioned removal steps can be accomplished with distilled, pyrogen-free, microbe-free (e.g., sterile) or tap water, the latter which is particularly convenient for the user when employed in the first bulk soil removal step. Alternatively, the first bulk soil removal rinsing step can be optionally performed with either an enzyme-containing or non-enzyme detergent. The latter type of detergent would be more preferred over an enzyme detergent. Additionally, the first removal step can be preceded by a manual soil removal step employing a sponge, cloth or towel. The final solution removal step can be followed by storage of the device in a way which prevents recontamination either by microorganisms or soils. The final solution removal step can also be followed by a drying step with air or other means. This step in turn can be followed by storage of the device in a way which prevents recontamination either by microorganisms or soils. The final solution removal step can also be followed by an alcohol rinse step, which in turn can be followed by a drying step with air or other means and thereafter by storage in a way that prevents recontamination. An even simpler medical device reprocessing regimen includes: a) contacting the medical device with a solution comprising a per-compound oxidant in an amount effective to achieve decontamination and an enzyme in an amount effective for cleaning the device, wherein the decontaminating and cleaning occur simultaneously; and

[0053] b) removing the solution from step a) from the device. An even simpler medical device reprocessing regimen includes only step a) above. These simplified steps can be preceded or followed by additional reprocessing steps for soil removal, drying, alcohol rinsing and storage to prevent recontamination as above. All of the foregoing steps can be repeated to reprocess a plurality of devices such as medical devices. These foregoing methods provide simplified, convenient reprocessing regimens for devices. The compositions of the present invention, employing the methods of the present invention, can extend solution reusability for a plurality of devices, preferably between about one-five days, although solution reuse for much longer periods of up to thirty days is possible, the latter especially wherein a non-enzyme detergent is utilized for the first bulk soil removal rinsing step.

[0054] A third advantage is that the combination of enzyme plus decontaminant may include a corrosion inhibitor which is compatible with both the enzyme and the disinfectant. The corrosion inhibitor may be necessary when using the compositions and methods of the present invention for devices containing metal parts and adhesives.

[0055] Another advantage of the present invention is that the combination of enzyme plus disinfectant can also be prepared as a concentrate designed to be diluted with distilled, tap or other water to a final use dilution just prior to use. Additionally, either the enzyme-containing solution or the decontaminant solution can be prepared as a concentrate and then mixed with the other and with distilled, tap or other water as needed just prior to use.

[0056] The use of concentrates reduces product shipping and storage requirements, making the system more convenient.

[0057] An additional advantage is the cost savings over existing chemical reprocessing solutions. For example, the compositions and methods of the present invention for kidney dialyzer reprocessing can extend dialyzer life two-five times or more. A two-fold improvement in dialyzer life extends the average reuse to 30 times, reducing the amortized dialyzer cost per reuse to $0.68-1.00 and yielding an equal amount saved. Also, there are savings on hazardous waste disposal of $0.25-$0.28 (($0.50/2)-($0.55/2)). Lastly, savings of $0.23 would be obtained due to elimination of the labor cost of manual cleaning/dislodging of clots. Thus, total cost savings per reuse ranges from $1.16 (15-18% of the total reuse cost range) to $1.51 (20-23%). Similarly, cost savings incurred with a four-fold improvement in dialyzer life ranges from $1.63 (21-25%) to $2.14 (28-32%) per reuse. Moreover, considering that these cost savings are achieved with the new reprocessing solutions of the present invention, a higher product price can be justified. An additional price of $1.07 or more can be justified (one-half or more of the cost savings). Combined with the 1998 cost average of $1.09 for prior art reprocessing solutions per use, a total product price of $2.16 or more can be justified. Thus, the world market for reprocessing solutions can be doubled from the current forecast of $125 million in the year 2005 to $250 million.

[0058] Still another advantage of the methods and compositions of the present invention is that efficient cleaning of a dialyzer can take place safely during decontamination, such that the biocompatibility of the dialyzer is maintained or even enhanced, rather than compromised as when cleaned with sodium hypochlorite bleach. The compositions and methods of the present invention efficiently remove cellular debris and protein deposits while leaving at least a partial monomolecular layer of protein remaining to mask immune reaction sites located on the dialyzer membrane surface. At the same time, this thin layer of remaining protein does not compromise dialyzer ultrafiltration performance due to its thinness.

[0059] Still another advantage of the methods and compositions of the present invention is that, unlike the present cleaning compositions and methods utilizing bleach, dialyzer fibers will not be damaged with solution exposure times exceeding a few minutes.

[0060] Still another advantage is that the preferred human enzymes for dialyzer cleaning will not provoke an immune response if they enter the circulatory system in amounts arising from desorption from the dialyzer fibers following the final rinsing step employed just prior to dialyzer use, and the preferred non-human enzymes will not provoke an immune response because they have little to no interaction or binding with the dialyzer fibers, resulting in an insufficient amount of material desorbing from the fibers into the blood during dialysis to provoke an immune response. Additionally, it may be advantageous to clean a dialyzer by contacting only the ends of the fiber bundle, which are readily isolated from the remaining portion, with the simultaneous cleaning and decontaminating solution. This may limit an immunogenic response in comparison to a response produced by contacting the entire portion of the fiber bundle with the solution.

[0061] An additional advantage is that the solution does not have the capability to transform organic compounds within the dialyzer into carcinogenic haloforms as does chlorine bleach.

[0062] The following provides a detailed description of the invention regarding specific elements of the compositions, methods of use and applications.

[0063] Composition

[0064] Enzymes

[0065] Source

[0066] A single enzyme or a mixture of several enzymes may be employed in the present invention. At least one enzyme used herein is preferably proteolytic in nature, that is, it has at least partial capability to hydrolyze peptide-amide bonds, which in turn reduces proteinaceous material deposited on a device or instrument to smaller water-soluble peptide or amino-acid subunits. Proteolytic enzymes may be endoproteases or exoproteases or a combination of both types. Other enzymes exhibiting amylolytic or related carbohydrase activities and/or lipolytic or lipase activities may also be employed. Enzymes may exhibit alkaline, neutral or acidic pH activity profiles and may additionally be thermally stable. Enzyme raw materials may exhibit some lipolytic, amylolytic or related activities associated with the proteolytic activity. Enzymes may be derived from any plant or animal source, including human, other mammalian sources and microbial sources, as long as they meet all of the requirements of the particular application within the scope of the present invention, i.e., human enzymes are preferred for kidney dialyzer reprocessing applications to minimize foreign enzyme immunoreactivity.

[0067] A thermally stable or thermophilic enzyme is stable and active at temperatures >50° C. or even >100° C. One such heat stable protease is thermolysin and others may be obtained at pages 642-650 of Perlmann et al., “Proteolytic Enzymes,” Methods in Enzymology, Volume XIX, Academic Press (1970), which pages are specifically incorporated by reference herein.

[0068] Examples of suitable proteolytic enzymes include but are not limited to pancreatin, trypsin, chymotrypsin, collagenase, keratinase, carboxylase, aminopeptidase, elastase, aspergillo-peptidases A and B, pronase E (from S. griseus),. dispase (from Bacillus polymyxa) and mixtures thereof. Metalloproteases, enzymes which contain a divalent metal ion such as calcium, magnesium or zinc bound to the protein, and acid proteases such as pepsin may also be utilized.

[0069] The preferred group of proteolytic enzymes for non-dialyzer applications are microbially derived such as those derived from Bacillus, Streptomyces and Aspergillus species. Microbially derived enzymes are disclosed in U.S. Pat. No. 4,690,773 which is expressly incorporated herein by reference in its entirety. Reference is also made to Keay, L, Moser, P W and Wildi, B S, “Proteases of the Genus Bacillus. II Alcaline Proteases,” Biotechnology and Bioengineering, Vol. XII, pp. 213-249 (1970) and Keay, L and Moser, P W, “Differentiation of Alkaline Proteases from Bacillus Species”, Biochemical and Biophysical Research Comm., Vol. 34, No. 5, pp. 600-604 (1969). Most preferred are the Bacillus derived alkaline proteases generically called subtilisin enzymes.

[0070] The subtilisin enzymes include subtilisin A and subtilisin B sub-classes. Subtilisin A includes enzymes derived from such species as B. subtilis, B. licheniformis and B. pumilis and produce little or no neutral protease or amylase. Subtilisin B includes enzymes from such organisms as B. subtilis, B. subtilis var. amylosacchariticus, B. amyloliquefaciens and B. subtilis NRRL B3411, and produce neutral proteases and amylases on a level about comparable to their alkaline protease production. Generally, the preferred enzymes are active proteolytic enzymes, with the most preferred being subtilisin A for use in non-dialyzer applications, e.g., endoscope and other semicritical medical devices. A particularly preferred commercial subtilisin A product is Alcalase® (Novo Nordisk Biochem North America, Inc., Franklinton, N.C.), available in granular as well as liquid product varieties. A particularly preferred variety of Alcalase® is a liquid concentrate known as Alcalase® 2.5 L, having a specific enzyme activity of 2.5 Anson units (A.U.) per gram. Alcalase® 2.5 L has a density of 1.056 g/ml, therefore the enzyme activity of Alcalase® 2.5 L can also be expressed as 2.64 A.U./ml. Other preferred enzymes available from the same source for non-dialyzer applications are Savinase®, a serine protease of the subtilisin class which is produced by submerged fermentation of a genetically modified alkalophilic species of Bacillus, Durazyme® 16.0 L, Type EX, a first generation protein-engineered variant of Savinase®, and Everlase®, a second generation protein-engineered variant of Savinase®.

[0071] Yet another preferred enzyme available from the same source for selected non-dialyzer applications is Neutrase®, a bacterial protease produced by a selected strain of B. amyloliquefaciens. Neutrase® contains only the neutral part of B. amyloliquefaciens proteases, whereas most other commercial preparations also contain the alkaline protease. Neutrase® is a zinc metalloprotease which is stabilized with Ca²⁺ and consequently inhibited by EDTA. Neutrase® can be used for selected applications requiring a more neutral pH, as it has optimal activity between pH 5.5-7.0. Neutrase® has limited application as it is 100% inactivated in a solution containing 0.2% ^w/v PAA and 1.08% ^w/v H₂O₂ It is also inactivated by zinc and calcium chelating agents. A particularly preferred Neutrase® raw material is designated Neutrase® 0.5 L. The specific enzyme activity of Neutrase® 0.5 L is 0.5 A.U./gram, with a solution density of 1.226 g/ml.

[0072] Human enzymes from the serine protease class at a neutral or alkaline pH are preferred for kidney dialyzer applications. The reason for this is that the preferred human enzymes will not provoke an immune response if they enter the circulatory system in small amounts arising from desorption from the dialyzer fibers following the final rinsing step employed just prior to dialyzer use. Human trypsin and chymotrypsin are examples of suitable human enzymes for use at a neutral or alkaline pH. Enzymes such as recombinant human tissue plasminogen activator, anisoylated plasminogen streptokinase activator complex (APSAC; anistreplase), streptokinase and urokinase can also be used for kidney dialyzer applications. Additionally, preferred non-human enzymes for kidney dialyzer applications are enzymes which will not provoke an immune response because they have little to no interaction or binding with the dialyzer fibers, resulting in an insufficient amount of material desorbing from the fibers into the blood during dialysis to provoke an immune response. Enzyme interaction with dialyzer surfaces such as fibers can be measured with classical enzyme-substrate assays which can detect bound or desorbed active enzyme. A preferred substrate for use with a serine protease for this purpose is benzoylarginine ethyl ester (BAEE). The ethanol which is produced can be detected in very small amounts using gas chromatography. Total protein analyses can also be performed on dialyzer materials, provided that non-enzyme protein, if present, can be subtracted from the total.

[0073] Acid-acting enzymes are useful when it is desirable not to change the pH of an acid stabilized disinfecting solution such as acid stabilized peracetic acid (PAA)—H₂O₂. This situation may arise when a more alkaline pH for either of the latter solutions may be detrimental to the biocompatibility of the device or the shelf stability of the solution. H₂O₂—PAA solutions are typically stabilized between pH 1.9-3 to achieve a reasonable shelf-life of one to two years. Alternatively, it may be desirable to avoid adding a large amount of alkaline buffer due to buffer solubility limitations in a liquid enzyme formula. Thus, preferably, the acid-acting enzymes are effective between about pH 2-5. Specific examples of acid-acting enzymes which may be employed in the present invention include pepsins such as penicillopepsin from Penicilliumjanthinellum, gastricsin, chymosin (rennin), Cathepsin D, genetically engineered enzymes such as subtilisins with acid pH activity profiles, fungal acid proteases such as aspergillopeptidase A from Aspergillus saitoi and the like, acid-acting enzymes disclosed in U.S. Pat. No. 5,630,884, which is expressly incorporated herein by reference in its entirety, and mixtures thereof. Two additional acid-acting enzymes designated NS16009 and NS16010 (Novo Nordisk) are acid endoproteases from A. niger var. aculeatus. Acid-acting enzymes are also useful for kidney dialyzer reprocessing applications because of the need to minimize exposure to any residual active enzyme which may desorb from the dialyzer during use. In this situation, the desorbed enzyme would be exposed to the physiological pH of the circulating blood and thus would inherently be inactivated by the pH 7.45 environment. Additionally, an acid-acting enzyme in combination with an acid peroxide-containing disinfectant will not result in oxygen gas formation as occurs with neutral or alkaline pH, which can burst dialyzer fibers due to pressure build-up. A human acid-acting enzyme such as human pepsin or pepsinogen, the precursor of pepsin, would be most preferred for dialyzer applications.

[0074] While proteolytic enzymes are preferably utilized, it is also preferred to utilize a proteolytic enzyme in combination with one or more enzymes from the amylase, cellulase or lipase classes, especially for non-dialyzer applications. The use of an amylase along with a protease is particularly preferred. Preferred commercial amylases from Novo are Duramyl™ and Termamyl® 300L. Duramyl™ is a protein-engineered alpha-amylase produced by submerged fermentation of a genetically modified species of Bacillus. Termamyl® 300L is an alpha-amylase produced by a selected strain of Bacillus licheniformis. Both amylases are endoamylases which hydrolyze 1,4-alpha-glycosidic linkages in amylase and amylopectin. Starch is therefore rapidly broken down to soluble dextrins and oligosaccharides. Both enzymes may be used in alkaline, neutral or acidic formulas. Other enzymes which can be utilized, particularly in combination with a protease include cellulases such as Celluzyme® and lipases such as Lipolase™ and Lipolase™ Ultra (all from Novo).

[0075] Methods for enzyme identification, separation and purification are well established. Many techniques exist in the general scientific literature for the isolation and identification of enzymes, including enzymes having proteolytic and mixed proteolytic/amylolytic or proteolytic/lipolytic activity. The enzymes contemplated by the present invention can be readily obtained by known techniques from plant, animal or microbial sources.

[0076] With the advent of recombinant DNA techniques, new sources and types of proteolytic enzymes have become available. Such enzymes, as well as enzymes produced through a combination of recombinant DNA or site-directed mutagenesis techniques with chemical modifications, should be considered to fall within the scope of this invention so long as they meet the criteria set forth herein. See Japanese laid open application No. J60030-685 (production of proteases by recombinant DNA from Bacillus subtilis), International Patent Application No. PCT/DK94/00274 and International Patent Application No. PCT/DK91/00103 (production of chemically-modified proteases by a combination of recombinant DNA or site-directed mutagenesis, such proteases being incorporated herein by reference). Additionally, native or wild-type enzymes which are subsequently chemically modified should also be considered to fall within the scope of this invention so long as they meet the criteria set forth herein.

[0077] Effective and Efficacious Amounts

[0078] The present invention generally employs an effective and efficacious amount of enzyme to clean the device or instrument, e.g., medical devices such as endoscopes or kidney dialyzers. An effective and efficacious amount is that which (1) removes, in a reasonable time a substantial portion of the proteinaceous or other deposits which occur during the use of the medical device, (2) does not decrease the efficacy of the decontaminating agent(s) when combined with the latter agent(s) in the working solution, and (3) allows the decontaminating agent(s) to achieve the standards, e.g., high-level disinfection and sterilization, required for the reprocessing of the particular device or instrument.

[0079] The precise amount of enzyme required to produce an effective and efficacious cleaner will depend on several factors including enzyme activity and purity, the amount of proteinaceous and other matter deposited, the desired soaking period and temperature, the nature and concentration of the decontaminating agent(s), the specific medical device or instrument, the delivery form of the enzyme and its related shelf stability, the presence of surfactants and other solution components known to enhance the activity of the particular enzyme, as well as other known factors.

[0080] Activity

[0081] The precise amount of enzyme used by weight will vary with the purity and specific activity of the enzyme, normally expressed as Anson Units per gram, (A.U./g), which can vary on a lot-by-lot basis. Additionally, the expected enzyme activity loss during shelf storage and during the solution reuse period, if any, must be considered. It is standard practice with proteolytic enzyme cleaning products to expect about a 40-50% enzyme activity loss during a two year shelf-life and to properly plan for this by formulating with a higher level of enzyme than is needed. Activity losses during the reuse period may also be about 50%. The temperature of the combined cleaning and decontaminating solution is also a critical determinant of the amount of enzyme to utilize, as is the regimen soak time. The regimen soak time for reprocessing endoscopes and other semicritical medical devices utilizing the present invention is generally between about 10-30 min, preferably between about 10-20 min although shorter soak times can be employed for all applications and longer soak times for kidney dialyzers. The regimen soak time for reprocessing kidney dialyzers can be as short as 1 min with ultrasonic energy input, to 10-20 min and as long as or longer than 43 hours, which is almost as long as the 48 hour interdialysis period. Generally, the longer the soak time, the less enzyme is necessary for cleaning if activity losses are the same. Enzyme activity loss from exposure to the decontaminant is greater with longer regimen soak times. Thus, these two effects need to be balanced in the final enzyme formula.

[0082] If the enzyme cleaning component is formulated as a separate solution from the decontaminating component, such that the two must be mixed prior to use, consideration must be made for dilution of the enzyme. Current stand-alone (i.e., no decontaminant) proteolytic enzymatic precleaners for semicritical medical devices utilize generally between about 0.0012 and 0.0031 A.U./ml final soak volume following a dilution of 1 ounce into 1 gallon (total volume 129 ounces). Thus, the current stand-alone enzymatic precleaners are concentrates with enzyme activity generally between 0.154-0.397 A.U./ml. The diluted solutions are designed for the most part to be used for 10 min with warm water at 50° C. In contrast, the combined cleaning and decontaminating solutions of the present invention are designed to be used primarily for about 10-30 min at 20° C., with a preferred soak time of 20 min for endoscope reprocessing. Since there is a nominal eight-times rate enhancement of enzyme activity at 50° C. over 20° C., eight times the amount of enzyme would be necessary at 20° C. to achieve the same cleaning. Also, half the enzyme amount can generally achieve the same cleaning in twice the soak time (20 min versus 10 min).

[0083] Surprisingly, the combined cleaning and decontaminating solutions of the present invention achieve significantly greater cleaning per unit of enzyme than prior stand-alone enzyme solutions, therefore the aforementioned amounts of enzyme can be reduced. Taking under consideration the enhancement of cleaning, which can in many cases make up for expected storage losses, the combined cleaning and decontaminating solutions of the present invention should contain between about 0.0048 (4×0.0012) and 0.0124 (4×0.0031) A.U./ml of final working solution to ensure that the same cleaning effect is achieved for semicritical medical devices. This activity range is multiplied by 129 in the case wherein the enzyme is kept separate from the decontaminant prior to use and is diluted 1 ounce into 1 gallon (128 ounces) to achieve a final working solution. Thus, the enzyme activity range for the separate enzyme concentrate would be about 0.619-1.600 A.U./ml. Similar considerations need to be taken in determining the appropriate amount of enzyme to be utilized for kidney dialyzer reprocessing.

[0084] Taking all factors under consideration, the final working solution should contain sufficient proteolytic enzyme to provide between about 0.00001-10 A.U./ml, preferably between about 0.001-0.10 A.U./ml, and more preferably between about 0.0048-0.0124 A.U./ml soak volume.

[0085] Where an amylase enzyme is employed, similar considerations need to be taken in determining the appropriate amount of enzyme. Thus, the recommended amounts of amylase enzyme to utilize are the same, however they are expressed in Kilo Novo units/ml, wherein for every 2.5 A.U., 300 Kilo Novo units are substituted. Thus, the aforementioned recommended range limits for proteolytic enzymes are multiplied by (300/2.5)=120. Therefore, the more preferred proteolytic enzyme range of 0.0048-0.0124 A.U./ml is translated to 0.58-1.49 Kilo Novo units/ml for an amylase enzyme. One Kilo Novo alpha-amylase unit is the amount of enzyme which breaks down 5.26 g starch/h in Novo's standard analytical method AF-9 for the determination of alpha-amylase. Also, as previously taught, both types of enzyme may be employed separately, together or combined with another type of enzyme. Where both a proteolytic and amylolytic enzyme are employed together, each is recommended to be used within their above respective ranges.

[0086] Enzyme activity is pH dependent and for any given enzyme, there is an optimum pH range as determined by techniques known to one skilled in the art. It is preferred, but not required, to manipulate the working solution to an optimum pH range for a given enzyme. Generally, it is preferred that the enzyme be selected to have activity between pH 6-9, even more preferably between pH 6.5-8.5, and most preferably between pH 7-8.5. These pH ranges avoid acidic solutions which can be corrosive to metal-containing devices and instruments. Strongly alkaline solutions >pH 9 are also generally undesirable as they impart chemical instability to per-compound based disinfectants such as H₂O₂ plus PAA. This chemical instability may also cause a too-rapid breakdown of medical devices. One of the preferred commercially available proteolytic enzymes for endoscope cleaning applications, Alcalase® 2.5 L , has an optimal activity profile between about pH 6-12, with a peak cleaning activity between about pH 8-8.5. A preferred amylase enzyme such as Duramyl™ has high activity between about pH 5-9 and optimal activity between about pH 6-7, whereas another preferred amylase enzyme, Termamyl®, has high activity between about pH 5-9 and optimal activity between about pH 6-8. Also preferred are enzymes that have activity between pH 1.5-3.0, where most per-compounds are stable. These compositions would also preferably include a metal corrosion inhibitor for non-dialyzer applications.

[0087] Formulation

[0088] The enzyme component may be employed in liquid or solid form such as tablets, pills, capsules, granules and the like which is introduced into the liquid medium. Due to the time constraints for medical device reprocessing which currently exist to control labor costs and maximize profits, it is desirable to utilize a delivery form for the enzyme which is most efficient for the user of the reprocessing system. In this context, a liquid enzyme concentrate which can be rapidly diluted into a high-level decontaminating solution is most preferred, especially for endoscope reprocessing with soak times as short as 20 min. The disinfecting solution in this context can be a concentrate itself, wherein distilled, pyrogen-free, microbe-free or tap water would also be added before, at or after the time of addition of the enzyme solution to achieve the final use-dilution concentration of the combined enzyme and decontaminant solution. Alternatively, enzymes may be formulated in rapidly dissolving effervescent granules to minimize reprocessing time. Effervescing agents are typically employed when the enzyme component is provided in solid form. Examples of suitable effervescing agents include tartaric or citric acid used in combination with a suitable alkali metal salt such as sodium carbonate. In addition, binders, lubricants, carriers, and other excipients normally used in producing tablets may be used when enzyme component-containing tablets are employed. The decontaminating per-compound may also be included in the enzyme solid dose form such as a tablet.

[0089] Stabilizers

[0090] The shelf-life stability of enzymes useful in the present invention can be achieved or improved with standard methods such as adding calcium ions for subtilisin in a liquid formulation or producing a liquid formulation of low water content. One way of reducing water in the formulation is to use propylene glycol, other glycols, as well as other polyols such as various sugars, e.g., sorbitol. Concentrations of glycols between ₂₀% ^w/v-70% ^w/v are preferred, although lower or higher concentrations may be employed. Other conventional means for stabilizing a liquid enzyme formulation can also be employed. The aforementioned considerations do not generally apply to solid state enzyme formulations, the latter which can be stabilized by conventional means, e.g., using very dry raw materials or other known methods.

[0091] Decontaminating Agents

[0092] The disinfecting or sterilizing agent(s), collectively termed decontaminating agents, may be any of one or more per-compounds which produce active oxygen in solution and which can achieve high- or intermediate-level disinfection or sterilization. Examples of such compounds include inorganic peroxides, peracids (which exist in equilibrium with a certain amount of H₂O₂) such as PAA and H₂O₂ plus PAA, with PAA and mixtures of H₂O₂ and PAA being preferred. PAA or other peracids, alone or in combination, may also be used. Peracids produced from C₁-C₄ mono or dicarboxylic acids, such as performic acid and perpropionic acid may be used. Peracids produced from carboxylic acids with linear, saturated or unsaturated hydrocarbon chains having 5-20 carbon atoms may also be used. Alkali metal persulfates, alkali metal carbonate peroxide, diperisophthalic acid, peroxydiphosphate salts and sodium aluminum aminohydroperoxide are examples of compounds which can also be used in combination with one or more peracids or one or more solid peracid precursors, such as tetraacetylethylenediamine (TAED) or acetylsalicylic acid. Collectively, these compounds are all per-compound oxidant-based anti-microbial agents.

[0093] Existing high-level disinfecting solutions such as the 7.5% ^w/w solution of H₂O₂ (and an unspecified peracid) marketed as Sporox® (Reckitt and Colman, Montvale, N.J.) and the mixture of 1.0% ^w/w H₂O₂ and 0.08% ^w/w PAA marketed as Cidex PA® (Johnson and Johnson, Irvine, Calif.) or Peract 20® (Minntech Corp, Minneapolis, Minn.) also may be utilized in the compositions and methods of the present invention. Renalin® Dialyzer Reprocessing Concentrate (Renal Systems Division, Minntech Corp.) may also be utilized.

[0094] Additionally, hydrogen peroxide-peracid compositions disclosed in U.S. Pat. No. 4,518,585, (which inherently generate a peracid), which is expressly incorporated herein by reference in its entirety, may be employed in the present invention provided they meet the decontamination standards herein.

[0095] Effective and Efficacious Amounts

[0096] The identity, concentration and contact time of the selected agent(s) will vary depending on the extent of decontamination; that is, whether high- or intermediate-level disinfection or sterilization is desired. In many applications, only high-level disinfection is sought. The disinfecting or sterilizing contact time is also a function of the concentration of the decontaminating agent(s) employed. Hence, the contact time or the concentrations of the disinfecting agent(s) may be adjusted accordingly. As an example, Sporox® has a high-level disinfection time of 30 minutes at 20° C. and a sterilization time of 6 h at 20° C. Cidex PA® has a high-level disinfection time of 25 min at 20° C. and a sterilization time of 8 h at 20° C. The identity and concentration of selected decontaminating agents is also dependent upon the type of device requiring cleaning and decontamination. Devices containing metals, especially soft metals, require non-corrosive solutions. Peracetic acid solutions which minimize acetic acid contant will minimize metal corrosion. Given the known reaction between hydrogen peroxide and acetic acid to produce peracetic acid and the equilibrium relationship between the three molecules, it is desireable to maximize the concentration of hydrogen peroxide and minimize the concentration of acetic acid to produce a desired amount of peracetic acid for a minimally corrosive solution. For example, a hydrogen peroxide concentration of about 7.5% ^w/w in combination with varying amounts of acetic acid is optimal for producing varying amounts of peracetic acid in the less hazardous concentration range of less than 8.0% ^w/w H₂O₂. Also, since in decontaminating agent solutions containing both hydrogen peroxide and peracetic acid, the sporicidal activity is contributed to significantly by hydrogen peroxide, it is often preferable to maximize the concentration of hydrogen peroxide as much as possible. Another factor is the inherent instability of both peracetic acid and hydrogen peroxide. Moreover, peracetic acid stability is dependent upon hydrogen peroxide stability. If hydrogen peroxide is neutralized or decays, the peracetic acid will rapidly decay to acetic acid and hydrogen peroxide owing to the equilibrium relationship noted above. Thus there is a need for stabilization of hydrogen peroxide in such solutions.

[0097] A single peroxide concentration cannot apply to all peroxides as the percentage of active oxygen varies substantially among peroxides. The preferred concentration range for H₂O₂ in combination with a peracid such as peracetic acid is between about 3.0-8.0% ^w/w, with a more preferred range between about 6.0-8.0% ^w/w. However, concentrations ≧8.0% ^w/w are generally undesirable as concentrations of H₂O₂ between 8.0-16% ^w/w are classified by the United States Department of Transportation as significantly more hazardous than lower concentrations and must therefore be handled accordingly, imposing costlier shipping requirements. Concentrations of H₂O₂ <6.0% ^w/w are less preferred since these require significantly longer high-level disinfection and sterilization times. Lower or higher concentrations of H₂O₂ may be employed in combination with a peracid from about 0.5-50% ^w/w.

[0098] The preferred concentration range for PAA is between about 0.05-0.30% ^w/w, with an even more preferred concentration range between about 0.08-0.22% ^w/w. Concentrations higher than about 0.30% ^w/w are generally undesirable as they are too corrosive to adhesives and other components of devices. However, concentrations as high as 5% can be employed provided an appropriate level of anticorrosive agent(s) is also present. PAA at concentrations <0.05% ^w/w has not been shown to be an effective high-level disinfectant or sterilant with contact times of 30 minutes or less, with 0.05% ^w/w PAA stated as being the minimum effective concentration for high-level disinfection at 25 minutes contact at 20° C.

[0099] The appropriate concentrations of any given peroxide will be a matter to be determined through routine laboratory testing as known to one skilled in the art.

[0100] Stabilizers

[0101] Decontaminating agents such as PAA or mixtures of H₂O₂ and PAA are inherently unstable above pH 4-5, rapidly decaying to water and oxygen in the case of H₂O₂ and additionally to acetic acid in the case of PAA. Thus, such solutions are pH stabilized, preferably between about pH 1-3, more preferably between about pH 1.5-3, and even more preferably between about pH 1.6-2.0. This is achieved with an acidic compound such as an inorganic or organic acid, typically at a concentration between 0.001-10%. Additionally, transition metals such as iron, manganese and copper also destabilize peroxides. Such metal-induced destabilization can be prevented with sequestrants such as hydroxy-ethylidene diphosphonic acid (Dequest 2010™M,Monsanto Co.), or with the use of conventional chelating agents. Preferred concentrations for Dequest 2010™ are between 0.10 and 1.0% ^w/v. Other conventional means of stabilizing peroxides can also be employed, such as nitrilotrismethyl-, methyl- and other phosphonic acids and nonionic surfactant stabilizers.

[0102] Formulation

[0103] The decontaminating agent may be employed in liquid or solid form. Solid forms include tablets, pills, capsules, granules and the like which are introduced into a liquid diluent such as water. Due to the current time constraints for medical device reprocessing to control labor costs and maximize profits, it is desirable to utilize a delivery form for the decontaminant which is most efficient for the user of the reprocessing system. In this context a liquid form, either concentrated or already diluted to its final use dilution, is most preferred. If the disinfecting agent is utilized in a liquid concentrate designed to be diluted with tap water to its final use-dilution prior to use, it needs to be compatible with water hardness cations (CA²⁺ and Mg²⁺) up to between about 500 and 1000 ppm CaCO₃ equivalence, and ideally between about 800 and 1000 ppm. Compatibility in this context includes the performance that the concentrate will not form a precipitate with calcium or magnesium or other entities in the tap water. This can be achieved by avoiding the use of formulation excipients that will form precipitates with calcium or magnesium or alternatively, by using sufficient chelators to complex calcium and magnesium. These same considerations apply to enzyme concentrates which may be mixed with tap water. Lastly, other forms of purified water such as sterile water may also be utilized to dilute a concentrated disinfectant. Alternatively, decontaminating agents may be formulated in rapidly dissolving effervescent granules to minimize reprocessing time.

[0104] Additional Components

[0105] Buffering agents, chelating agents, anticorrosive agents, surfactants, antifoam agents, soil antiredeposition agents, preservatives, tonicity adjusting agents, indicator dyes, fragrances and the like can be employed in the composition of the invention.

[0106] Buffering Agents

[0107] A buffer should be added to a separate enzyme formulation to maximize the enzyme activity if the pH of the combined decontaminating and cleaning solution is designed to be different than that of decontaminating formulation alone. Additionally, each device, when added to the combined enzyme and disinfectant solution, may carry over an amount of rinse water or other rinse liquid into the combined solution, adversely changing its pH and/or buffer capacity. A neutral or alkaline buffer used to provide a proper working pH for a corresponding neutral or alkaline enzyme can also serve to buffer the combined enzyme and disinfectant solution so that it can be re-used to clean and disinfect a plurality of devices over one or more days.

[0108] Usually, a large amount of buffer is required in the enzyme formulation so that the pH of the very acidic H₂O₂ and/or PAA solutions at which the latter are stable can be enhanced with much smaller quantities of enzyme formulation (i.e., 1 gallon PAA/H₂O₂:1 ounce liquid enzyme formulation is a preferred volume ratio). However, many buffers are either insoluble or unstable at the required high concentrations in liquid enzyme formulations which contain a large amount of organic solvents, such as propylene glycol, to stabilize the enzyme to achieve a reasonable shelf life. For example, buffers disclosed in the prior art such as 0.5-1 M phosphate buffer and borate buffer solutions in the presence of 23% ^w/v propylene glycol, 15% ^w/v Macon 10 and 10.7% ^w/v Alcalase® form two liquid phases and a precipitate, respectively. It was discovered that large amounts of buffering agents can be dissolved in low water content enzyme solutions if proper hydrophobicity of the buffering agent molecules is carefully selected. If such a buffering agent in a required amount with high hydrophobicity is still not soluble, one can use organic counter ions with sufficient hydrophobicity to replace the lower hydrophobicity counter ions. Examples of qualified buffering agents are 2-amino-2-hydroxymethyl-1,3-propanediol/acetic acid, 2-amino-2-methyl-1-propanol, ethanolamine/lactic acid and triethanolamine (TEA). High tris(hydroxymethyl) aminomethane (Tris) buffer concentrations sometimes can cause the solution to be unstable if the pH of the mixed enzyme solutions is adjusted with inorganic acid, thus the Tris inorganic acid buffer compositions taught in the aforementioned U.S. Pat. No. 5,356,555 are unsuitable for concentrated enzyme solutions. Tris is preferably paired with an organic counter ion such as acetate from acetic acid or borate from boric acid. However, inorganic or combined inorganic-organic buffers can be used in the enzyme formulation if the enzyme solution either does not require its own strong buffer (e.g., in the case wherein an acid-acting enzyme is employed with an acid per-compound) or in the case wherein a solid enzyme formula is used which does not require a polyol enzyme stabilizing agent. Inorganic or organic buffers can be used as part of per-compound formulas, whether the latter are solids, concentrated solutions or solutions already diluted to their final working concentration. Examples of suitable buffers for use in the present invention are also presented in Tables 7c and 14a. In general, a concentration of buffer between about 2 mM and about 2 M can be used in compositions of the present invention. Preferably, between about 0.25 M and 1.5 M of completely organic buffer is used in an enzyme solution concentrate designed to be diluted 129-fold with a liquid disinfectant. However, lower or higher concentrations can be used.

[0109] Chelating Agents

[0110] Transition metal ions such as Fe(III), Fe(II), Cu(II) and Ni(II) usually exist in oxidant disinfecting and sterilizing solutions as the result of metal corrosion occurring in the process of instrument disinfection and sterilization. Fe(III) and Fe(II) are also introduced into a disinfecting and sterilizing solution by blood remaining on a medical instrument to be disinfected orsterilized. It is known that trace levels of transition metals can participate in the metal-catalyzed Haber-Weiss reaction or superoxide-driven Fenton reaction, which causes H₂O₂ to decompose into O₂ (Buettner, Radiation Research 145, 532-541 (1996)). Also, the destabilization of H₂O₂ will cause the destabilization of PAA or other peracids derived from H₂O₂ when the two (e.g., H₂O₂ and PAA) are combined. It is, therefore, necessary to sufficiently stabilize H₂O₂ in reusable oxidant-based disinfecting or sterilizing solutions.

[0111] Chelating agents have been used extensively to study the catalytic nature of metals in free radical oxidative processes. It was shown in the literature that EDTA enhances the reactivity of Fe(III) toward superoxide, forming Fe(II) (Buettner, Photochem. Photobiol. 28, 693-695 (1978)), and results in an accelerated H₂O₂ degradation process. It was also shown that diethylenetriamine pentaacetic acid (DETAPAC) drastically slows the reactivity of Fe(III) toward superoxide forming Fe(II). Regardless of the opposite roles of chelating agents in stabilizing or destabilizing H₂O₂ as described in the prior art, we discovered unexpectedly that some chelating agents such as EDTA which are reported in the literature to accelerate the Fe(III) conversion to Fe(II) superoxide-driven Fenton reaction can actually stabilize H₂O₂ when their concentrations are low. For such a chelating agent-H₂O₂-transition metal system there exists a maximum chelating agent concentration at which H₂O₂ in a disinfecting or sterilizing solution can reach a maximum stability. Chelating agents such as EDTA, DETAPAC and similar compounds may be employed in an amount at which the stability of H₂O₂ is at maximum. It has been found the Na₂EDTA concentrations in the solution between about 0.05% ^w/v and 0.35% ^w/v are preferred. The optimal amount of chelating agent or agents can be determined with the methods and teachings herein.

[0112] Anticorrosive Agents

[0113] Copper or brass corrosion inhibitors such as triazoles, azoles, benzoates, or five-membered ring compounds may be added to the decontaminant formula, as long as they do not interfere with the activity of the enzyme or disinfectant. A preferred corrosion inhibitor within this class is 1,2,3-benzotriazole, at a concentration between about 0.10 and 1.0% ^w/v. Commonly known corrosion inhibitors for aluminum and stainless steel such as borates, phosphates and tungsdates may also be included as long as they do not interfere with the activity of the enzyme or disinfectant. Preferably, the corrosion inhibitors do not form a deeply colored complex in the final solution, which would be unacceptable to the user. For example, it was found that the common corrosion inhibitor molybdate forms an unacceptably dark solution at pH 8.5. Typical concentrations for corrosion inhibitors are between about 0.001%-10% ^w/v.

[0114] Surfactants

[0115] The surface of a used medical instrument usually forms hydrophobic domains as the result of surface adsorption of the organic components in body fluid. Thus, a surfactant may be added to the decontaminant solution and/or to the cleaning solution as another cleaning agent. A surfactant can effectively remove most organic soils from the surface of a used instrument, and can stabilize the organic soils in the solution through the formation of emulsions or solubilized micelles. Another function of surfactants in a disinfecting/sterilizing solution is to solubilize the large molecular weight enzyme-hydrolyzed proteins and therefore to prevent them from forming precipitates.

[0116] The choice of surfactants significantly influences the rate of denaturation of enzymes. Anionic surfactants are generally denaturizing, whereas nonionic surfactants are neutral or even stabilizing to enzymes. Therefore the ratio of anionic:nonionic surfactants should not be too high, preferably below 1, and most preferably below 0.5. On the other hand, O₂ gas is constantly generated in H₂O₂ and/or PAA decontaminant solutions due to either disinfection or solution instability in the presence of organic soils. O₂ gas bubbles can anchor onto the hydrophobic domain surface of the instrument, which can effectively prevent the decontaminant solution from contacting the instrument surface and consequently defy the disinfection/sterilization action of the solution. Thus, wetting agents such as nonionic or anionic surfactants may be added to minimize O₂ gas bubbles from adhering to the used instrument surface.

[0117] Surfactants which are incorporated directly into high-level disinfecting solutions such as 7.5% H₂O₂ in combination with PAA should be stable in the solution and not interfere with disinfection. Such surfactants can be selected with conventional screening techniques. Makon 10 and Makon NF-12 are examples of suitable nonionic surfactants.

[0118] A preferred concentration range for surfactants in the final working solution is 0.001-1.0% ^w/v, with an even more preferred range of 0.01-0.25% ^w/v. Lower or higher concentrations may be used, however, depending upon the expected level of soils that need to be solubilized.

[0119] Antifoam Agents

[0120] Antifoam agents or defoamers may be added to either the cleaning component or the disinfectant component. Antifoam agents prevent excessive foam formation arising from agitation of any surfactant in the solution during dilution of the cleaning or decontaminant formula or during reprocessing of the device, e.g., during manual brushing of the narrow channels and other surfaces of an endoscope during the combined simultaneous cleaning and disinfecting step. Antifoam agents such as dimethyl polysiloxane (Antifoam C) (Dow Corning Corp., Midland, Mich.) may be utilized at a concentration <0.26 ml/liter or between about 0.005 and 0.05% ^w/v in a concentrate designed to be diluted 1:128 with water. The particular antifoam agent employed will depend upon the amount and type of surfactant utilized.

[0121] Soil Antiredeposition Acients

[0122] Soil antiredeposition agents or antisoils may be added to either the cleaning or disinfectant component to prevent redeposition of removed soil onto the cleaned device such as a medical device. Such agents are preferably used with a reusable medical device reprocessing system, which will be exposed to more soil than a single-use system. Thus, such agents are preferably used with reprocessing of multiple endoscopes in the same solution and are generally not useful for reprocessing kidney dialyzers with single-use solutions. Agents such as polyacrylic acid, carboxymethylcellulose and polyvinylpyrrolidone may be employed in conventional amounts as antiredeposition agents.

[0123] Preservatives

[0124] Preservatives may be added to the formulas of the present invention, particularly to a separate liquid enzyme formula to preserve the solution against contamination from microorganisms such as bacteria, yeasts and fungi. Conventional preservatives such as thimerosal may be employed, as long as they do not interfere with the performance of the cleaning and decontaminating system. EDTA may be employed, with a preferred concentration of 0.05%-0.10% ^w/v in the formula requiring preservation. EDTA is preferably used in combination with sorbic acid or potassium sorbate, the latter at a concentration of 0.10-0.20% ^w/v. Other common preservatives such as benzalkonium chloride (about 0.0044-0.0100% ^w/v) or methyl paraben (methyl 4-hydroxy benzoate about 0.1% ^w/v) may be employed. Other parabens such as benzyl-4-hydroxybenzoate, ethyl-4-hydroxybenzoate, propyl-4-hydroxybenzoate and butyl-4-hydroxybenzoate can also be employed as preservatives. Dowicide® 1 (Dow Chemical USA) is another example of an acceptable preservative. Dowicide® 1 is o-phenylphenol and is typically used at a concentration of 0.10-0.20% ^w/v. Preservatives may be used alone or in combination.

[0125] Tonicity Adjusting Agents

[0126] A tonicity adjusting agent or agents may be incorporated into either the cleaning component or decontaminating component to adjust the osmotic value of the formula or final cleaning and decontaminating solution to achieve greater chemical stability or to optimize another performance parameter. Tonicity adjusting agents are more important in some cases for kidney dialyzer reprocessing applications, since the dialyzers may contain dialysis fibers or membranes which respond to changes in solution osmolarity, changing their membrane characteristics and hence their filtration characteristics. Conventional tonicity adjusting agents such as simple electrolytes, e.g., sodium and potassium chloride, etc. and other non-ionic agents such as simple sugars, e.g., glucose, etc. may be employed.

[0127] Indicator Dyes

[0128] Indicator dyes can be added to enhance user compliance with the reprocessing steps and additionally to serve as an indicator of completion of the required decontamination soak time if desired. The dyes may be added to either the separate cleaning formula, a separate decontaminant formula, both separate formulas or to a combined cleaning plus decontaminant formula to differentiate the formula from water or other reprocessing solutions. An indicator dye such as FD&C blue dye #1 may be used for both purposes.

[0129] The dye is added to the formula at a concentration sufficient either to produce the desired color or to provide an indication of completion of decontamination soak time through a controlled redox reaction with the oxidant-based decontaminant. The latter reaction proceeds through a basic dye-bleaching mechanism, wherein the initially colored dye is bleached to a new color or to a colorless state with one of the components of an oxidant-based decontaminant, such as H₂O₂, at a defined rate which can be controlled through conventional control of dye-bleaching reactions. FD&C blue dye #1 or other dyes such as Pyla-Cert Mx3 (Grass Green) or FD&C green dye #1 are typically used at a concentration of between about 0.001 and 0.010% ^w/v, preferably 0.004% ^w/v in an enzyme concentrate formula designed to be diluted 1:128 in water.

[0130] Indicators, which are colorants which indicate the presence or absence of H₂O₂ or PAA or other peroxides may also be incorporated into these formulations.

[0131] Fragrances

[0132] Fragrances such as peppermint oil may be added to mask disagreeable odors or to create an agreeable fragrance. Fragrances may be added to either the separate cleaning formula, a separate decontaminant formula, both separate formulas or to a combined cleaning plus decontaminating formula. A typical concentration for peppermint oil is 0.204 ml/l in an enzyme concentrate formula designed to be diluted 1:128 in water. Concentrations between about 0.01 and 0.04% ^w/v are preferred.

[0133] Method

[0134] In use, a solution of per-compound and enzyme is prepared and one or more devices such as a medical device is contacted with this solution, preferably by immersion in the solution. The device may first be treated to remove loosely adhering soil, then contacted with this solution. The device is maintained in contact with this solution long enough so that substantially all protein and other soils are removed from the device surfaces and the device is disinfected or sterilized. The solution may then be removed from the device.

[0135] The sequence of combining the essential components for the solution which contacts the device will vary with the physical characteristics of the component employed, but the order of addition is not critical to the practice of this invention.

[0136] There is no particularly preferred form for manufacturing these materials. The two essential components, i.e. the cleaning and decontaminating components, may be formulated as separate components in dry or aqueous form, may be combined in a single solid form, or either one may be in a dry form while the other is an aqueous solution.

[0137] Other energy input may be employed to potentiate the solution's cleaning and decontaminating effect. For example, ultrasonic devices are known to potentiate the speed at which enzymes work in such circumstances in cleaning and may be employed.

[0138] The practice of this invention is not to be limited by temperature except by those temperature extremes which would substantially inactivate the capability of the enzymes employed, as is known to one skilled in the art.

[0139] It is also contemplated that certain components may be separately prepared in a manner to effect the timed release of that component or to prevent interaction of the components during tablet, granule or powder preparation and subsequent storage. For example, in certain instances it may be appropriate to separately prepare the per-compound and the enzyme in a manner to prevent or reduce their interaction in a tableting or granulation process and upon subsequent storage thereafter.

[0140] In addition, solutions or powders may contain agents for detoxifying residual per-compound as part of the overall process of cleaning, decontaminating and ultimately removing residual per-compound. Enzymes which catalyze the conversion of per-compounds to oxygen and water can be included in these formulations to remove residual per-compound in anticipation of reinserting devices into the body. For example catalases, which are organic enzymes that catalyze the degradation of per-compounds, can be incorporated particularly into tablets, granules and powders, more particularly in a time-release form. Additionally, metals such as the heavy metal transition elements which catalyze the conversion of per-compound to oxygen and water can be included preferably in a powder, granule or tablet form, again preferably in some delayed release form to provide a method for reducing any residual per-compound remaining in the solution to a non-toxic level after a given time interval. The use of transition metal catalysts for decomposing peroxides in a disinfecting solution is disclosed in U.S. Pat. No. 3,912,451, which is expressly incorporated herein by reference in its entirety.

[0141] The compositions and methods of the present invention can also be utilized with existing or suitably modified medical device reprocessing machines and their associated per-compound disinfection chemistries, in addition to the manual methods previously described. The Steris® System 1™ Sterile Processing System and its associated per-compound chemistry (Steris Corp., Mentor, Ohio) for example, can be utilized with the compositions and methods of the present invention. The Steris® System 1™ and associated chemistry is disclosed in U.S. Pat. No. 4,731,222: 4,892,706; 5,037,623; 5,077,008 and 5,091,343; Canadian patent Nos 1,273,774; 1,321,137; 1,320,030; Japanese patent Nos 1,745,511; 1,852,815; Austrian EP 0,397,352; 0,322,310 and EP 0,232,170, all of which are incorporated herein by reference.

[0142] The Steris® System 1™ and associated chemistry can be utilized with the compositions and methods of the present invention with or without modifying any of the machine hardware or software components or sterilant chemistry package currently in use. The latter package is a two compartment cup for powdered reagents which interact in water to form an antimicrobial solution. The presently marketed chemistry package utilizes sodium perborate and acetylsalicylic acid as two powdered reagents in a two compartment cup. A cleaning enzyme of the present invention such as subtilisin-A in the form of Novo Alcalase® 2.5 L can be combined with either the sodium perborate or the acetylsalicylic acid. The enzyme may be additionally protected from premature inactivation by either the perborate or acetylsalicylic acid during storage and prior to use by utilizing a coated granulation enzyme formulation. Upon dissolution with water according to the above Steris® patents and in accordance with the present invention, the combined enzyme, perborate and acetylsalicylic acid solution will produce a solution of sodium metaborate, a high-level disinfecting amount of PAA, salicylic acid and subtilisin-A. This solution can be delivered to the medical device according to the current Steris® System 1™ process wherein only a high-level disinfection step occurs over approximately a 12 min period at between about 43-48° C., however, with the composition and method of the present invention a simultaneous cleaning and disinfection step occurs over this same time period and at this same temperature. Following this step, the same machine processing steps would occur as without the enzyme, e.g., several rinsing cycles (the presently marketed instrument utilizes four rinsing cycles) and final processing steps. It is also possible to utilize other simultaneous cleaning and disinfection time intervals and temperatures, as long as the enzyme or enzymes utilized effect cleaning.

[0143] Preferred Compositions

[0144] The following are preferred compositions of the present invention, with applications, preferred delivery forms, components and concentration ranges:

Reusable Composition for Endoscone and Other Semi-Critical Medical Device Reprocessing

[0145] Aqueous solution of enzyme, to be dissolved 1 ounce into 1 gallon (128 ounces) of disinfecting agent solution. 1

[0146] Aqueous solution of disinfecting agent 2

[0147] In the above combined solution, the pH can be between 5-9. Another preferred reusable composition for endoscope and other semi-critical medical device reprocessing uses the same disinfecting solution as above along with the enzyme solution which follows: 3

[0148] In this system, the pH of the disinfecting solution can be between 1-3 where the disinfectants are stable and active and the pepsin is also active. The pH of the enzyme solution is also adjusted to between 1-3 with H₃PO₄ prior to adding it to the disinfectant solution. Thus, an additional buffer is not required to be added to the enzyme solution.

[0149] Another preferred composition for reprocessing dental instruments in an ultrasonic cleaning bath containing about 2000 ml water at 50° C. follows: 4

[0150] In the above example, the H₂O₂ from the monopersulfate combines with the peracetic acid precursor to produce sufficient peracetic acid to decontaminate the instruments. Sodium perborate monohydrate at about 1% ^w/v can also be used as a source of hydrogen peroxide. Combinations of Na₂HPO₄ (0.12% ^w/v) and NaH₂PO₄ (0.08% ^w/v) can be used to buffer the solution at pH 7.5 if desired. Alternatively, an acid-acting protease can be utilized at a pH preferably around 3-3.5 together with a suitable buffer.

[0151] In this system, the pH of the disinfecting solution can be between 1-3 where the disinfectants are stable and active and the pepsin is also active. The pH of the enzyme solution is also adjusted to between 1-3 with H₃PO₄ prior to adding it to the disinfectant solution. Thus, an additional buffer is not required to be added to the enzyme solution.

[0152] Single use Composition for Kidney Dialyzer Reprocessing:

[0153] Enzyme tablet formula, to be dissolved 1 tablet into each 250 ml of disinfecting/sterilizing agent solution just prior to use (combined solution pH range 1-6): 5

[0154] 6

[0155] The above composition is illustrative for kidney dialyzer applications in that it contains few components other than per-compounds and enzyme, which is preferable to minimize potential patient exposure to chemicals. Alternatively, the enzyme may be contained in a liquid formula, preferably stabilized with a sugar-based polyol such as sorbitol and an appropriate buffer. It is also preferable for dialyzer applications to utilize a 10-35 fold concentrate for the disinfecting/sterilizing solution, which is diluted with suitable purified water just prior to mixing with the enzyme and prior to use.

[0156] The following examples are presented to illustrate, but not limit, the scope of this invention. Alcalase® 2.5L was obtained from Novo Nordisk. Azocasein, a protein substrate for Alcalase®, was obtained from Sigma. Abs₃₉₀=absorbance measured on a spectrophotometer with wavelength set at 390 nm. A.U.=Anson Units. The following examples which incorporate H₂O₂ alone are not illustrative of the scope of the invention, since H₂O₂ alone does not achieve high- and intermediate-level decontamination. However, it is necessary to sufficiently stabilize H₂O₂, since destabilization of H₂O₂ will cause destabilization of peracids such as PAA or other peracids derived from an equilibrium mixture of H₂O₂ and a precursor organic acid.

EXAMPLE 1

Sporicidal Activity of Combined Proteolytic Enzyme and Hydrogen Peroxide (H₂O₂) Solutions.

[0157] The Association of Official Analytical Chemists (AOAC) test for Sporicidal Activity of Disinfectants, AOAC Official Methods of Analysis, 15th edition, 1995, was employed to evaluate the sporicidal activity of combinations of proteolytic enzyme and solutions containing H₂O₂.

[0158] No organic soil load was used for this example. Clean porcelain penicylinders (O.D. 8 mm±1 mm, I.D. 6 mm±1 mm, length 10 mm ±1) were sterilized in a 180° C. air oven for 2 h. Carriers were immersed for 15 min in a 72±4 h old broth culture containing spores of Bacillus subtilis (ATCC #19659) in soil extract-nutrient medium at a ratio of one carrier/ml broth culture, and carriers were placed into a glass petri dish matted with two layers of filter paper. The contaminated carriers were transferred in a vacuum desiccator containing CaCl₂ and the vacuum was drawn to 69 cm (27 inches) Hg for 20 min The contaminated carriers were dried in a desiccator for 42 h.

[0159] Test solutions of proteolytic enzyme and H₂O₂ were placed into duplicate test tubes and equilibrated to 20° C. in a water bath for 10 min. Five contaminated porcelain penicylinder carriers were placed into each of two tubes containing the individual test solutions. After a defined period of contact representing the sterilization time, the carriers were individually removed by hook needle to a subculture medium of Fluid Thioglycollate Medium USP. After completion of subcultures, each carrier was re-transferred to a fresh tube of the same subculture medium and incubated for 21 days at 37±2° C. After 21 days the tubes were examined for growth as determined by turbidity. Tubes demonstrating growth were subcultured onto agar medium for confirmation of the test organism. Tubes demonstrating no growth of the test organisms were heat shocked for 20 min at 80±1° C. and reincubated for 72±4 h at 35-37° C. Tubes without growth following the heat shock treatment were considered negative.

[0160] Two concentrations of H₂O₂, 6.5 and 15.0% ^w/w were utilized for this test. These were prepared from a 35.1% ^w/w solution of H₂O₂ in water (Aldrich Chemical Company, lot #06004 diluted with the required amount of deionized water. The proteolytic enzyme utilized for this test was subtilisin-A from B. licheniformis (Novo Nordisk A/S in Bagsvaerd, Denmark, hereinafter Novo). The subtilisin-A was prepared in 135.4 mg tablets containing 0.4 mg subtilisin raw material representing 0.012 A.U. enzyme activity/ tablet. One tablet was dissolved in 10.0 ml of one of the aforementioned H₂O₂ solutions for each test, 16 min prior to exposure of the contaminated penicylinder to the test solution. Thus, the final enzyme concentration in the H₂O₂ solutions representing each of the test solutions in Table 1 was 0.0012 A.U./ml. Each enzyme tablet also contained 40 mg sorbitol, 52 mg sodium carbonate, 7 mg tartaric acid, 30 mg N-acetyl cysteine and, 6 mg polyethylene glycol 3350 for formulating purposes. The enzyme tablets contained sodium carbonate as an alkaline buffer which increased the pH of the H₂O₂ solutions to the final pH indicated in Table 1. The final pH varied with the concentration of H₂O₂ since the original concentrated H₂O₂ contained acid to maintain a low pH for stability. The final pH values reported in Table 1 are within the known pH activity range of subtilisin-A. Deionized water was used as a negative control. 7

[0161] The results, shown in Table 1, indicated that while none of the solutions achieved complete sterilization, an increasing exposure time for a given H₂O₂ concentration resulted in fewer positive penicylinders. It is also clear hat a higher H₂O₂ concentration achieves a greater reduction in positive penicylinders in the same exposure time than a lower concentration. The results of Example 1 were utilized to design a second microbiology experiment to test the capacity of a combination H₂O₂-containing and subtilisin-A solution to achieve sporicidal activity within the 20-hour time limit imposed by the Food and Drug Administration (FDA) as a sterilization standard for high-level disinfection solutions.

EXAMPLE 2

Sporidal Activity of Combined Proteolytic Enzyme and Hydrogen Peroxide Solutions.

[0162] The microbiology test method, test solutions and enzyme tablets were the same as in Example 1 but the H₂O₂ concentrations and exposure times were different. Control H₂O₂ solutions without subtilisin-A were adjusted from the original pH 3.9 to either pH 5 with potassium dihydrogenphosphate and dipotassium hydrogen phosphate, or to pH 7 with sodium carbonate and potassium dihydrogenphosphate. The same concentration of subtilisin-A (0.0012 A.U./ml of H₂O₂) was utilized for each test solution except the control H₂O₂ solutions.

[0163] The results are presented in Table 2. Subtilisin-A solutions containing 7.5%, 4.9% and 2.5% H₂O₂ were sporicidal (no positive penicylinders) at 3, 8 and 10 h, respectively. Solutions of 7.5% H₂O₂ alone at a pH in the range of about 3.9-7 were all sporicidal within 4 h. The inherent sporicidal activity of H₂O₂ was not reduced with the addition of subtilisin-A (compare test number 7 with numbers 10-12). The difference in pH is not considered significant here, as it has been reported that sporicidal activity of H₂O₂ does not differ between pH 6.5 and 8.0 and increases at pH 5. The bactericidal. fungicidal and sporicidal properties of hydrogen peroxide and peracetic acid (Baldry, M. G. C., J App Bact, 1983; 54:417-423). This demonstrates that a combination of a proteolytic enzyme such as subtilisin-A with H₂O₂ can achieve sterilization of spores on an object and thus can meet one of the primary standards for a high-level disinfectant or sterilant for medical devices. 8

EXAMPLE 3

[0164] Effect of pH on Protease Activity in 7.5% ^w/w H₂O₂

[0165] Alcalase® 2.5 L, 0.8 grams (Novo Nordisk) was mixed with 1 liter of 7.5% ^w/w H₂O₂ (diluted from 35% H p 2 Aldrich) solution at room temperature, thus yielding an initial enzyme activity of 0.002 A.U./ml. At each time point, an aliquot of the mixed solution was removed and assayed for enzyme activity using an Azocasein substrate method (Novo Nordisk). The enzyme solution was allowed to hydrolyze azocasein for 30 min at 40° C. Undigested protein was precipitated with trichloroacetic acid and digested protein was quantitated by spectrophotometry. Standards and samples were prepared using Alcalase® 2.5 L with 0.2 M trishydroxymethylaminomethane (Sigma, “Trizma Base”) as a diluent. One ml sample or enzyme standard was added to test tubes and equilibrated to 40° C. for approximately one min. At precisely timed intervals, 5 ml substrate solution was added to each tube. The substrate solution consisted of 0.6% ^w/v Azocasein powder (Sigma), 5% ^w/v urea solution, 0.2 M tris(hydroxymethyl)aminomethane buffer at pH 8.5, and the final solution was adjusted to pH 8.5 with dilute H₂SO₄. After adding substrate, the solutions were mixed using a vortex mixture and returned to the 40° C. water bath for exactly 30 min, after which time 5 ml 10% ^w/v trichloroacetic acid (Aldrich) was added to each tube. After room temperature incubation for 15-20 min, the solutions were gravity filtered using Whatman #3 filter paper into clean test tubes and the absorbance of each solution was determined at 390 nm (Abs₃₉₀) on a Shimadzu UV-1601 spectrophotometer using a standard blank. A calibration curve was generated using the standard absorbances, and then linear regression was used to determine the sample enzyme concentrations.

[0166] Table 3 shows the enzyme activity in the mixture of 0.8 mg/ml Alcalase® 2.5 L and 7.5% ^w/w H₂O₂ solution at room temperature at pH 3.9. There is considerable loss in enzyme activity almost immediately due to the stressed condition of 7.5% H₂O₂ (data not shown). At 240 min, however, there is partial recovery of enzyme activity.

[0167] After the initial 30 min, the mixed enzyme/H₂O₂ solution was split into two parts; one part remained unchanged and 2 M Tris buffer was added to the other part in a volume ratio of 9:1 to achieve 0.2 M Tris at pH 8.5. As shown in Table 3, recovery of enzyme activity was more striking in 0.2 M Tris buffer at pH 8.5 than at pH 3.9. This phenomenon was also observed in a mixed solution of Alcalase®/PAA-H₂O₂. It is yet to be determined if the enzyme activity recovery occurred in the enzyme/H₂O₂ mixed solutions or during the enzyme activity assay. This example, however, demonstrated the pH effect on enzyme activity in the presence of a high concentration of hydrogen peroxide. 9

EXAMPLE 4

[0168] Effect of pH on Protease Activity in 7.5% ^w/v H₂O₂ Solutions

[0169] The enzyme activity lost under stressed conditions, such as in H₂O₂ or PAA solutions at concentrations capable of achieving sporicidal activity (H₂O₂) or high-or intermediate-level disinfection and sterilization (PAA), respectively, can be partially recovered after an aliquot of the solution is diluted with 0.2 M Tris buffer (as in the enzyme manufacturer's recommended method for determining enzyme activity). Thus, in this example, an in-situ protease activity assay method was employed which deviated from the manufacturer's method but better mimicked the real cleaning process.

[0170] Solutions of 7.5% ^w/v H₂O₂ at various pH levels were prepared by adjusting the pH of 35% ^w/w H₂O₂ solution (Degussa) with 10 mM K₃PO₄ in a volumetric flask in the presence of K₂HPO₄ (phosphate buffer, PB). The solution was diluted with 10 mM KH₂PO₄ solution of the same pH. The 7.5% ^w/v H₂O₂ solutions were then mixed with Alcalase® 2.5 L in a volume ratio of 1280:1 and thus, had an initial enzyme activity of 0.002 A.U./ml. Azocasein powder was added to the H₂O₂/Alcalase® mixed solutions to yield 0.4% ^w/v azocasein. After 30 min at 23° C., 5 ml azocasein solution was mixed with 5 ml 10% ^w/v TCA solution. Precipitates were removed after 15 min by filtration, and the supernatant Abs₃₉₀ was measured. As a comparison for the cleaning efficacy, 0.4% ^w/v azocasein was also added to 10 mM potassium phosphate buffer solutions mixed with Alcalase® 2.5 L in the same volume ratio of 1280:1 but without H₂O₂. The absorbance of two other azocasein solutions (0.4% ^w/v) containing 7.5% ^w/v H₂O₂/phosphate buffer and phosphate buffer alone, respectively, without Alcalase® 2.5 L were measured as controls. While azocasein dissolves within a few minutes with stirring in all of the above solutions at pH 8 and pH 5, it virtually did not dissolve at pH 2. The results are given in Table 4 as absorbance of hydrolyzed azocasein solution at 390 nm containing 7.5% ^w/v H₂O₂ and Alcalase® 2.5 L at various pH values at 23° C. The volume ratio of H₂O₂ solution or 10 mM potassium phosphate buffer to Alcalase® 2.5 L was 1280:1. 10

[0171] Although the azocasein substrate can be dissolved in the solutions at pH 8 and pH 5, the Abs₃₉₀ of the solutions without enzyme remained as low as that of the solution at pH 2. In contrast, the Abs₃₉₀ with enzyme solutions increased about 40-fold. Table 4 also shows that the azocasein hydrolysis rate had no significant change within 30 min in the presence of enzyme when azocasein was poorly dissolved in the solutions at pH 2. At pH 8 and pH 5 respectively, the azocasein hydrolysis rate was 1.6 and 1.9 times higher in 7.5% ^w/v H₂O₂ solutions containing enzyme than in the corresponding phosphate buffer PB solutions containing enzyme, indicating the existence of synergism in protein hydrolysis/cleaning efficiency between H₂O₂ and enzyme.

EXAMPLE 5

Compatibility of Various Protease Enzymes With H₂O₂ Solutions Over Time

[0172] The initial H₂O₂/enzyme mixed solutions were prepared following the procedures described in Example 4. One half hour prior to various time points (listed in Tables 5a-d), azocasein powder was added to the solution in an amount of 0.4% of the total solution weight. Abs₃₉₀ of the solution was measured as in Example 4 except that the supernatant was diluted 6-7 fold to avoid saturation of the UV spectrophotometer. The results are presented in Tables 5a-5d for the protease enzymes Alcalase, Neutrase, chymotrysin and Savinase respectively. 11

[0173] The results in Table 5a are obtained as follows: (dilution factor)×(Abs₃₉₀) hydrolyzed azocasein solution containing _7.5% ^w/v H₂O₂ and Alcalase® 2.5 L at 23° C. at either pH 5.0 or pH 8.0. The mixing volume ratio of 7.5% ^w/v H₂O₂/PB to Alcalase® 2.5 L was 1280:1.

[0174] The cumulative enzyme activity with 7.5% H₂O₂ solution was not only about 1.6-2.0 higher than that without H₂O₂, but was either constant or even slightly increased over the 8 hour period. These results implied that Alcalase® was stable in and compatible with 7.5% H₂O₂ solutions for at least 8 h at pH 5-8. This demonstrated the potential reusability of a combination cleaning and high- or intermediate-level disinfecting or sterilizing solution containing high concentrations of hydrogen peroxide in combination with a peracid. Although the absolute enzyme activity at pH 5 was slightly lower than at pH 8, the relative enzyme activity of the solution with H₂O₂ to that without H₂O₂ was higher at pH 5.

[0175] Table 5b shows results from an additional experiment employing the same method but with the enzyme Neutrase™ 0.5 L. These results are presented, normalized with the dilution factor as Abs₃₉₀ hydrolyzed azocasein solution containing _7.5% ^w/v H₂O₂ and Neutrase™ 0.5 L at pH 5.6 at 23° C. The mixing volume ratio of 7.5% ^w/v H₂O₂/PB to Neutrase™ 0.5 L was either 1280:1 or 2560:1, and the nominal enzyme activity was 0.00048 and 0.00024 A.U./ml, respectively. 12

[0176] These results demonstrated the feasibility of utilizing an enzyme with a more neutral or even slightly acidic pH profile.

[0177] An additional experiment employing the same method was conducted with human chymotrypsin. The results are presented in Table 5c as Abs₃₉₀ hydrolyzed azocasein solution by human chymotrypsin with and without H₂O₂ at 23° C. and at pH 7.75. 13

[0178] Lower enzyme activity was seen in the presence of the H₂O₂ solution than in the phosphate buffer solution alone. However, substantial enzyme activity remained and even increased over time.

[0179] Table 5d. Absorbance (normalized with dilution factor) of hydrolyzed Azocasein solution at 390 nm containing 7.5% ^w/v H₂O₂, 0.2% ^w/v Savinase™ 16.0 L and/or 1% ^w/v benzotriazole at pH 8.5 at 23° C. All the solutions contained 7.4 mM Na₃PO₄, 1% ^w/v Azocasein and the solution pH was adjusted to 8.5 with HCl or NaOH solutions. 14

[0180] These results show that the corrosion inhibitor benzotriazole alone or H₂O₂/benzotriazole mixed solutions do not have significant proteolytic activity and that benzotriazole is compatible with the mixed systems of H₂O₂/Savinase™ 16.0 L. It is expected from these results that benzotriazole will also be compatible with mixed enzyme and H₂O₂ plus peracid solutions, which would be capable of meeting high- or intermediate-level disinfection requirements.

EXAMPLE 6

[0181] Na₂EDTA Stabilization of H₂O₂ in Enzyme/Bovine Hemoglobin Solutions

[0182] Table 6 shows the H₂O₂ concentration change over a period of 28 hours in solutions containing H₂O₂, Na₂EDTA, Alcalase® 2.5 L and bovine hemoglobin (Sigma, St. Louis, Mo.). The concentrations, other than that at 28 h, were initial concentrations after mixing 7.5% ^w/w H₂O₂ with Na₂EDTA powder, 15% ^w/w bovine hemoglobin, 10.7% ^w/v Alcalase® 2.5 L formulation (see Table 7c formula D), and NaOH to adjust pH. Initial Alcalase® activity was 0.092-0.094 A.U./ml. The assay was performed at room temperature. H₂O₂ concentration measurement was based upon a standard method utilizing a KMnO₄ titration. 15

[0183] Hemoglobin significantly reduced H₂O₂ concentration, with a 13 fold faster reduction with hemoglobin present (solution 3) than with hemoglobin absent (solution 2). However, in the presence of 0.11% ^w/v Na₂EDTA, H₂O₂ was as stable with hemoglobin as without hemoglobin (compare solutions 4 and solution 2). With further increases in Na₂EDTA concentration, however, the stability of H₂O₂ decreases (solutions 5 and 6).

[0184] While the scientific literature indicates that Na₂EDTA consistently accelerates Fenton-reactions such as the hemoglobin-mediated degradation of H₂O₂, these results surprisingly show that a solution of enzyme and H₂O₂ can be stabilized against a principle component of blood with the proper level of Na₂EDTA. This further demonstrates the reusability of simultaneous cleaning and disinfecting solutions of the present invention containing high concentrations of hydrogen peroxide.

EXAMPLE 7

[0185] Effect of Chelating and Other Stabilizing Agents on H₂O₂ Stability in Enzyme/Blood Solutions

[0186] Fifty ml of 7.5% ^w/w H₂O₂ solution, prepared by diluting 35% ^w/w H₂O₂ solution (Degussa), was mixed with 1 ml of chelating agent (in solution at various concentrations), 1.95 ml of the enzyme formulation containing 10.7% ^w/v Alcalase® 2.5 L (see formula D, Table 7c), and 1.1 ml duck blood. Na₂EDTA and phenanthroline chelating agents were tested. Na₂EDTA solutions were adjusted to pH 8.1 using Tris buffer, and Alcalase® 2.5 L formulations were similarly adjusted to pH 8.5. The pH of phenanthroline solutions was not adjusted. Table 7a lists the concentrations of H₂O₂, chelating agents, Alcalase® 2.5 L and duck blood, as well as pH values after mixing. For mixed solutions without chelating agent and/or duck blood, 1 ml or 2.1 ml distilled water was added so that the resulting concentrations of H₂O₂ and Alcalase® 2.5 L were the same. The initial Alcalase® enzyme activity was about 0.0098 A.U./ml. The H₂O₂ concentrations of the mixed solutions were measured using a standard KMnO₄ titration method and traced for 5 days. The results are listed in Table 7a. 16

[0187] H₂O₂ was very unstable in the absence of chelating agents (solution 5), losing 16% and 72% over 1 and 5 days, respectively. Addition of phenanthroline at pH 7.8 and 6.47 (solutions 3 and 4) causes H₂O₂ to be even more unstable. In the presence of Na₂EDTA, however, H₂O₂ stability was significantly enhanced, losing only 7% and 22-32% for 1 and 5 days, respectively (solutions 1 and 2). These results demonstrated that a solution of enzyme and H₂O₂ can be stabilized to retain activity even in the presence of blood with the proper concentration of Na₂EDTA, and further demonstrate the reusability of simultaneous cleaning and disinfecting solutions of the present invention containing high concentrations of hydrogen peroxide.

[0188] Table 7b shows the results of using H₂O₂ stability seven days after 100 ml _7.5% ^w/v H₂O₂ solution was mixed with 0.775 ml enzyme formulation and various amount of organic soils (duck blood or calf serum) at 23° C. The pH of the mixed solution was 8.0±0.1. 17

[0189] 18

[0190] These results demonstrated that enzyme formulations with various buffer systems can maintain H₂O₂ concentrations.

[0191] Another example, which follows, illustrates the utility of a boric acid (H₃BO₃) buffer in the compositions of the present invention. 19

[0192] it can be seen that in the presence of duck blood, only sample 1 containing 80 mM boric acid maintained hydrogen peroxide concentration at 6%.

[0193] The following examples illustrate the effect of boric acid on hydrogen peroxide stability in a combined enzyme-peroxide formulation in the presence of an anticipated amount of whole blood which would be expected to be present under real use conditions in a solution system designed for reprocessing multiple endoscopes over an 8 day period. 20

[0194] 21

[0195] The preceding examples of H₂O₂ stabilization in the presence of varying amounts of blood indicate that amounts of boric acid between about 1 and 80-100 mM, or 0.006 and 0.62% ^w/v in the final solution can prevent degradation of H₂O₂ over eight days. The results of examples 6 and 7 also indicate that amounts of Na₂EDTA between about 0.05-0.35% ^w/v in the final solution can prevent degradation of H₂O₂.

EXAMPLE 8

Proteolytic Enzyme Cleaning Efficacy in Peracetic Acid (PAA) Solutions at Various pH

[0196] Solutions of 1.08% ^w/v H₂O₂/0.2 PAA were prepared by diluting a stock of 4.9% ^w/w PAA (Degussa, density 1.12 g/ml) and pH values were adjusted with 1 N NaOH in the presence of 10 mM K₂HPO₄ solution. The PAA/H₂O₂ solutions were then mixed with Alcalase® 2.5 L in a volume ratio of 1280:1, yielding a solution with a nominal enzyme activity of 0.0021 A.U./ml. Azocasein powder was added to the H₂O₂/Alcalase® mixed solutions to yield an Azocasein concentration of 0.4% ^w/v. After 30 min incubation at 23° C., 5 ml of the Azocasein solution was mixed with 5 ml of 10% TCA solution. After 15 min, the precipitates were filtered, and the supernatant absorbance was measured at 390 nm using a UV-visible spectrophotometer. As a comparison for the cleaning efficacy, Azocasein 0.4% ^w/v was also added to 10 mM potassium phosphate buffer solutions mixed with Alcalase® 2.5 L in the same volume ratio of 1280:1 but without PAA/H₂O₂. The absorbance of two other Azocasein solutions (0.4% ^w/w) containing 0.2% PAA/1.08% H₂O₂/phosphate buffer and phosphate buffer alone, respectively, without Alcalase® 2.5 L were measured as a control. While it can be dissolved in all the above solutions at pH 8 and 5 within a few minutes with stirring, Azocasein virtually does not dissolve at pH 2.

[0197] The results show the Abs₃₉₀ of hydrolyzed Azocasein solution containing 1.08^w/v % H₂O₂, 0.2^w/v % PAA and Alcalase® 2.5 L at various pH values at 23° C. The volume ratio of H₂O₂/PAA solution or 10 mM potassium phosphate buffer to Alcalase® 2.5 L is 1280:1. 22

[0198] Comparing absorbance data in Tables 4 and 8 demonstrate that the mixed solutions of 0.2% PAA/1.08% H₂O₂ without enzyme can hydrolyze 4-5 times more Azocasein than a solution of 7.5% ^w/v H₂O₂ without enzyme in the same time period at pH 5 and pH 8. Furthermore, although 0.4% Azocasein was poorly dissolved in the PAA/H₂O₂ solutions at pH 2, the partially dissolved Azocasein molecules are hydrolyzed about two times faster than in the 7.5% H₂O₂ solutions, with or without enzyme. Table 8 shows that the absorbance of hydrolyzed Azocasein solutions in the mixed solutions of PAA/H₂O₂ and enzyme is higher than in the corresponding phosphate buffer solutions containing enzyme at pH 5 and pH 8. The data indicate that synergism in cleaning efficacy exists between PAA/H₂O₂ and protease at pH 8, but not at pH 5.

EXAMPLE 9

[0199] Proteolytic Enzyme Compatibility With Peracetic Acid Solutions

[0200] A solution of 0.2% ^w/v PAA/1 .08% ^w/v H₂O₂ was adjusted to pH 5.6 with 1 N NaOH prior to mixing with Neutrase™ 0.5 L in a volume ratio of 1280:1. The mixed Neutrase™/0.2% PAA/1.08% H₂O₂ solution, with a nominal enzyme activity of 0.00048 A.U./ml was then mixed with Azocasein powder to result in 2% ^w/w Azocasein at 23° C. Three ml of the above solution were removed at each time point and mixed with 3 ml of 10% TCA solution. The precipitates were filtered after 15 min, and the supernatant absorbance was measured at 390 nm using a UV-visible spectrophotometer. As a comparison for the cleaning efficacy, 2% ^w/w Azocasein was also added to 10 mM potassium phosphate buffer solutions mixed with Neutrase™ in the same volume ratio of 1280:1 but without PAA/H₂O₂, and also at pH 5.6. The absorbance of 2% ^w/w Azocasein solutions containing 0.2% PAA/1.08% H₂O₂/10 mM phosphate buffer, pH 5.6, without Neutrase™ 0.5 L was measured as a control.

[0201] Azocasein/TCA solutions were diluted 6-7 fold to avoid saturation of the UV spectrophotometer. The results show absorbance of hydrolyzed Azocasein solution at 390 nm containing 1.08% ^w/v H₂O₂/2.0% PAA and NeutraseT™ 0.5 L at pH 5.6 at 23° C. 23

[0202] It can be seen that Neutrase™ 0.5 L was 100% inactivated in the solution containing 0.2% PAA and 1.08% H₂O₂. Neutrase™ 0.5 L maintained its activity in the presence of 7.5% ^w/v H₂O₂ in example 5b, indicating that enzyme activity in the solutions of the present invention is difficult to predict.

EXAMPLE 10

Enzyme Activity in 0.368% ^w/v PAA/2% ^w/v H₂O₂, Assayed With Novo Method

[0203] In a 100 ml volumetric flask, a solution was prepared using 7.5 ml of 4.9% ^w/w (Degussa) PAA, 1 ml enzyme, and diluted with 0.2 M tris(hydroxymethyl)aminomethane to control the pH at 8.5 to yield a final concentration of 0.368% ^w/v PAA/2% ^w/v H₂O₂. Two of these solutions were prepared using two different enzymes resulting in a 1% ^v/v dilution of both Alcalase® 2.5 L and Savinase® 16.0 L. The nominal initial enzyme activity of Alcalase® was 0.0264 A.U./ml in this example. The nominal initial enzyme activity of Savinase® was 0.0529 A.U./ml. The Novo Nordisk assay method described previously was used with a slight modification. A timer was started as soon as the combinations of enzymes and PAA/H₂O₂ were mixed. At periodic intervals, aliquots of the enzyme/PAA/H₂O₂ solutions were removed and diluted 64 times to react with the 0.6% ^w/w Azocasein substrate solution in a water bath at the modified temperature of 30° C. for 30 min. After stopping the reaction with 10% TCA and filtering the precipitate, the supernatants were analyzed on a spectrophotometer at 390 nm. 24

[0204] A 1% solution of Alcalase® had greater stability and proteolytic activity than the 1% Savinase® solution in the presence of 0.368% PAA/2% H₂O₂ combination after 6.8 hours. Alcalase® showed a 13% decrease in activity after 7.5 hours and Savinase® showed a 47% decrease after 6.8 hours. These results indicate that Alcalase® is a better candidate than Savinase® for enzyme solutions involving a longer term exposure to high- or intermediate-level disinfecting concentrations of PAA and H₂O₂. These data also indicate that the stability of an enzyme in such solutions cannot be predicted a priori, since both enzymes are closely related alkaline serine proteases.

EXAMPLE 11

[0205] PAA Stability in Proteolytic Enzyme Solution With Organic Load

[0206] Two ml of an enzyme solution containing 10.7% Alcalase® 2.5 L at pH 8.5 (formula D, Table 7c, with additional boric acid (0.018^w/v %)) was mixed to a final volume of 50 ml with a solution of 0.2^w/v % PAA/1 .0^w.v % H₂O₂ at 23° C. The resulting solution was pH 8.0 and had a nominal enzyme activity of 0.0107 A.U./ml. Twenty-four hours after bovine hemoglobin powder was added to the above solution, the PAA content was measured using a redox titration method as follows: The sample was weighed (10-15 grams) and transferred to a 250 ml erlenmeyer flask containing 50 ml of 1 N ice-cooled sulfuric acid. After adding two drops of ferroin indicator (Sigma), the H₂O₂ content in the sample was reacted with 0.1 N ceric sulfate (Fisher) in a redox titration until the disappearance of a salmon color and the appearance of a light blue color. Ten ml of 2.5 N potassium iodide was added in excess so that the hydroiodic acid formed in the acidic environment would react with PAA to liberate iodine. A standard solution of 0.0206 N sodium thiosulfate was used to titrate the liberated iodine. A few ml of starch indicator were added just after the solution turned brown-yellow, and then additional sodium thiosulfate was added until the solution maintained a salmon color for 15 seconds. The endpoint of the titration was used to calculate the PAA content. Samples without Alcalase or without hemoglobin were also prepared and the PAA content was assayed for comparison. 25

[0207] Table 11 shows that both Alcalase® and bovine hemoglobin cause a decrease in PAA concentration. The mechanism of action of Alcalase and hemoglobin in reducing PAA concentration may be entirely through the reduction of H₂O₂ concentration in the solution, which in turn shifts the equilibrium between H₂O₂ and PAA toward H₂O₂. Alternatively, since the original PAA/H₂O₂ solution used herein was a dilution of a more concentrated solution of PAA and was used immediately following dilution, the equilibrium between PAA and H₂O₂ may have taken time to re-establish, resulting in the appearance of a loss of concentration of PAA. Nonetheless, the PAA concentration after 24 hours in this system was still above 0.052%, which is above the minimum effective concentration of 0.05% for PAA for a reasonable high- or intermediate-level disinfection time of 25 min at 20° C.

EXAMPLE 12

Peracetic Acid and H₂O₂ Disinfecting and Sterilizing Efficacy in Enzyme/Blood Systems

[0208] Sporicidal activity was tested using the method of Example 1 except that 17 days of culture was employed instead of 21 days. Various solutions containing H₂O₂ or a mixture of H₂O₂ and PAA, with or without Na₂EDTA, with or without an Alcalase® 2.5 L enzyme formula and with or without added whole sheep blood were tested. Except where indicated, whole sheep blood was added to the solution just prior to the start of the microbiology experiment. Exposure time was varied according to the concentration of sterilant. Table 12a presents the results. 26

[0209] 27

[0210] The results show that a solution containing 7.5% H₂O₂, Alcalase® and 2% ^v/v whole blood can achieve sterilization against B. subtilis in 6 h. The results also demonstrate that when whole blood is added to the solution five days prior to the start of the test, the H₂O₂ solution is sufficiently destabilized such that sterilization is not achieved. This is believed to be caused by the Fenton-reaction mediated degradation of H₂O₂ by hemoglobin. However, with addition of Na₂EDTA to the H₂O₂ solution, sterilization is achieved. This result was unexpected, as it has been reported in the literature that Na₂EDTA accelerates the Fenton-reaction. The results additionally demonstrate that solutions containing 0.2% PAA with H₂O₂ between 1 and 7.5%, Alcalaseo and 2% ^v/v whole blood can achieve sterilization in various times. The results further demonstrate that a solution containing PAA alone, with Alcalase® and whole blood can achieve sterilization. The results still further demonstrate that existing high- or intermediate-level disinfection and sterilization products such as Peract-20™ and Sporox® can still achieve sterilization in the presence of whole blood when combined with an enzyme formula of the present invention.

[0211] The results of examples 10, 11 and 12 indicate that the compositions and methods of the present invention can achieve simultaneous cleaning (e.g., protein removal, etc.) and high-level disinfection or sterilization.

EXAMPLE 13

Enzyme Activity of Human Enzyme/Phosphate Buffer/PAA/Hydrogen Peroxide (H₂O₂) Mixed Systems With Azocasein as an Organic Soil Model

[0212] Table 13a lists the enzyme- activity data of human trypsin (Sigma) in 10 mM phosphate buffer and in 0.2% PAA/1.05% H₂O₂ solution at 23° C. at pH 7.5. The activity for the PAA/H₂O₂ solution alone as a control is also given. The initial trypsin/phosphate buffer solution was prepared as follows: 124 μg human trypsin was dissolved in ml of 10 mM K₃PO₄ at pH 7.5. A volume of 4.05 ml of 10 mM K₃PO₄ buffer was combined with 0.45 ml initial trypsin buffer solution and 45 mg Azocasein to prepare the trypsin/phosphate buffer solution for the test.

[0213] A 0.22% ^w/v PAA/1.17% ^w/v H₂O₂ solution was prepared using a dilution of Degussa's 4.9% ^w/w PAA/26.6% ^w/w H₂O₂ in a volumetric flask. A volume of 4.05 ml of the latter solution was mixed with 0.45 ml initial trypsin/phosphate buffer solution and 45 mg Azocasein powder. The final concentrations were 12.4 mg/mL trypsin, 0.2% ^w/v PAA, 1.05% ^w/v H₂O₂ and 1% ^w/v Azocasein.

[0214] As a comparison, the solution to test the activity of PAA/H₂O₂ alone on the Azocasein substrate was prepared in the same manner as the solution above, without added trypsin, using a dilution of Degussa's 4.9% ^w/w PAA/26.6% ^w/w H₂O₂ in a volumetric flask for a concentration of 0.20% PAA/1.05% H₂O₂. Azocasein powder was added to the 0.2% PAA/1.05% H₂O₂ solution which resulted in a 1% ^w/v Azocasein protein.

[0215] At 2.8 and 7 h, 1.5 ml sample aliquots were removed from the above three solutions and reacted with 1.5 ml of 10% ^w/v trichloroacetic acid stop reagent. After incubating for 15-20 min, the solutions were filtered using Whatman #3 filter paper with a syringe and a Gelman 13 mm filter holder. The filtered solutions were then analyzed with a Shimadzu UV-visible 1601 spectrophotometer Abs₃₉₀. 28

[0216] The results demonstrate that trypsin is active in the PAA/H₂O₂ solution. The data further indicate that there is not a large difference in the proteolytic activity between trypsin alone and trypsin in 0.2% PAA/1.05% H₂O₂ solution. An additional example with human chymotrypsin follows. The solutions were similarly prepared as above. 29

[0217] The results show that human chymotrypsin retains significant activity in a PAA/H₂O₂ solution suitable for high- or intermediate-level disinfection or sterilization in a reasonable regimen time. Moreover, since it has been previously demonstrated herein that the presence of a proteolytic enzyme such as trypsin or chymotrypsin will not interfere with sterilization and high- or intermediate-level disinfection of an oxidant-based solution such as PAA, the results of Examples 13a and 13b demonstrate that the compositions and methods of the present invention can achieve the requirements for a simultaneous protein removal (cleaning) and high- or intermediate-level disinfection or sterilization system for endoscope or kidney dialyzer reprocessing.

EXAMPLE 14

pH Adjustment of H₂O₂ and/or PAA Solutions by Adding Enzyme Formulations

[0218] To effectively increase the pH of an acidic disinfectant solution, such as a solution containing a large amount of concentrated H₂O₂ (e.g., 7.5% ^w/w) in combination with a low concentration of peracid (e.g., 0.2% ^w/w) or a H₂O₂/PAA solution, to within a range of 5-9 when mixed with an enzyme formulation, a large amount of buffering agent(s) must be incorporated in the enzyme formulation. This is not only due to the low pH values of acidic H₂O₂ and/or PAA solutions at which they are stable, but also because large mixing ratios of H₂O₂ and/or PAA solutions to enzyme formulation (i.e., 1 gallon H₂O₂containing solution:1 oz liquid enzyme formulation are required in many practical applications for the compositions and methods of the present invention, especially those applications involving endoscope reprocessing. However, many buffering agents are either insoluble or unstable at the required high concentrations in liquid enzyme formulations which contain a large amount of organic solvents such as propylene glycol to stabilize the enzyme to achieve a reasonable shelf life. For example, prior art buffers such as 0.5-1 M phosphate buffer and borate buffer solutions in the presence of 23% propylene glycol, 15% Makon 10 and 10.7% Alcalase® form two liquid phases and a precipitate, respectively. It was discovered that large amounts of buffering agents can be dissolved in low water content enzyme solutions if proper hydrophobicity of the buffering agent molecules is carefully selected. A tromethamine or Tris buffer, 2-amino-2-hydroxymethyl-1,3-propanediol, paired with an organic acid such as acetic acid, and 2-amino-2-methyl-1-propanol, mono-, di- and triethanolamine (TEA) paired with either an inorganic or organic acid are some of the qualified buffering agents. High Tris buffer concentration sometimes can cause the solution to be metastable if the pH of the mixed enzyme solutions is adjusted with inorganic acid, thus the Tris buffer compositions of the '555 patent are unsuitable. Examples of suitable buffers are presented in Table 14a. 30

[0219] Alcalase®, the enzyme utilized in these examples, exerts maximum protein removal efficacy in combination with H₂O₂ at a pH greater than about 7 to 7.5. The results of these examples show that all of the buffers evaluated will produce an acceptable initial pH for Alcalase® activity. It is generally desired for solution reuse purposes to have a solution which can be used for at least 1-5 days, especially for endoscope and other semicritical medical device reprocessing. These results show that buffers in accordance with the present invention can achieve the desired pH stability over a one day use period, which is for practical purposes 16 hours (two 8-hour work shifts in a hospital or clinic). It is also clear from the foregoing examples that these buffers could maintain a proper pH for a neutral protease such as Neutrase™ (pH optimum 5.5-7) for an even longer period, provided they were adjusted to a somewhat lower initial pH. The effect of various buffers on enzyme activity over typical solution reuse times as well as short term shelf stability times was evaluated in the examples in Table 14b. 31

[0220] 32

[0221] The results presented in Table 14b show that in comparison to the Tris buffer alone (solution C), a 2 M TEA buffer used alone (solution A) achieves maximum stability of enzymatic activity. The results also show that of the combination boric acid buffers evaluated, the combination of boric acid and Tris buffers in solutions D & E achieves maximum enzyme activity stability.

EXAMPLE 15

[0222] Effect of Enzyme and Antifoam Agent on Blood-Derived Foam

[0223] Table 15a compares foam volume versus reaction time of bovine blood with 7.5% H₂O₂ solutions at 23° C. The solution contained 40 ml of 7.5% ^w/w H₂O₂ (Degussa), 20 mg Antifoam C (Dow Corning), 200 mg Alcalase® 2.5 L and 0.8 ml whole bovine blood which were mixed in a 1 L Tripour beaker. Table 15b illustrates the relationship between foam volume and reaction time of bovine blood with 0.2% PAA/1.08% H₂O₂ solutions at 23° C. The solution contained 40 ml of 0.2% PAA/H₂O₂ (Degussa), 20 mg Antifoam C, 200 mg Savinase® 16.0 L and 0.8 ml bovine blood which were mixed in a 250 ml Tripour beaker. The pH of 7.5% ^w/w H₂O₂ and 0.2% PAA/H₂O₂ solutions were adjusted to 7.7 with 2 M Tris buffer prior to mixing with other ingredients. As a comparison, data for the mixed solution systems without enzymes and/or Antifoam C were also included. The foam volumes were measured immediately after the blood was added to the solutions. The formation of foam reflects the redox reaction between Fe(II) in the hemoglobin of the blood and H₂O₂ alone or H₂O₂/PAA and the catalytic degradation of H₂O₂ or H₂O₂/PAA by Fe(II) and Fe(III). One result of these reactions was released from the solutions in the form of gas bubbles which is likely O₂ gas. The bubbles when formed were immediately coated with bleached blood proteins to form foam. Tables 15a and b show that the foam is much less stable in the presence of enzymes, which implies that a considerable part of the blood protein molecules are hydrolyzed into small molecules by enzymes immediately after the blood was added to the solution. The results also show the utility of adding an antifoaming agent to the solution to reduce foam formation. 33

[0224] 34

EXAMPLE 16

[0225] Integrated Disinfection Regimen Soak Time Indicator

[0226] The enzymatic precleaner MetriZyme® (Metrex Research Corp.), which contains 10.94% ^w/v Alcalase® 2.5 L and 1.367% ^w/v Savinase® 16.0 L proteolytic enzymes and 0.004% FD&C blue dye #1, was mixed with 7.5% ^w/v H₂O₂ solution in a volume ratio of 1:128. The 7.5% H₂O₂ solutions at various pH were prepared by diluting 35% ^w/w H₂O₂ solution (Aldrich) and adjusting with 0.2 M Tris buffer. The initial colors of the MetriZyme/H₂O₂ mixed solutions were blue and faded with time. The color fading times measured with the naked eye for the MetriZyme®/H₂O₂ solutions at various pH are listed in Table 16. 35

[0227] MetriZyme® solution is blue when mixed with tap water in a 1:128 volumetric ratio. The disappearance of blue color over time in the MetriZyme®/H₂O₂ mixed solution suggests the occurrence of an oxidative bleaching reaction between H₂O₂ and dye. Surprisingly, such an oxidative reaction may not occur or can be too slow to be observed with the naked eye in a regular cleaning and disinfection time period of about 10 to 30 min if the solution pH is too low. It was also observed that at pH>8.5, the color of the mixed MetriZyme®/H₂O₂ solution can fade in less than 2 min, which is too short to be used as a solution soaking time indicator. Table 16 illustrates that the color fading time at pH 7-8.5 ranges between 8-15 and 45-75 min. These time ranges bracket the preferred 10-30 min soak times for high- or intermediate-level disinfection and enzymatic cleaning in the market. Thus, an H₂O₂ bleachable dye at a proper solution pH can be used as an integrated soak time indicator for simultaneous cleaning and disinfecting solutions containing at least H₂O₂ as one component of the disinfecting chemistry.

EXAMPLE 17

[0228] Kidney Dialyzer Cleaning Efficacy of H₂O₂/Enzyme Mixed Solution

[0229] Three used kidney dialyzers obtained from a dialyzer reprocessing center were utilized for cleaning efficacy tests of a H₂O₂/enzyme mixed solution of the present invention. These dialyzers had been processed to the end of their useful life with a prior art reprocessing chemistry (Renalin® PAA solution without an enzyme). The blood chamber fiber bundle volumes of the used dialyzers were measured as follows: After both blood chamber and dialysate chamber of a dialyzer were filled with tap water, the dialyzer was tapped repeatedly to rid the chambers of air bubbles and all four outlets/inlets of the dialyzer were plugged to prevent water loss. The dialyzer was weighed before and after the water in the blood chamber was evacuated by blowing compressed air through it. The blood chamber (fiber bundle) volume was determined as the difference between the two weight values divided by the density of water. The used dialyzer initial volumes, V_used (before treatment with the cleaning/disinfecting solution of the present invention), measured with the above method are consistent with the labeled minimum volumes (measured by the dialyzer processing center) which are about 80% of the new dialyzer volumes before the fiber bundles were clotted with patient blood. The cleaning/disinfecting solution in this example consisted of 128 parts of 7.5% ^w/v H₂O₂ and 1 part of Metrizyme® enzyme solution (Metrex Research Corp.) which contains 12% ^w/v Alcalase® 2.5 L+Savinase® 16.0 L combined. This solution has an initial proteolytic enzyme activity of 0.0031 A.U./ml. The pH of the mixed cleaning/disinfecting solution was adjusted to 8.5 with NaOH. Both blood and dialysate chambers of the dialyzers were then soaked with the above cleaning/disinfecting solution after the water in the dialysate chambers was removed. Multiple soaking intervals were utilized, with measurements of fiber bundle volume taken after each interval. The figure shows the relationship between the calculated percentage fiber bundle (blood chamber) volume recovery towards the 100% new dialyzer value and the cleaning/disinfecting solution soak time. The percentage fiber bundle volume recovery (P) was determined with the equation:

P=400× Fiber Volume Increase/V_used

[0230] wherein V(new) is assumed to be 1.25×V(used), and % recovery P is also=(Vol. incr./ (V(new)−V(used)))×100

[0231] As can be seen from the figure, from 60 to 90% of the clotted fiber bundle volume was recovered within 30 h of soaking with the cleaning/disinfecting solution of the present invention, which is well within the average Monday to Wednesday or Wednesday to Friday 43 h interdialysis interval. This example clearly indicates that the enzyme/H₂O₂ combined solution of the present invention can effectively remove the blood proteins and cellular debris clotted in the used dialyzer fiber bundles or alternatively, prevent blood proteins or debris from building up onto the dialyzer fiber bundle wall through regular treatment of the dialyzer following each dialysis procedure. Additionally, from the foregoing examples, it is expected that a solution comprising a proteolytic enzyme, H₂O₂ and PAA would be even more efficacious in cleaning a used dialyzer.

EXAMPLE 18

Simultaneous Enzymatic Cleaning and Disinfecting in One Step in Automated Instrument Disinfecting Systems

[0232] The following powder formula in Table 18a was mixed with 92.5 g of 40° C. water. This mixture is equivalent to reprocessing chemistry compositions utilized in the Steris® System 1™ Sterile Processing System from the Steris Corporation. This mixture is known to produce a high-level disinfecting amount of PAA from the reaction between perborate and acetylsalicylic acid. The concentration of proteolytic enzyme in this example was initially about, 0.02 A.U./ml. The temperature of the solution was kept at 40° C. using a temperature control bath. This temperature is essentially equivalent to the temperature used in the Steris® System 1™. During the mixing, 1.85 g of Azocasein was also added as a substrate for enzyme activity measurement (see the above in-situ protease activity assay method). At each time point listed in Table 18b, 5 ml of the above mixed solution was taken and mixed with 5 ml of 10% ^w/v TCA solution. The resulting precipitates of the TCA-containing solutions were removed using filtration after 15 min, and the absorbance of the supernatant was measured at 390 nm using a UV-visible spectrophotometer. As a control for the cleaning efficacy measurement, a mixed powder containing all the ingredients except Alcalase® 2.5 L was also mixed with Azocasein and water at 40° C. 36

[0233] 37

[0234] In this example, the Alcalase® 2.5 L-containing solution shows significant hydrolysis of Azocasein, indicating substantial cleaning activity, in the presence of the perborate/acetylsalicylic acid mixture. The hydrolysis of Azocasein has reached a maximum at or before the 11 min sampling time, indicating that the enzyme has been inactivated thereafter by exposure to the combination of PAA, 40° C. solution temperature and other excipients in the solution. Despite this subsequent inactivation, the very high enzyme activity observed over the first 11 min is representative of substantial cleaning activity. The currently marketed Steris® System 1™ utilizes a 12 min disinfection time, with the remainder of the total 30-35 min automated reprocessing time being allocated to the 4 following rinsing cycles and other automated processing cycles. Thus, the results of this example indicate that enzymatic cleaning can be simultaneously combined with disinfecting in one step in automated instrument disinfecting systems.

EXAMPLE 19

[0235] Tuberculocidal Effectiveness of Hydrogen Peroxide (H₂O₂).

[0236] A quantitative tuberculocidal test (suspension test) designed to determine the tuberculocidal effectiveness of a disinfectant/sterilant, following the EPA Guidelines for the Quantitative Tuberculocidal Procedure, was utilized.

[0237] Several H₂O₂ solutions were formulated without any additional preservative or other antimicrobial agents and without surfactants, chelators or organic buffers. The following formulas were tested, with H₂O₂ purchased from FMC Corp.: 38

[0238] Two sterile glass test tubes to which an organic soil (2% bovine serum albumin) had been added, containing 18 ml of a particular test formula at 20° C., were each inoculated with 2 ml of a standardized suspension of Mycobacterium bovis (ATCC 35743) containing approximately 1.0×10⁶ colony forming units (CFU)/ml. At contact times of 10 min for the formula at pH 6.3, and at 30 min for all formulas (pH 8.5, 6.3, 5.0), aliquots of the test suspension were removed and diluted in a neutralizer containing catalase to inactivate the H₂O₂. Serial tenfold dilutions of the neutralized suspension were then made in sterile saline. Each dilution was filtered through a 47 mm membrane filter with 0.45 micron porosity under a vacuum. Each filter was removed and aseptically placed on the surface of a Middlebrook 7H11 agar plate. The plates were incubated at 37° C.±2° C. for 15 to 25 days. Colonies were counted and the average CFU/ml was calculated for each contact time. The log₁₀ reduction was determined by subtracting the log₁₀ survivors at each contact time from the log₁₀ at time zero CFU/ml.

[0239] Tuberculocidal Activity Test Results. 39

[0240] The tests indicate that H₂O₂ solutions alone, without any additional preservative or other antimicrobial agents, cannot achieve in a short time interval one of the fundamental antimicrobial efficacy requirements for a high- or intermediate-level disinfectant, namely tuberculocidal activity. The omission of the enzyme in this example is not significant, as the presence of the enzyme does not change the test result. The prior art compositions and methods (for example, U.S. Re. 32,672) do not disclose or teach tuberculocidal activity and high- or intermediate-level disinfection and sterilization.

[0241] Further evidence that hydrogen peroxide alone cannot achieve high- or intermediate-level disinfection is derived from experiments conducted with Sporox®, disclosed in Greene et al., U.S. Pat. Nos. 4,518,585 and 4,557,898. Sporox® product labeling states that the minimum effective hydrogen peroxide concentration for high-level disinfection is 6.0% ^w/v. Greene '585 discloses one part aqueous acidic disinfecting and sterilizing compositions containing hydrogen peroxide, a surfactant, an organic or inorganic acid, an organic triazole corrosion inhibitor and an aqueous alcoholic mixture of a tertiary amine and a fatty acid alkanolamide such as linoleic diethanolamide. Suitable cationic surfactants include quaternary ammonium compounds such as alkyl dimethyl benzyl ammonium chloride and dialkyl quaternary ammonia compounds, which are known antimicrobial agents. Useful alcohols include the water-miscible alcohols such as isopropyl alcohol, also a known antimicrobial agent. Compositions of the invention have a pH below about 5, preferably about 3, and even more preferably about 1.8. An organic triazole corrosion inhibitor such as benzotriazole is thus disclosed to be necessary to prevent metal corrosion. Additionally, such one-part solutions are inherently unstable above pH 3, and even more so at pH 4-5 where metal corrosion is much less. Interestingly, in an attempt to understand the corrosive nature of Sporox®, the solution was tested for the presence of peracids using a recently developed sensitive HPLC assay for this purpose (Pinkernell et al., Anal. Chem., 1997, 69:3623-3627). The assay is very sensitive, however, it is a general peracid detection assay and cannot determine specific peracids. This assay showed the presence of an unspecified peracid. It is believed this peracid in perlinoleic acid derived from the perhydrolysis of the amide bond in linoleic diethanolamide. Tuberculocidal activity tests were run according to the above procedure on Sporox® versus a pure solution of 6.0% ^w/v hydrogen peroxide at the same pH of 1.8 and containing only peroxide stabilizers present in the raw material. A concentration of 6.0% ^w/v hydrogen peroxide was selected, as this is the claimed minimum effective concentration of hydrogen peroxide for tuberculocidal activity and high-level disinfection in Sporox®. Another commercially available product, Cidex PA® was also tested. The results are shown in the following table: 40

[0242] These tests show in fact that while Sporox® kills 6.74 logs of M. bovis in 20 minutes, the 6.0% ^w/v solution of stabilized hydrogen peroxide kills only 2.21 logs in 20 minutes. Moreover, the amount of kill with the 6.0% ^w/v hydrogen peroxide solution does not increase with increasing time between 5 and 20 minutes. Thus, the tuberculocidal activity claim of Sporox®, which is based upon killing 6 logs in the specified regimen soak time of 30 minutes, is due to the presence of the peracid and perhaps other antimicrobial agents and cannot be achieved without it/them. These tests demonstrate again that hydrogen peroxide alone is not a high-level disinfectant.

[0243] An example of the compositions of the present invention which demonstrates tuberculocidal activity follows.

EXAMPLE 20

Tuberculocidal tests of H₂O₂ Plus Peracetic Acid Solutions

[0244] Several H₂O₂ plus peracetic acid solutions were formulated and their tuberculocidal effectiveness was evaluated with a quantitative tuberculocidal test (suspension test), as in the previous example. 41

[0245] Tuberculocidal Activity Test Results: 42

[0246] The tuberculocidal activity test results show that all of the solutions, representing compositions of the present invention, tested at all time intervals exhibited maximum tuberculocidal activity, with a 6 log₁₀, reduction, or total kill, of the entire initial inoculum challenge. Again, the omission of the enzyme in this example is not significant as the presence of the enzyme and its associated pH do not materially change the test result.

[0247] An example of a H₂O₂-containing solution of the present invention which does not contain peracetic acid and which also meets the tuberculocidal standards of high-level disinfection is disclosed in U.S. Pat. No. 4,518,585, which has previously been expressly incorporated herein by reference in its entirety.

[0248] It should be understood that the simultaneous cleaning and decontaminating compositions and methods of the present invention shown and described in the specification are only preferred embodiments of the inventors who are skilled in the art and are not limiting in any way. Various changes, modifications or alterations to these embodiments may be made or resorted to without departing from the spirit of the invention and the scope of the following claims.