Compositions for protein stabilization and related methods

Abstract

Enzymatic compositions having enhanced thermal stability comprise an enzyme, a polymeric charged molecule having a charge opposite a charge of the enzyme, and a zwitterionic stabilization agent. Methods for enhancing thermal stability of an enzyme are also provided and include steps of combining an enzyme with one or more of a polymeric charged molecules having a charge opposite a charge of the enzyme and a zwitterionic stabilization agent. Methods for treating a wound are also provided and include administering to a subject an effective amount of an enzymatic composition.

Description

COMPOSITIONS FOR PROTEIN STABILIZATION AND RELATED METHODSRELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 63 / 718,313, filed November 8, 2024, the entire disclosure of which is incorporated herein by this reference.TECHNICAL FIELD

[0002] The presently-disclosed subject matter generally relates to compositions and methods for protein stabilization. In particular, the presently-disclosed subject matter relates to compositions and methods for protein stabilization that make use of, in both medical and nonmedical applications, in particular, a thermally-stabilized enzyme, such as a Hathewaya histolytica (HH) collagenase having an acidic isoionic point.BACKGROUND

[0003] Proteins are long chains of amino acids linked through covalent bonds between the alpha amino group of one amino acid to the alpha carboxyl group of another amino acid to form a covalent amide bond. Segments of sequence then interact to form elements of secondary structures which are identified as helical, beta sheets, or beta turns. These elements then, in turn, fold to form a compacted tertiary structure. For some proteins, these compacted tertiary structures then bind with themselves or other folded proteins to form a quaternary folded protein. These tertiary and quaternary structures, however, are the functional form of a protein, with properly folded and assembled proteins being capable of having many functions for a given cell or organism. For instance, proteins can be structural in nature providing rigidity to a cell, tissue, or organism, and / or a protein can be protective in nature and protect a cell or organism from an invading cell, virus, or foreign molecule, such as an antibody or agglutin. In this regard, one particular form proteins are enzymes, which are proteins that are generally capable of linking molecules together (synthases) or breaking them apart (lyases). Maintaining the elements of secondary, tertiary, and quaternary structure though is essential for the maintenance of protein and, in particular, enzyme function.110527945:v2

[0004] Proteases are enzymes which fragment other proteins. Proteases can be very specific, such as many of the enzymes in the blood clotting or complement cascades, mammalian collagenases that only proteolyze collagen at specific locations in the collagen molecule for tissue remodeling, or bacterial collagenases that only proteolyze native or denatured collagen (gelatin). Alternatively, proteases can be general proteases where their only selectivity is in the peptide bonds which they fragment. Examples of such general proteases include: trypsin, which predominately cleaves proteins at lysine and arginine residues; chymotrypsin, which predominately cleaves at hydrophobic amino acids like leucine and phenylalanine; and elastase, which predominately proteolyzes proteins at glycine, alanine, and serine residues. These enzymes vary in size from very small to very large, have molecular weights that can range from less than 20,000 Daltons to greater than 100,000 Daltons, and have structures that can range from very compact to including a number of loosely connected domains. One characteristic that is common to all proteases, however, is that they are often susceptible to self-cleavage (autolysis) or cleavage by other proteases. This susceptibility can have several consequences. First, it can convert an inactive precursor protease into its active form. Second, it can fragment an active protease into an inactive form (metabolic control). And third, it can have no biological function and just be the result of the biological system. This susceptibility though can have a significant impact on enzyme selectivity and stability.

[0005] Proteolytic enzymes, because of their varied activities, also have a wide variety of applications in industrial and medical applications. For industrial applications, the most common use for proteases is in food processing to modify the texture and flavor properties of animal and plant proteins, to convert protein waste into nutritionally valuable supplements, and as stain removal ingredients in laundry detergents. For medical applications, proteases have found several uses to treat several health-related conditions. These include the use of animal pancreatin to replace the digestive enzymes lost in patients who have undergone total pancreatectomy. Crude Hathewaya histolytica (HH) collagenase has also been used in petrolatum to accelerate the healing of burns and skin ulcers and can be used in clinical and nonclinical environments. More recently, purified HH collagenase has been used as a nonsurgical treatment for Pyrone’s disease and Dupuytren’s contracture. In addition, purified HH collagenase, when blended with other proteases, has been shown to be effective in recovering pancreatic islets for transplant into type I diabetics to reduce or eliminate the need for insulin injections, as well to recover a variety of210527945:v2cells from different tissues for different experimental medical applications. These procedures are typically done in a laboratory or clinical setting under controlled conditions for the storage and use of the collagenase reagents.

[0006] Another major medical collagenase and protease application is for topical wound healing and debridement. There are currently three medical strategies for wound healing and debridement. The first is to cover the wound to protect it from infection and allow the body to heal itself. The second option is to use enzymes to proteolyze the matrix proteins at the base of the eschar, the dead tissue, and allow it to slough off. The third option is to surgically remove the eschar. This last option requires trained medical personnel and often damages the underlying tissue, thus slowing the healing and repair process. Two products are often currently used for such enzyme debridement, namely Nexobrid™ (Vericel Corporation, Cambridge, MA) and Santyl™ (Smith & Nephew, Ft. Worth, TX) Ointment. Nexobrid™ is a partially purified bromelain product which can rapidly degrade necrotic tissue but causes extreme pain to the patient and can only be used in a medical facility where the pain can be managed. Santyl™ Ointment is an older product that has been on the market unchanged since 1965. It is composed of a partially purified cell culture supernatant from the fermentation of Hathewaya histolytica (HH) in a protein-based culture media. It is an impure preparation containing collagenase, Clostripain, neutral protease, and other secreted enzymes along with cell debris and culture media that is lyophilized and blended with petrolatum. Its mode of action is very slow, often taking weeks to have a significant clinical outcome.

[0007] With that in mind, recent literature has identified the enzyme responsible for the bulk of the debridement as collagenase rather than the Clostripain or neutral protease also found in the crude collagenase API. These references also noted that higher concentrations of the purified collagenase applied as a powder or dispersed in petrolatum resulted in more rapid debridement and healing of the damaged tissue. However, there was a saturation effect with diminishing rates of clinical improvement above a threshold level and, to date, these findings have not been implemented into an improved product. Indeed, because of this slow action and inconvenient delivery format, the Santyl™ product is regarded as suboptimal for most debridement needs.

[0008] Accordingly, a stabilized high activity protease, such as a HH collagenase product, that could be formulated in stabilized delivery formats to address wound healing and debridement medical situations and may be handled by first responders, in nonclinical settings or310527945:v2in military combat situations, and with or without the need for medical treatment facilities, would be both highly desirable and beneficial.SUMMARY

[0009] The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0010] This summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.

[0011] The presently-disclosed subject matter includes compositions and methods for protein stabilization and, in particular, compositions and methods for protein stabilization that include or make use of a thermally-stabilized enzyme. In some embodiments of the presently-disclosed subject matter, an enzymatic composition having enhanced thermal stability is provided that comprises an enzyme, a polymeric charged molecule having a charge opposite a charge of the enzyme, and a zwitterionic stabilization agent. In some such compositions, the composition has a pH greater than an isoionic point of the enzyme, and the polymeric charged molecule has a positive charge. In other such embodiments, the composition has a pH less than an isoionic point of the enzyme, and the polymeric charged molecule has a negative charge.

[0012] In some embodiments of the presently-disclosed enzymatic compositions, the enzyme is a protease. In some embodiments, the enzyme is selected from the group consisting of a collagenase, an elastase, trypsin, bromelain, and papain. In some embodiments, the protease is a collagenase such as, in certain embodiments, a collagenase that is derived from Hathewaya histolytica by natural or recombinant fermentation. Such collagenase can comprise collagenase Type I, Type II, or combinations thereof, and can further be a natural or a recombinant collagenase Type I and / or Type II. In certain embodiments, the collagenase is enriched prior to410527945:v2inclusion in the composition. Tn some embodiments that make use of collagenase Type IT, the collagenase Type IT is provided in the enzymatic composition in an amount of from about 10% to about 60% of the total weight of the collagenase in the composition. In some embodiments, the collagenase and the polymeric charged molecule are provided in the enzymatic composition at a mass ratio of from about 1 : 1 to about 1 :3.

[0013] With regard to the polymeric charged molecule, in some embodiments, the polymeric charged molecule is an amine containing molecule or a resin-based amine derived synthetic polymer. For example, in some embodiments, the amine containing molecule is selected from the group consisting of: a chemically derivatized cellulose; a linear, branched, or cross-linked dextran; a derivatized polyacrylate or polymethacrylate having charged groups selected from primary, secondary or tertiary amines; diethyl amino ethyl (DEAE); DEAE Dextran; poly- epsilon-lysine; poly-lysine; poly-arginine; poly-ornithine; poly delta ornithine; poly 2,3-diamino propionic acid; poly beta 2,3 diamino propionic acid; poly 2,4-diamino butyric acid; poly gamma 2,4 diamino butyric acid; poly 2,5 diamino valeric acid; poly delta 2,5 diamino valeric acid; DEAE cellulose; DEAE Sephadex; DEAE Sepharose; QAE Sephadex; and Q Sepharose. In some particular embodiments, the amine containing molecule is DEAE Dextran or poly-epsilon- lysine.

[0014] A number of metal salts useful for enzyme stabilization (i.e., a thermally-stabilizing metal salt) can also be included in an enzymatic composition of the presently-disclosed subject matter and, in certain embodiments, are selected based on the impact of the metal ion on the stability and function of the enzyme included in an exemplary enzymatic composition. In some embodiments, the thermally-stabilizing metal salt is a non-chelated calcium salt that, in certain embodiments, is selected from the group consisting of calcium chloride, calcium bromide, and calcium acetate. In some embodiments, the non-chelated calcium salt is provided in an amount of from about 0.1% to about 2% of the total weight of the composition.

[0015] Turning now to the zwitterionic stabilization agent, in some embodiments, the zwitterionic stabilization agent is an amino acid or an amino acid-peptide blend. For example, in some embodiments, the zwitterionic stabilization agent is an amino acid that is selected from the group consisting of glycine, alanine, serine, threonine, and combinations thereof. In some embodiments, the amino acid is glycine.510527945:v2

[0016] In some embodiments, the exemplary enzymatic compositions can also be formulated for use in a variety of applications. In some embodiments, the composition is formulated such that the composition has a pH of from about 6.0 to about 8.0. In some embodiments, the composition is spray dried or lyophilized such that the spray dried or lyophilized form of the composition can, in certain embodiments and implementations, be admixed with a suitable carrier or bulking agent, if desired.

[0017] In some exemplary embodiments of the enzymatic compositions described herein, an enzymatic composition having enhanced thermal stability and formulated for use in wound healing and debridement is provided that comprises from about 5% w / w to about 20% w / w enriched collagenase, from about 5% w / w to about 30% w / w DEAE dextran or poly-epsilon- lysine, from about 0.1% w / w to about 2% w / w calcium salt, and from about 48% w / w to about 89.9% w / w free amino acids. In some such embodiments, the enzymatic composition includes about 10% w / w enriched collagenase, about 10% w / w DEAE dextran or poly-epsilon-lysine, from about 0.5% w / w to about 2% w / w calcium salt, and from about 78% w / w to about 79.5% w / w free amino acids. In other such embodiments, the enzymatic composition includes about 10% w / w enriched collagenase, about 10% w / w DEAE dextran or poly-epsilon-lysine, about 1% w / w calcium salt, and about 79% w / w free amino acids.

[0018] Further provided, in some embodiments of the presently-disclosed subject matter, are methods for enhancing the thermal stability of an enzyme. In some embodiments, a method for enhancing thermal stability of an enzyme comprises an initial step of providing an enzyme in need of thermal stabilization, and then mixing the enzyme with a polymeric charged molecule having a charge opposite a charge of the enzyme and a zwitterionic stabilization agent in accordance with the presently-disclosed subject matter to produce a thermally-stable enzyme. In some implementations, the methods further comprise a step of further mixing the enzyme, the polymeric charged molecule, and the zwitterionic stabilization agent with an enzyme-stabilizing metal salt. In some implementations, the enzyme is collagenase and the collagenase is enriched prior to inclusion in the composition. In some implementations, subsequent to combining the components, the methods include a further step of dissolving the composition at a pH of from about 6.0 to about 8.0. In some implementations, the dissolved composition is then dried by spray drying or lyophilization.610527945:v2

[0019] Still further provided, in some embodiments and implementations of the compositions and methods described herein, are methods for treating a wound. In some implementations, a method for treating a wound comprises administering to a subject an effective amount of an enzymatic composition of the presently-disclosed subject matter such as, in certain implementations, an enzymatic composition comprising collagenase. In some implementations, administering the composition comprises administering an amount of the composition sufficient for wound healing and / or debridement. In some such implementations, the composition is in a spray dried or lyophilized form, and is applied directly to a wound or is placed in a water permeable pouch prior to being applied to a wound. In some implementations, the composition is applied to a substrate that is then applied to a wound. For instance, in some implementations, the substrate is a solid support, such as, in certain implementations, a medical gauze.

[0020] Further features and advantages of the present invention will become evident to those of ordinary skill in the art after a study of the description, figures, and non-limiting examples in this document.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows a representative Mono Q chromatographic profile of a Collagenase Gold starting product. The bulk solution was diluted prior to application to the Mono Q column. The two small peaks eluting at 2 to 3 minutes are buffer salts. The small peaks eluting at 9 to 10 minutes are related substance peptides from the parent collagenase I and II proteins. The large peak at about 11 minutes is intact Collagenase II enzyme. The shoulder at about 12 to 13 minutes is proteolyzed Collagenase I and II related substances which have had their collagen binding domain removed. They are fully active gelatinases but have little collagen degrading activity. The peak at about 15 minutes is a Collagenase I related substance which has lost both collagen binding domains. It is a fully active gelatinase but with little collagen degrading activity. The large peak at about 18 minutes is intact Collagenase I enzyme. The shoulder eluting on the backside of the Collagenase peak at 19 to 20 minutes is composed of two collagenase I related proteins which have had one of the collagen binding domains proteolytically removed. They are both active gelatinase and collagenases. The peak at 25 minutes contains aggregated forms of the collagenase enzymes.710527945:v2

[0022] FTG. 2 shows a representative Mono Q chromatographic profile of a sample of collagenase Gold that was prepared and lyophilized as the samples in the stability study 231004 but maintained as a negative control at -20°C. The dashed trace is a vial of the lyophilized collagenase gold enzyme maintained at +45°C for 22 weeks in the same experiment.

[0023] FIG. 3 shows a representative Mono Q chromatographic profile of the Lyophilized Collagenase Gold control (solid trace) and Formulation #01 (dashed trace) both maintained at 45°C for 22 weeks in experiment 231004.

[0024] FIG. 4 shows a representative Mono Q chromatographic profile of the Lyophilized Collagenase Gold control (solid trace) and Formulation #02 (dashed trace) both maintained at 45°C for 22 weeks in experiment 231004. The large peak eluting at 2-3 minutes in the Formulation #02 chromatogram is the DEAE Dextran in the formulation.

[0025] FIG. 5 shows a representative Mono Q chromatographic profile of the Lyophilized Collagenase Gold control (solid trace) and Formulation #06 (dashed trace) both maintained at 45°C for 22 weeks in experiment 231004. The large peak eluting at 2-3 minutes in the Formulation #06 chromatogram is the DEAE Dextran in the formulation.

[0026] FIG. 6 shows a representative Mono Q chromatographic profile of the Lyophilized Collagenase Gold control (solid trace) and Formulation #09 (dashed trace) both maintained at 45°C for 22 weeks in experiment 231004. The large peak eluting at 2-3 minutes in the Formulation #09 chromatogram is the DEAE Dextran in the formulation.

[0027] FIG. 7 shows a representative Mono Q chromatographic profile of the Lyophilized Collagenase Gold control (solid trace) and Formulation #12 (dashed trace) both maintained at 45°C for 22 weeks in experiment 231004. The large peak eluting at 2-3 minutes in the Formulation #12 chromatogram is the DEAE Dextran in the formulation.

[0028] FIG. 8 shows a representative Mono Q chromatographic profile of the Lyophilized Collagenase Gold control (solid trace) and Formulation #15 (dashed trace) both maintained at 45°C for 22 weeks in experiment 231004. The large peak eluting at 2-3 minutes in the Formulation #15 chromatogram is the DEAE Dextran in the formulation.

[0029] FIG. 9 shows a representative Mono Q overlay of the purified Collagenase I and II proteins used in experiment 241014. The solid trace is of the Collagenase II enzyme, and the dashed trace is the Collagenase I enzyme.810527945:v2

[0030] FTG. 10 shows a representative Superdex 200 overlay of the purified Collagenase I and II proteins used in experiment 241014. The solid trace is of the Collagenase I enzyme, and the dashed trace is the Collagenase II enzyme.

[0031] FIG. 11 shows a representative Superdex 200 chromatographic overlay of the Collagenase II samples in experiment 241014 at 46 weeks. The solid trace is the sample stored at -20°C, the dashed trace is the sample at +45°, and the doted trace is the sample at +55°C.

[0032] FIG. 12 shows a representative Superdex 200 chromatographic overlay of the Collagenase I samples in experiment 241014 at 38 weeks. The solid trace is the sample stored at -20°C, the dashed trace is the sample at +45°, and the doted trace is the sample at +55°C.

[0033] FIG. 13 shows a representative Superdex 200 chromatographic overlay of the Collagenase II samples in experiment 250327 at 17 weeks. The solid trace is the sample stored at -20°C, the dashed trace is the sample at +45°C, and the doted trace is the sample at +55°C.

[0034] FIG. 14 shows a representative Superdex 200 chromatographic overlay of the Collagenase II samples in experiment 250327 at 22 weeks. The solid trace is the sample stored at -20°C, the dashed trace is the sample at +45°C, and the doted trace is the sample at +55°C.

[0035] FIG. 15 shows a Superdex 200 chromatographic overlay of the Collagenase II samples in experiment 250327 at 26 weeks. The solid trace is the sample stored at -20°C, the dashed trace is the sample at +45°C, and the doted trace is the sample at +55°C.DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0036] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0037] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.910527945:v2

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0039] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites, non-patent literature, and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0040] Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0041] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9): 1726-1732).

[0042] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0043] The present application can “comprise” (open ended), “consist of’ (closed ended), or “consist essentially of’ the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0044] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0045] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are1010527945:v2approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0046] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0047] As used herein, ranges can be expressed as from “about” one particular value, and / or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0048] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0049] Stabilization of proteins is an empirical art due to the variability and complexity of protein structures. Most three-dimensional protein structures are maintained by hydrophobic, hydrogen binding, and salt bridge interactions, and maintaining these interactions is important for the maintenance of enzymatic function. Enzyme denaturation (inactivation)) occurs when the kinetic energy of the environment (temperature) is sufficient to break some of these noncovalent interactions and allow portions of the protein to become less compact. Usually the native conformation reforms but sometimes a nonnative structure can form, which can then lead to a loss of enzyme activity or further unfolding with resultant activity loss. The presently-disclosed subject matter, however, is based, at least in part, on the discovery of a systematic approach to protein stabilization based upon biochemical principles to increase the energy of activation needed to begin the unfolding process. In particular, bulking agents have previously been used to spread the protein molecules apart to reduce the potential for unfolding, aggregation, and denaturation. That prior approach was commonly done using sugars, dextran, starches, or salts,1110527945:v2but had limited efficacy at much above room temperatures. In contrast, and without wishing to be bound by any particular theory or mechanism, the presently-disclosed subject matter demonstrates that a different mechanistic approach can provide enhanced protein temperature stability. In particular, and as described in further detail herein, the presently-disclosed subject matter includes enzymatic compositions that include or make use of a polymeric molecule with a charge opposite that of the enzyme that is mixed with the enzyme to stabilize the protein, including in solution. By ionic interactions, the enzyme is covered by the polymeric molecule, which then reduces the potential for protein aggregation. In some embodiments, and as also described herein below, stabilization can then be enhanced by the addition of a zwitterionic stabilization agent that further covers the surface of the protein through ionic and, in some cases, hydrogen binding and hydrophobic interactions. If the enzyme requires metal salt for stabilization, a low concentration of such a metal salt is further added, in some embodiments, to ensure that the binding sites are fully saturated. In this way, and again without wishing to be bound by any particular theory or mechanism, it is believed that the enzyme is placed in a tighter structure that, in turn, increases the energy of activation necessary for unfolding and denaturation to occur.

[0050] Moreover, as the stabilization effect provided in accordance with the presently- disclosed subject matter is based, in part, on charge-charge interaction, it is believed that the compositions and methods described herein are capable of use with proteins without regard for whether the protein is negatively charged and the polymer is positively charged or whether the protein is positively charged and the polymer is negatively charged as the stabilizing interaction would be expected to be the same. For example, and as also described herein below, work performed with two enzymes at a pH above their isoionic points showed that it could be scientifically expected that all proteins at a pH above their isoionic point would have enhanced thermal stability when formulated in a similar manner. Conversely, this work also showed that it would be scientifically expected that proteins in solution at a pH below their isoionic point would have enhanced thermal stability when formulated with a positively charged polymer. In this regard, and although certain embodiments of the presently-disclosed subject matter are described herein with particular reference to collagenase, it is believed that the compositions and methods of the present invention can readily be adapted for any number of enzymes or other proteins having a particular charge.1210527945:v2

[0051] In some embodiments of the presently-disclosed subject matter, an enzymatic composition is thus provided that has enhanced thermal stability. In some embodiments, such an enzymatic composition having enhanced thermal stability comprises an enzyme; a polymeric positively charged molecule; and a zwitterionic stabilization agent.

[0052] Thermal stability, as used herein, refers to a composition or molecule’s ability to resist chemical or physical changes when exposed to higher temperatures and, more particularly, in the case of enzymes and proteases, the ability to resist chemical or physical changes that may alter the ability of the enzyme or protease to break down proteins and polypeptides, as described further below. Reference to high temperature stability, as used herein, is thus generally used to refer to stability at any temperature above that in which the material or object will typically be maintained. For example, frozen materials are usually maintained at -20°C, refrigerated materials are usually maintained at +2 to +8°C, and room temperature materials are usually maintained at about 20°C, such that a reference to a high temperature stability can be defined, in some instances, as stability at the next higher temperature range. In other words, for room temperature storage materials, for instance, high temperature stability is usually defined as an extended stability at a temperature of 37°C or above. For hot environment materials, and as another example, high temperature stability can often considered to be about +45°C or above. In some other embodiments where there is an absence of designated control stability temperature for a given material, the temperatures can often get significantly higher and short-term stability at +55°C is desirable. In some embodiments, reference to a thermally-stable enzyme or protease refers to an enzyme or protease that retains some or all its catalytic ability when exposed to a temperature increase above a maintenance temperature of about 5 °C, about 10 °C, about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, or about 40 °C. In some embodiments, such a measurement of activity is performed with reference to that of the same enzyme or protease at a control maintenance temperature.

[0053] In some embodiments of the presently-disclosed subject matter, the enzyme being thermally stabilized is a protease. The term “protease,” as used herein and as described above, is used to refer to various enzymes that break down proteins and polypeptides into smaller fragments or, in other words, catalyze proteolysis such that the proteins and polypeptides are broken down into small peptides or even individual amino acids. Typically, such proteases function by enzymatically cleaving individual peptide bonds through a hydrolysis reaction. Such1310527945:v2proteases are inclusive of serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, and metalloproteases. These enzymes can have a broad specificity as to the bonds which they can proteolyze such as chymotrypsin, thermolysin, papain, as some examples, while others have restricted cleavage sequences, such as the enzymes in the coagulation cascade or collagenases. In addition these enzymes can either proteolyze a broad range of proteins as a substrate, such as trypsin or bromelain, or can have a restricted substrate specificity, such as collagenase for native collagen.

[0054] The term “collagenase” is used herein to refer to a type of protease that breaks the peptide bonds of collagen, a component of the extracellular matrix in most animals. Almost all collagenases are of animal or bacterial origin. These enzymes are predominantly used as digestive enzymes to proteolyze collagen as a food source or for tissue remodeling and repair. The collagenases obtained from Hathewaya histolytica, in particular, have been extensively used for the recovery of cells from tissue. Indeed, these collagenases have been utilized as a therapeutic enzyme to treat multiple diseases and medical problems, including cartilage repair, Dupuytren’s disease (DD), cellulite therapy, glaucoma diseases, keloid disease, and the like. Collagenases for use in such therapeutic application can be obtained or derived from a number of natural sources, including both animal and bacterial species, and artificially, such as through recombinant technologies.

[0055] In some embodiments of the presently-disclosed subject matter, the collagenase comprises collagenase Type I and / or Type II. In some embodiments, the collagenase is a natural or a recombinant collagenase Type I and / or Type II. In some embodiments, the collagenase is enriched prior to inclusion in the composition to thereby increase the purity of the collagenase included in the composition.

[0056] In some particular embodiments of the presently-disclosed subject matter that make use of collagenase in an enzymatic composition, the collagenase is derived from Hathewaya histolytica (HH) by natural or recombinant fermentation. In some embodiments, the HH collagenase can be prepared by fermentation of the cells in a protein-based culture media and purified by a variety of published procedures. In some embodiments, the HH collagenase can be obtained, however, from commercially-available sources, as GMP grade HH collagenase products are currently available from several suppliers including SERVA Electrophoresis GmbH (Heidelberg, Germany), Roche Diagnostics (Indianapolis, IN), and VitaCyte LLC (Indianapolis,1410527945:v2IN). VitaCyte LLC offers three GMP grade Collagenase products. Collagenase HA which is a highly purified blend of intact Collagenase G (Type I) and H (Type II) in a defined ratio, Collagenase rHA which is a recombinant blend of Collagenase G and H in a defined ratio, and Collagenase Gold plus which is a highly enriched natural collagenase product containing about 85% intact Collagenase G and H with the remaining proteins being proteolyzed collagenase forms. It contains very low levels of Clostripain and protease activities. In some embodiments, Collagenase Gold Plus from VitaCyte LLC is utilized; however, in certain embodiments, other HH collagenase products such as Collagenase HA, rHA, purified natural or recombinant Collagenase G or H or collagenase from other suppliers could be substituted without departing from the spirit and scope of the subject matter described herein.

[0057] HH collagenase is not a single enzyme but is two unique related enzymes (Col G (Type I) and Col H (Type II)). They are homologous in structure but proteolyze collagen differently. Both proteins are large multi domain enzymes with an activation and catalytic domain, one or two linking domains and one or two collagen binding domains. Each domain is a tightly folded structure connected to the adjacent domains by unstructured segments of sequence which are susceptible to proteolysis. It has been determined that the collagen binding domains of these enzymes must first bind to the collagen molecule prior to the catalytic domain proteolyzing the collagen molecule. Thus, once all the exposed collagen has been bound by the collagenase enzymes, no additional binding and catalytic proteolysis can occur explaining the saturation effect observed in wound healing and debridement. For bum, wound healing, and debridement applications, very little collagenase will therefore be required for a medically effective dose. This requirement for the uniform delivery of small amounts of active pharmaceutical ingredient (API) to potentially large surface areas place stringent requirements on the excipients to stabilize the active enzyme. It is appreciated that in the public literature (Yoshida and Noda, Biochim. Biophys. Akta , vol. 105, pages 562-574, 1965.), it was described that upon long term exposure of collagen fibers to purified intact Collagenase G (I) or Collagenase H (II) would result in complete dissociation of the fibers to soluble peptides. For most medical applications one or the other single enzyme may be sufficient for positive clinical results.

[0058] In some embodiments that make use of an enzyme such as collagenase in an enzymatic composition of the presently-disclosed subject matter, the enzyme (e g., collagenase)1510527945:v2and the polymeric positively charged molecule are provided in the composition at a mass ratio of from about 1 : 1 to about 1 :3. In some embodiments, an exemplary composition comprises about 5% w / w to about 20% w / w of the enzyme and about 5% w / w to about 30% w / w of the polymeric charged molecule. The phrase “polymeric charged molecule” is used herein to refer to an organic molecule comprised of individual monomeric subunits that are covalently linked together in a chain or network and have a net positive or net negative charge.

[0059] In some embodiments, the polymeric charged molecule is an amine containing molecule or a resin-based amine derived synthetic polymer, where the amine is incorporated into a resin structure to create a polymeric network. In some embodiments, the polymeric charged molecule is an amine containing molecule that is selected from the group consisting of a chemically derivatized cellulose; a linear, branched, or cross-linked dextran; a derivatized polyacrylate or polymethacrylate having charged groups selected from primary, secondary, or tertiary amines; diethyl amino ethyl (DEAE); DEAE Dextran; poly-lysine; poly-epsilon-lysine; poly-arginine; poly-omithine; poly delta ornithine; poly 2,3-diamino propionic acid; poly beta 2,3 diamino propionic acid; poly 2,4-diamino butyric acid; poly gamma 2,4 diamino butyric acid; poly 2,5 diamino valeric acid; poly delta 2,5 diamino valeric acid; DEAE cellulose; DEAE Sephadex; DEAE Sepharose; QAE Sephadex; and QAE Sepharose. In some embodiments, the amine containing molecule is DEAE Dextran or poly-epsilon-lysine.

[0060] With regard to the enzyme-stabilizing metal salt used in the exemplary enzymatic compositions, in some embodiments, the metal salts are selected based on an understanding of the impact of the presence or absence of specific metal ions on the stability and function of each individual protein (i.e., each enzyme). In this regard, reference to an “enzyme-stabilizing metal salt” is used to refer to salts of a particular metal, where the metal included in the salt is useful for stabilizing the structure and / or function of the enzyme. In the case of HH collagenase enzymes, for instance, calcium ions are important for enzyme stability and are thus utilized. In some embodiments, the metal salt is a non-chelated calcium salt. In some such embodiments, the calcium salt is selected from the group consisting of calcium chloride, calcium bromide, and calcium acetate. In some embodiments, the calcium salt is provided in an amount of from about 0.1% to about 2% of the total weight of the composition. In some embodiments that make use of calcium ions for enhanced stability, it is believed that these calcium ions usually form salt bridges between aspartyl and glutamyl side carboxylate groups reducing ionic repulsion between1610527945:v2the groups and forming a more tightly ordered tertiary structure, which is believed to increase the energy of activation required to begin the unfolding process. Secondarily, for many proteins, it is believed that these bound calcium ions often protect segments of the enzyme which are susceptible to proteolysis. Indeed, calcium ion stabilization is seen in the proteases secreted by the pancreas into the small intestine for food digestion. In some other embodiments and for some proteins, magnesium ions perform the same function (e.g., nucleases). Many other proteins, however, do not specifically bind metal ions for stabilization and in embodiments that make use of such proteins (e.g., enzymes), the addition of enzyme-stabilizing metal salts may not be required.

[0061] Turning now to the zwitterionic stabilization agent, the phrase “zwitterionic stabilization agent” is used herein to refer to various excipients having both positively- and negatively-charged functional groups and that can be further added to an exemplary composition of the presently-disclosed subject matter and utilized to enhance the thermal and overall stability of the exemplary compositions. In some embodiments, the zwitterionic stabilization agent is added to the composition in an amount of about 50% to about 90% w / w of the composition. In some embodiments, such a zwitterionic stabilization agent is an amino acid or an amino acid- peptide blend. In some embodiments that make use of amino acids, the amino acid is selected from the group consisting of: glycine, alanine, serine, threonine, and combinations thereof. In some embodiments, the amino acid is glycine. Indeed, without wishing to be bound by any particular theory or mechanism, it is believed that by making use of such amino acids, the presently-disclosed subject provides an entirely novel approach where the presence of amino acids can additively increase the thermal stability of a lyophilized enzyme. In some embodiments, for those applications not requiring a bulking agent, the concentration of the zwitterionic stabilization agent can be significantly reduced. In some embodiments, and again without wishing to be bound by any particular theory or mechanism, it is believed that such zwitterionic stabilization agents can have a positive impact upon enzyme stabilization through several processes. First, because the zwitterionic stabilization agent has both positive and negatively charged portions, the zwitterionic stabilization agent is believed to be capable of bridging positive charged side chains such as lysine, arginine, and histidine to negative charged side chains in aspartic and glutamic residues that are too far apart spatially to naturally form a salt bridge. This salt bridge again increases the energy of activation required to disrupt the1710527945:v2secondary and tertiary structure of the enzyme. A second mode of action for the zwitterionic stabilization agent can, in some embodiments, be the replacement of single charged salts, such as sodium chloride or water, on the surface of the protein with a larger molecule with lower thermal mobility, again reducing the protein’s flexibility and thereby increasing the energy of activation needed for denaturation to occur. Lastly, because the zwitterionic stabilization agents are zwitterionic, it is believed that the zwitterionic stabilization agents can align themselves in layers on the surface of the enzyme, thereby spatially separating the enzyme molecules and thus reducing the potential for aggregation.

[0062] By making use of the above-described components, each of the various exemplary enzymatic compositions described herein can be formulated for use in a variety of applications. For example, in some embodiments, the exemplary compositions are formulated for use in wound healing and debridement. In some embodiments, the compositions can be formulated such that the composition is dissolved at a pH of from about 6.0 to about 8.0. In some embodiments, such a dissolved composition is formed by using a spray-dried or lyophilized form. In some embodiments, the spray dried or lyophilized composition is admixed with a suitable carrier. In other embodiments, the suitable carrier or bulking agent is combined with the composition and spray-dried or lyophilized such that, in certain embodiments, the spray dried or lyophilized composition can be applied directly to a wound. As would be recognized by those skilled in the art, the term “carrier” is generally used to refer to materials useful for transporting and delivering a substance, while the term “bulking agents” is often used to refer to substances utilized to increase the volume or weight of a particular composition and, in the case of proteins, can be useful in spreading the protein molecules apart to reduce the potential for unfolding, aggregation, and denaturation. It is appreciated, however, that the materials encompassed by the terms “carrier” and “bulking agents” may overlap with one another such that the terms “carrier” and “bulking agents” may be used interchangeably in some embodiments and implementations of the presently-disclosed subject matter. In some embodiments, potential carriers or bulking agents capable of use in accordance with the presently-disclosed subject matter can be in a liquid, semi liquid or dry format. A liquid format, in some embodiments, can be a suspension of almond oil or similar therapeutic oil, while a semi liquid format can include petrolatum, as is the case with Santyl™ ointment but at a more effective dose. In some embodiments and1810527945:v2implementations, a solid format is provided that makes use of a powder blend with a hydrogel powder, dextran, soluble starch, or other similar non-irritating solid matrix.

[0063] In some other embodiments, however, the dissolved composition or compositioncarrier admixture is applied to a suitable substrate and dried. For instance, in some embodiments, the composition can be applied to a substrate where the substrate is a solid support. In some embodiments, such a solid support is a medical gauze that can be applied to a wound for therapeutic applications, as described in further detail below. In some embodiments, the spray-dried or lyophilized composition or composition-carried admixture can be placed into a water permeable pouch which can subsequently be applied to the wound, as described below.

[0064] As a further refinement to the exemplary formulations described herein, in some embodiments, the enzymatic compositions are provided that have enhanced thermal stability and are formulated for use in wound healing and debridement. In some embodiments, such an enzymatic composition is provided that comprises: from about 5% w / w to about 20% w / w enriched collagenase; from about 5% w / w to about 30% w / w DEAE dextran or poly-epsilon- lysine; from about 0.1% w / w to about 2% w / w calcium salt; and from about 58% w / w to about 89.9% w / w free amino acids. In another embodiment, an enzymatic composition for wound healing and debridement is provided that comprises: about 10% w / w enriched collagenase; about 10% w / w DEAE dextran or poly-epsilon-lysine; from about 0.5% w / w to about 2% w / w calcium salt; and from about 78% w / w to about 79.5% w / w free amino acids. In a further embodiment, an enzymatic composition for wound healing and debridement is provided that comprises: about 10% w / w enriched collagenase; about 10% w / w DEAE dextran or poly-epsilon-lysine; about 1% w / w calcium salt; and about 79% w / w free amino acids.

[0065] In some embodiments in which an enzymatic composition is formulated particularly for use with wound healing and debridement, the free amino acids included in the compositions are selected from the group consisting of: glycine, alanine, serine, and threonine. In some of those embodiments, the free amino acid is glycine. Moreover, in some enzymatic compositions for wound healing and debridement, the enriched collagenase comprises collagenase Type I and Type II (e.g., either native or recombinant) and, if utilized, the collagenase Type II comprises from about 10 wt% to about 60 wt% of the collagenase in the composition. In some embodiments, the recombinant collagenase utilized has a sequence identity of at least 95% to1910527945:v2native Hathewaya histolytica collagenases, as can be determined by methods well known to those skilled in the art.

[0066] Further provided, in some embodiments of the presently-disclosed subject matter, are methods for enhancing the thermal stability of an enzyme, including methods of making such enhanced enzymatic compositions. In some embodiments, a method for enhancing thermal stability of an enzyme, comprises an initial step of providing an enzyme in need of thermal stabilization, such as an enzyme that has or is expected to exposed to increased temperatures. Once provided, the enzyme is then mixed or otherwise combined with a polymeric charged molecule having a charge opposite that of the enzyme and a zwitterionic stabilization agent. In some implementations, that mixture is further combined with an optional enzyme-stabilizing metal salt that can be selected based on the stability needs of the enzyme. In some implementations, the polymeric charged molecule is an amine containing molecule or a resinbased amine derived synthetic polymer. In some embodiments, the amine containing molecule is selected from the group consisting of: a chemically derivatized cellulose; a linear, branched, or cross-linked dextran; a derivatized polyacrylate or polymethacrylate having charged groups selected from primary, secondary or tertiary amines; diethyl amino ethyl (DEAE); DEAE Dextran; poly-epsilon-lysine; poly-lysine; poly-arginine; poly-omithine; poly delta ornithine; poly 2,3-diamino propionic acid; poly beta 2,3 diamino propionic acid; poly 2,4-diamino butyric acid; poly gamma 2,4 diamino butyric acid; poly 2,5 diamino valeric acid; poly delta 2,5 diamino valeric acid; DEAE cellulose; DEAE Sephadex; DEAE Sepharose; QAE Sephadex; and Q Sepharose. In some implementations, the amine containing molecule is DEAE Dextran or poly- epsilon-lysine.

[0067] In some implementations of the methods of enhancing the thermal stability of an enzyme and / or making a thermally-stable enzymatic composition in accordance with the presently-disclosed subject matter, many enzymes have bound metal ions for increased stability with calcium ions being the most common. For these enzymes, additional calcium salts can be added to the formulation to ensure the binding site is saturated. In some implementations, the metal salt is a non-chelated calcium salt such as, in some implementations, a calcium salt selected from the group consisting of calcium chloride, calcium bromide, and calcium acetate. In some implementations, the calcium salt is provided in an amount of from about 0.1 to about 2% of the total weight of the composition. In some embodiments, calcium salts are utilized as most2010527945:v2prokaryotic and eukaryotic proteases contain stabilizing calcium ions, which, once bound, provide for enhanced stability and resistance to autolysis and inactivation.

[0068] In some implementation of the methods of enhancing the thermal stability of an enzyme and / or making a thermally-stable enzymatic composition in accordance with the presently-disclosed subject matter, the included zwitterionic stabilization agent is an amino acid or an amino acid-peptide blend or a zwitterionic organic molecule such as a “Goods” buffer salt. In some implementations and because of their good solubility, the amino acid is selected from the group consisting of glycine, alanine, serine, threonine, and combinations thereof. In some implementations, the amino acid is glycine.

[0069] Further, with regard to the enzymes utilized in the exemplary methods of enhancing the thermal stability of an enzyme and / or making a thermally-stable enzymatic composition in accordance with the presently-disclosed subject matter, in some implementations, the enzyme is a protease. In some implementations, the enzyme is selected from the group consisting of a collagenase, an elastase, trypsin, and papain. In some implementations, the enzyme is collagenase such as collagenase derived from Hathewaya histolytica by natural or recombinant fermentation. In some implementations, the collagenase comprises collagenase Type I and Type 11. In some implementations, the collagenase is a natural or a recombinant collagenase Type 11 that can be provided in an amount of from about 10 to about 60% of the total weight of the collagenase in the composition. In some implementations, the collagenase is enriched prior to inclusion in the composition. In some implementations, the collagenase Type 1 or Type II are used individually. In some implementations, the collagenase and the polymeric positively charged molecule at a mass ratio of from about 1 : 1 to about 1 :3.

[0070] In some implementation of the methods of enhancing the thermal stability of an enzyme and / or making a thermally-stable enzymatic composition in accordance with the presently-disclosed subject matter, the methods further include a step of dissolving the composition at a pH of from about 6.0 to about 8.0. In some implementations, the methods can include a step of drying the dissolved composition by spray drying or lyophilization.

[0071] Still further provided, in some embodiments of the presently-disclosed subject matter, are methods for treating a wound. In some embodiments, a method for treating a wound comprises administering to a subject an effective amount of a thermally-stable enzymatic composition of the presently-disclosed subject matter. In some implementations, administering2110527945:v2the composition comprises administering an amount of the composition sufficient for wound healing and debridement or, in other words, an amount of the composition sufficient for the removal of dead cells, cellular debris, and matrix proteins (eschar) from a wound site to allow for cell growth and tissue repair.

[0072] The terms “treatment” or “treating,” as used herein, refer to the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0073] The terms “reducing,” “reduction,” “inhibiting,” “inhibition” and grammatical variations thereof do not necessarily refer to the ability to completely inactivate all target biological activity in all cases. Rather, the skilled artisan will understand that those terms refer to decreasing biological activity of a target, such as can occur when a ligand binds a site of the target, a protein in a biochemical pathway of the target is blocked, a non-native complexes with a target, or the like. Such decrease in biological activity can be determined relative to a control, wherein the control can be representative of an environment in which an inhibitor is not administered. For example, in some embodiments, a decrease in activity relative to a control can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease.In some embodiments, the increases and / or decreases described herein can be in reference to a control subject that has not been treated with one of the presently-disclosed compositions. In other embodiments, the increases and / or decreases described herein can be in reference to a2210527945:v2baseline obtained in a subject that is in need of treatment, but has not yet began a particular therapeutic regimen.

[0074] In some embodiments, administration of the compositions in accordance with the presently-disclosed subject matter reduces or treats one or more of the underlying causes and / or symptoms associated with a wound. Measurement of such foregoing reductions can be performed using routine procedures known to those of ordinary skill in the art.

[0075] In some embodiments of the therapeutic methods described herein, the use of a high temperature stabilized enzyme (e.g., collagenase) is believed to be useful in the treatment of wounds in a manner that, again without wishing to be bound by any particular theory or mechanism, is believed to be faster acting than Santyl™ and be less painful and tissue damaging than Nexobrid™ for surgical debridement. In this regard, it is appreciated that, for such medical applications, collagenase or other protease compositions included in an exemplary compositions meets several stringent characteristics that allow their use in non-hospital settings by first responders, patients, or in military field operations. In particular, in some embodiments, the compositions are: 1) easy to use; 2) exhibit extended >35°C stability (> 2 years) for room temperature storage and short-term stability at higher temperatures for shipping stability; 3) are easily removed to apply a fresh treatment; and 4) do not inflict additional pain to the patient.

[0076] Several different formats can be used with the compositions described herein and are believed to be capable of meeting the foregoing requirements depending on the condition and environment of the subject and the condition being treated. For small burns, skin ulcers, or for scar reduction, a thermally-stabilized high activity enzyme in anhydrous hydrogel, petrolatum, or other suitable carrier can be the delivery mechanism of choice. For larger bums or injuries, a powder format which can be sprinkled onto the wound and covered by a bandage material can be a viable option. Another option is to impregnate the stabilized protease (e.g., collagenase) onto the bandage material for direct application onto the wound.

[0077] Regardless of the delivery vehicle, the exemplary enzyme compositions (e.g., a composition including a protease such as collagenase) of the presently-disclosed subject matter, in certain embodiments, are believed to also possess several properties beyond the abovedescribed temperature stability. The compositions hydrate rapidly in water or body fluids, have minimal hydroscopic properties for storage stability, and include excipients capable of being USP grade. Moreover, because, in certain embodiments, the collagenase or other protease or2310527945:v2enzyme binds tightly to the collagen or other molecule preset in a the wound bed, a reduced amount of enzyme is required for an effective dose.

[0078] Suitable methods for administering a therapeutic composition in accordance with the methods of the presently-disclosed subject matter include, but are not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, and / or intraarterial administration), oral delivery, buccal delivery, rectal delivery, subcutaneous administration, intraperitoneal administration, inhalation, intratracheal installation, surgical implantation, transdermal delivery, local injection, intranasal delivery, and hyper-velocity injection / bombardment. Where applicable, continuous infusion can enhance drug accumulation at a target site (see, e.g., U.S. Patent No. 6, 180,082). In some embodiments, the administration of the composition is via oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intraaural administration, rectal administration, intravenous administration, intramuscular administration, subcutaneous administration, intravitreous administration, subconjunctival administration, intracameral administration, intraocular administration or combinations thereof. In some embodiments that make use of the compositions for wound treatment, the compositions are administered topically.

[0079] Regardless of the route of administration, the compositions of the presently-disclosed subject matter are typically administered in amounts effective to achieve the desired response. As such, the term “effective amount” is used herein to refer to an amount of the therapeutic composition sufficient to produce a measurable biological response. Actual dosage levels of active ingredients in a therapeutic composition of the present invention can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject and / or application. Of course, the effective amount in any particular case will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. Preferably, a minimal dose is administered, and the dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art.2410527945:v2

[0080] For additional guidance regarding formulation and dose, see U.S. Patent Nos. 5,326,902; 5,234,933; PCT International Publication No. WO 93 / 25521; Berkow et al., (1997) The Merck Manual of Medical Information, Home ed. Merck Research Laboratories, Whitehouse Station, New Jersey; Goodman et al., (1996) Goodman & Gilman's the Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill Health Professions Division, New York; Ebadi, (1998) CRC Desk Reference of Clinical Pharmacology. CRC Press, Boca Raton, Florida; Katzung, (2001) Basic & Clinical Pharmacology, 8th ed. Lange Medical Books / McGraw-Hill Medical Pub. Division, New York; Remington et al., (1975) Remington's Pharmaceutical Sciences, 15th ed. Mack Pub. Co., Easton, Pennsylvania; and Speight et al., (1997) Avery's Drug Treatment: A Guide to the Properties, Choice, Therapeutic Use and Economic Value of Drugs in Disease Management, 4th ed. Adis International, Auckland / Philadelphia; Duch et al., (1998) Toxicol. Lett. 100-101:255-263.

[0081] The terms “subject” or “subject in need thereof’ refer to a target of administration, which optionally displays symptoms related to a particular disease, pathological condition, disorder, or the like. The subject of the herein disclosed methods can be a mammal. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In some embodiments, the term “patient” can be used to refer to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0082] The practice of the presently-disclosed subject matter can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Volumes I and II, Glover, ed., 1985; Oligonucleotide Synthesis, M. J. Gait, ed., 1984; Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984; Transcription and Translation, B. D. Hames & S. J. Higgins, eds., 1984; Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987; Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), A Practical Guide To Molecular Cloning; See Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer2510527945:v2Vectors For Mammalian Cells, J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory, 1987; Methods In Enzymology, Vols. 154 and 155, Wu et al., eds., Academic Press Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987; Handbook Of Experimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., 1986.

[0083] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.EXAMPLES

[0084] Materials and Methods for Examples 1 - 16.

[0085] Collagenase characterization - All chemicals used are ACS grade or better.

[0086] Mono 0 analytical chromatography - This characterization is performed according to the United States Pharmacopeia monographs 81.1 and 81.2. The Mono Q column 5 / 50 (Cytiva 17516601) is a strong anion exchange support separating the collagenase molecules by their net negative charge. It can resolve the intact Collagenase G and H forms as well as their related proteolyzed forms. The Mono Q column technology has been discontinued by Cytiva and replaced by a Capto HiRes Q 5 / 50 column. The following conditions were utilized and / or observed: Equilibration Buffer - 20 mM Tris, 1 mM Calcium Chloride pH 7.5; Limit Buffer - 20 mM Tris, 1 mM Calcium Chloride, 1,000 mM Sodium Chloride pH 7.5; and Flow Rate = 1.5 Milliliters / minute.Table 1. Column Gradient.2610527945:v2

[0087] Wunsch peptide activity analysis - The Wunsch assay is a peptide assay which predominately measures the Collagenase H activity. It is performed according to the United States Pharmacopeia monographs 89.1 and 89.2, and using the following reagents and reaction conditions: Assay Buffer - 100 mM Tris pH 7.1; 100 mM Calcium Chloride Solution; 25 mM Citric Acid Solution; 4-phenylazooxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (PZ Peptide); Methanol Ethyl Acetate; Anhydrous Sodium Sulfate.

[0088] For the substrate solution, 10 milligrams of PZ Peptide were placed into a test tube along with 0.20 milliliters of methanol. The solution was mixed until all the substrate had dissolved, and the volume was then brought to 10.0 milliliters with assay buffer. For the extraction tube, a 16 by 100 Pyrex test tube was used, and 1.0 milliliters of 25 mM Citric acid Solution and 5.0 milliliters of ethyl acetate was added, followed by capping to prevent evaporation. For the drying tube, a 16 by 100 mm Pyrex test tube was used and approximately 0.4 grams of anhydrous sodium sulfate crystals was added, followed by capping to maintain anhydrous conditions. Lastly, for the assay tube, a 13 by 100 mm Pyrex test tube was used, and 0.2 milliliter of 100 mM Calcium Chloride Solution and 1.0 milliliter of substrate solution was added before incubating the assay tubes at 25°C.

[0089] For the assay conditions, 0.1 milliliter of sample was added to the assay tube and incubated for 15 minutes. 0.5 milliliters of the assay solution was then transferred to the extraction tube and vortexed vigorously. The two phases were allowed separate and the organic phase (top layer) was transferred to the drying tube. The tubes were vortex mixed several times to insure complete drying of the sample, before measuring the absorbance at 320 nm.

[0090] Superdex 200 Size Exclusion Chromatography - The Superdex 200 (10 by 300 mm) column (Cytiva cat. No. 28-9909-44) is a gel permeation chromatography support that separates molecules by their molecular mass and shape. For this analysis, approximately 0.3 mg of Collagenase was applied to the column at a flowrate of 0.4 milliliters / minute. The buffer used was 10 mM Tris, 1 mM Calcium Chloride, 150 mM Sodium Chloride pH 7.5.

[0091] Soluble Collagen Degrading Assay - This assay measures the release of peptides from soluble native bovine collagen. This assay predominately measures the collagen degrading activity of Collagenase G. The following equipment was used: incubation Water Bath (30°C), color development water bath or dry heating bath (50°C); and UV / VIS Spectrophotometer. The2710527945:v2following materials were used: Soluble Collagen (6%) Nutragen (Advanced Biomatrix Cat. No. 5010-50mL); 2,4,6 Trinitrobenzene sulfonic Acid 5% in methanol (TNBSA); Assay Buffer - 100 mM Sodium Tetraborate, 1 mM Calcium Chloride pH 7.5; Color Development Buffer - 100 mM Sodium Tetraborate, 10 mM EDTA, 2.0% SDS pH 8.0; Quench Solution - 0.25 Molar Citric Acid.

[0092] 2 mg / mL TNBSA Color Development Solution - For each unknown, enzyme or leucine control sample to be analyzed, 160 microliters of 5% TNBSA was added to a final volume of 4 milliliters with color development buffer. This solution was made up immediately before use.

[0093] Substrate preparation For each sample to be analyzed, a total of 0.5 milliliters of Nutragen Collagen solution was mixed with 5.5 milliliters of Assay Buffer in an appropriate size container and allowed to equilibrate at room temperature. Into an appropriately sized Pyrex test tube (13 by 100 millimeter), 0.9 milliliters of the collagen solution was then dispensed. The tubes were then capped and placed in the 30°C water bath.

[0094] Leucine Color control Stock Solution (100 millimole s / Uter) - To 100 milliliters of water, 1.13 grams of L-Leucine free amino acid was added. With stirring and warming to completely dissolve the solids, the solution was dispensed into small aliquots before freezing at - 20°C until needed.

[0095] Leucine Control Assay Dilution - The following steps were utilized:1. Thaw an aliquot of the Leucine control solution and warm if necessary if insoluble material is observed.2. Dilute 100 microliters of Leucine control stock solution with 9.9 milliliters of assay buffer to prepare a 1 micromole / milliliter working stock solution.3. Further dilute the working solution with assay buffer to 1.0 milliliter in 13 by 100- millimeter Pyrex test tubes to prepare the assay samples according to the table below. The samples are now ready for color development.Table 2. Leucine Control Assay Solutions.2810527945:v2

[0096] Collagenase Assay Dilutions - The following steps were utilized for collagenase assay dilutions:1. Purified or enriched Collagenase Gold plus enzyme powders should be weighed and diluted to 1 mg / mL which will have an optical density at 280 nm of 1.41 optical density units. For excipient formulations this value needs to be modified dependent upon the concentration of the collagenase in the formulation. For example, for a formulation containing 5% Collagenase a total of 20 milligrams of formulation will need to be added to one milliliter of assay buffer to prepare a 1 mg / mL collagenase stock solution.2. A total of 100 microliters of the above stock solution is diluted with 4.9 milliliters of assay buffer to prepare a 20 microgram / milliliter working stock solution.3. Further dilute the working solution with assay buffer to 1.0 milliliter in 13 by 100- millimeter Pyrex test tubes to prepare the assay samples according to the table below. The samples are now ready for analysis. Because of the low concentration of the collagenase enzyme at these dilutions this step should be performed immediately prior to performing the assay.Table 3. Collagenase Assay Solutions.

[0097] Collagen Assay Procedure - To assay the collagen, the following steps were used:1. At the appropriate time remove the soluble collagen substrate tubes from the 30°C-water bath and uncap them. Transfer 100 microliters of the desired assay samples into the2910527945:v2appropriate tubes and recap them. Place the assay tubes into the water bath and allow the digestion to progress for 120 minutes (2 hours).2. To the assay and Leucine control tubes is added 0.5 milliliters of 2 mg / mL TNBSA Color Development Solution and incubated at 50°C for 60 minutes.3. At the end of the color development add 0.5 milliliters of quench solution.4. The samples are then diluted 1 :10 with water and the optical density at 340 nm is determined.

[0098] Activity Calculation - To assess the activity of the collagen, the absorbances from the sample dilutions and the Leucine dilutions are plotted. From the Leucine plot the A340 nm / pM amino group is determined. The sample dilution absorbances are then plotted against the enzyme concentration and the slope determined to obtain the A340 nm / pg enzyme. From these values the specific activity is calculated:Specific Activity = pM amino Group released / milligram enzyme-minute SA = [Sample Slope / Leucine slope] [1000ug / mg / 120 minutes]

[0099] Stabilization factors - The stabilization of lyophilized (dried) proteins is a multifactorial issue dependent upon a variety of factors including without intending any limitation: 1) the secondary, tertiary, and quaternary structure of the protein and the positive and negative impact of excipient molecules on these structures; 2) required cofactors such as active site and structure stabilizing metal ions; 3) pH stability range of the protein; and 4) effects of ionic strength around the shells of water surrounding the protein and its effect on protein-protein interactions and solubility.

[0100] Collagenase production - HH collagenase is produced by anaerobic fermentation of the microorganism in an amino acid rich media. Several media compositions have been reported in the public domain literature and in patents. The two collagenase forms are secreted into the culture media predominately during the late log early stationary phase of culture. The enzymes in the spent media are concentrated by salt or solvent precipitation or ultrafiltration / diafiltration or combinations of the above. After this initial processing, the enriched preparation can be further purified using ion exchange or hydrophobic interaction chromatographies as described.

[0101] HH Collagenase G & H (Collagenase Gold plus) - Collagenase Gold plus is a preparation composed primarily of two collagenolytic enzymes capable of proteolyzing native3010527945:v2and denatured collagen. Collagenase G is a five-subunit molecule comprised of an activation domain, a catalytic domain, a linking domain, and two collagen binding domains. The Collagenase H enzyme is homologous to the Collagenase G molecule having five subunits but with two linking domains and only one collagen binding domain. Both molecules are proteases containing a zinc ion in the active site and several calcium ions for stabilizing domain structure and domain-domain interactions. With these essential metal ions certain chelating molecules must be avoided including EDTA, EGTA, and histidine as examples.

[0102] This product is sold as a lyophilized protein powder for the dissociation of pancreas and the recovery of islets of Langerhans for research and transplantation into Type I diabetics. From an internal stability study, this lyophilized formulation lost about 20% of its collagen degrading activity when stored at 37°C for 52 weeks.

[0103] Bulk liquid Collagenase Gold Plus was used as the starting material. This material was recovered from spent culture supernatant of the fermentation of Hathewaya histolytica in a protein rich media. The clarified spent media was ultrafiltered and diafiltered to remove low molecular weight components, salts, and some pigments. This concentrate was then salt precipitated to remove additional pigments and acidic components. Finally, the collagenase preparation was bound to Q Sepharose Fast Flow resin (Cytiva) and step eluted to remove traces of pigment and endotoxin. This step elution could be performed as a single step to recover the two enzymes in a single pool or a two-step process to recover the two enzymes as separate pools. This material was ultrafiltered and diafiltered against 1 mM Calcium Chloride pH 6.5 solution and stored frozen at -20°C in aliquots. All development work was performed with material recovered in this manner. The objective was to identify potential excipient groups having the desirable characteristics of medical excipients.

[0104] Excipient Classes - A wide variety of different classes of compounds have been used as stabilizing and diluting agents. These fall into several general classifications including amino acids, peptides, peptones, and proteins. For this group amino acids were considered to be the best candidates as they can be chemically synthesized as defined molecules. Of the amino acids the best potential candidates are glycine, alanine, serine, and threonine as these are small and easily synthesized and have good solubility properties for formulation and are well characterized. Peptides are potentially less desirable as they tend to be more expensive, and3110527945:v2peptones are obtained from the hydrolysis of proteins which are heterogeneous. The use of proteins has several issues such as cost, purity, and potential immunogenicity.

[0105] Another potential candidate is carbohydrates including sugars, dextran, starches, cellulose, alginates, chitosan. Of these, sugars are problematic as many of them do not dry well and many of them are hydroscopic. Trehalose (a disaccharide) is an excellent protein stabilizing molecule, however when lyophilized it is very hydroscopic. USP grade Dextrans are available in many molecular weight forms in both underivatized and derivatized formats for medical applications and are considered as Generally Regarded as Safe (GRAS). Similarly, starches, cellulose, alginates, and chitosan have been used for medical applications. Of all the above materials dextran has the most extensive medical use.

[0106] Another potential excipient class is salts. Depending upon the enzyme many different salts have been used to improve enzyme stability. Many enzymes contain bound metal ions as either part of the active site or for protein stabilization such as calcium or magnesium ions. Because HH Collagenases contain bound calcium ions for stability calcium chloride will be included in certain formulations. However, and without wishing to be bound by any theory or mechanism, it is believed that any water-soluble calcium salt can be included.

[0107] Yet another candidate excipient class is detergents. For many injectable protein formulations (monoclonal antibodies as an example) include detergents to minimize aggregation and potential immunological response. However, because certain applications of this formulation are used topically on open wounds the use of detergent can have a pain issue and may be excluded from certain development sets.

[0108] Still yet another candidate excipient class is synthetic polymers. These include amino acid polymers, and natural and synthetic organic polymers such as the large family of hydrogels.

[0109] Formulation Characteristics - To be useful as a therapeutic product, and without wishing to be bound by any particular theory or mechanism, it was believed that for purposes of the experiments described herein, a formulation should have additional characteristics: 1) the enzyme will experience little to no changes in its physical, and enzymatic activities upon freezing and lyophilization or drying in the excipient mixtures; 2) the lyophilizate will be uniform and not experience any phase separation during the freezing and lyophilization process3210527945:v2which can result in product inhomogeneity; and 3) The lyophilized product will powder easily for admixture into the delivery vehicle.

[0110] Example 1 - Experimental design #! (Formulation 190315)

[0111] In this experiment, a synthetic formulation was developed to approximate the collagenase concentration in a crude collagenase in a defined format. It was initially developed as a reproducible replacement for crude collagenase preparations and to evaluate stability testing protocols. The composition was composed of 6% Collagenase Gold Plus, 1% Calcium Chloride, and 97% USP Glycine. These components were dissolved in water at a final concentration of 50 milligrams / milliliter and lyophilized in vials and in bulk. The resulting cake was easily powdered. Vials were stability tested at temperatures from -20°C to 45°C for durations of 104 weeks (-20°C and +4°C) to 12 weeks (+37°C and +45°C).

[0112] Results - At -20°C, no obvious Wunsch and collagen degrading activity loss was observed out to 104 weeks. At +04°C, possible 10±3% Wunsch and collagen degrading activity loss was observed out to 104 weeks. At +37°C, an initial 10% Wunsch and collagen degrading activity loss was observed at the first two-week time point and a minimal loss out to twelve weeks. At +45°C, a 10 to 20% loss of Wunsch and collagen degrading activity was observed after twelve weeks. From these analyses, it was concluded that glycine was an acceptable excipient as a carrier for HH Collagenase, but did not provide a significant improvement in thermal stability.

[0113] Example 2 - Excipient Screening - Peptones

[0114] Preliminary screenings were performed to characterize the effects of freezing and lyophilization on blends of excipients and Collagenase Gold plus. Each formulation was composed of 95% excipient and 5% Collagenase Gold plus. The formulations were dissolved in water at a concentration of 100 milligrams / milliliter. An aliquot was kept at +2-8°C, a second aliquot was frozen at -20°C and the bulk was frozen and lyophilized. The following candidate peptone excipients were evaluated: Peptone; Organo Techni Soy peptones; El 10, AM41, A2SC, A3 SC; Solabia Soy Peptones; Soy F, Papaic Soy, Papaic Soy USP, Soy Evolution; Fish gelatin peptone prepared by digestion of fish gelatin with Dispase TM; and Glycine USP grade.

[0115] Results - The Organo Techni peptones were unsuitable as they melted back upon lyophilization and dried as a glass-like solid. This was thus determined to be unsuitable for this application and not analyzed further. All other samples lyophilized well. The glycine and fish3310527945:v2gelatin peptone cakes had a white color and the Solabia peptones had a light tan color. Wunsch and collagen degrading activity analysis indicated that there was no loss of activity of the samples after freezing and lyophilization. Mono Q analysis of the glycine and fish gelatin peptone samples gave chromatograms identical to the original Collagenase Gold plus starting material. All the soy peptones had more complex Mono Q chromatograms with additional peaks making analysis more complex. It was thus concluded that complex mixtures of peptides do not have a negative effect upon the stability of Collagenase Gold Plus to lyophilization. From a manufacturing perspective these peptones were less suitable as an excipient because of their complexity and challenge to characterize.

[0116] Example 3 - Excipient Screening - Amino Acids

[0117] The following amino acid excipient candidates were considered: glycine, glycylglycine, sarcosine, and betaine. Formulations were prepared as described above, frozen and lyophilized.

[0118] Results - The sarcosine melted back upon lyophilization and dried as a glass film and was unsuitable for this application. The betaine and glycylglycine both dried as a hard cake, which could be powdered. Wunsch and Mono Q analyses indicated little to no differences between these formulations and the glycine formulation. It was thus determined that glycylglycine and betaine were potential suitable excipients.

[0119] Example 4 - Collagenase, Glycine, Low Calcium Chloride Lyophilization stability experiment 190111

[0120] The object of this experiment was to determine if a lower concentration of Calcium Chloride in the formulation would affect the temperature stability of the formulation. The formulation was 5.5% Collagenase Gold Plus, 0.1% Calcium Chloride, 94.4% Glycine. The concentration of the formulation was 100 milligrams solid per milliliter solution. The sample was then lyophilized.

[0121] Results - Pre and post lyophilization Mono Q analyses showed no obvious difference between the samples. The Wunsch activity of the lyophilized material showed a 3% loss of activity (well within the experimental error of the assay). The collagen degrading activity of the lyophilized material showed a 3% increase in activity over the liquid material. It was determined that the lower concentration of calcium chloride had no negative effect on the stability of the collagenase to lyophilization.3410527945:v2

[0122] Example 5 - Collagenase, Glycine, low Calcium Chloride Temperature stability study 100121

[0123] A short-term temperature stability study was then set up with samples from the 190111 -experiment maintained at -20°C, +2-8°C, and +45°C. Samples were analyzed at 2, 4, 6, 8 weeks of incubation.

[0124] Results - Little to no loss of Wunsch activity was observed across all samples. A slow loss of collagen degrading activity in the +45°C samples during the experiment was observed, consistent with the results seen in the 190315 experiment. It was thus determined that the 0.1% calcium chloride concentration was within experimental error as effective at stabilizing the collagen degrading activity of the formulation as the 1.0% concentration.

[0125] Example 6 - Sorbitol and mannitol lyophilization stability experiment 200827

[0126] The objective of this study was to evaluate non -reducing sugars such as sorbitol and mannitol as excipients. The formulation was 6% Collagenase Gold Plus, 0.1% Calcium Chloride, 93.9% Sugar (Sorbitol or Mannitol). The concentration of the formulation was 100 milligrams of solid per milliliter of solution. The solution was dispensed into petri dishes and lyophilized. After lyophilization was completed, the plates were placed out at room temperature and exposed to ambient air. All the sorbitol lyophilized cakes collapsed into a hard crusty solid and were not analyzed further. Samples of the unfrozen, frozen, and lyophilized mannitol formulation were analyzed by Mono Q anion exchange chromatography.

[0127] Results - Mono Q analysis revealed that freezing the mannitol formulation resulted in the loss of monomeric Collagenase II form. Lyophilization of this formulation resulted in the loss of the monomeric forms of both Collagenase I and II forms. These two nonreducing sugars were thus unsuitable as major excipients as they are either too hydroscopic, or they destabilize the monomeric Collagenase I and II forms.

[0128] Example 7 - Dextran and I-Carrageenan Lyophilization Stability Study Experiment 210325,

[0129] This study evaluated the solubility of three different dextran products and I- Carrageenan as potential collagenase excipients. The materials used included: Dextran - 6 KDa, 68 KDa, 500 KDa; and I-Carrageenan. To assess solubility, a total of 2.325 grams of each excipient was added into a container with enough water to bring the volume to 40 milliliters. The 6 and 68 KDa dextran materials dissolved with some mixing. The 500 KDa material3510527945:v2required some heating to completely dissolve but remained in solution upon cooling. The I- Carrageenan required heating in a boiling water bath and a complete solution could not be obtained. Upon cooling, the I-Carrageenan formed a gel. The I-Carrageenan was thus determined to not be suitable as an excipient, but the three dextran materials had acceptable solubility.

[0130] Example 8 - Blending and lyophilization.

[0131] To each dextran solution was added 150 milligrams of Collagenase Gold Plus and 25 milligrams of calcium chloride and the volume brought to 50 milliliters. This gives a solids composition of 6% Collagenase, 1% Calcium Chloride, and 93% Dextran. A total of two samples were removed from each preparation. One was kept at +2-8°C and the other frozen at - 20°C. The remaining material was frozen as a bulk and lyophilized. For analysis, Mono Q Strong Anion Exchange analytical chromatographies were performed on the unfrozen, frozen, and lyophilized preparations. Also, Wunsch activity analyses were performed on the three lyophilized preparations.

[0132] Results - Mono Q chromatographic overlays of the unfrozen, frozen, and lyophilized samples of each preparation showed no obvious change in the elution profiles for each formulation. Also, an overlay of the three lyophilized samples showed no obvious difference between the samples. The Wunsch specific activity of the three lyophilized preparations were identical within experimental error of the assay and consistent with the starting Collagenase Gold Plus.

[0133] The Dextran 6KDa lyophilizate dissolved very quickly, the 68 KDa lyophilizate dissolved slower and the 500KDa lyophilizate even slower. All three lyophilizate cakes were hard but they all could be powdered. The hardness followed the molecular weight of the dextran. These dextran products can be obtained as pharmaceutical grade.

[0134] Example 9 - Hydroscopic Properties.

[0135] For this study, experiments were undertaken to determine if the lyophilized dextran samples are hydroscopic and absorb moisture when exposed to air. The three dextran samples (6, 86, 500 KDa) were dissolved in water at 50 milligrams per milliliter, dispensed into weighed dishes and lyophilized. When dry, the dishes were exposed to room temperature air for four weeks and their weights monitored.3610527945:v2

[0136] Results - The net weight of the cakes immediately after lyophilization was consistent to the dry weight of the dextran dissolved in the water. This indicates that there was little water of hydration remaining in the lyophilized cakes. There was no change in the appearance of any of the cakes after four weeks exposed to atmospheric moisture. Also, all three cakes powdered consistent with the time zero sample. There was no significant change in the weight of the samples during the experiment’s duration. The dextran formulations were thus determined to be suitable for evaluation as excipients for enzyme stabilization.

[0137] Example 10 - Debridement Formulation experiment 220412

[0138] This experiment investigated the impact of DEAE Dextran on formulations of collagenase, glycine, dextran, and calcium chloride. The materials used included: Collagenase Gold Plus bulk liquid Lot 210312; Dextran 20 KDa alfa Aesar J61217 lot X01H007; DEAE Dextran Alfa Aesar J63781 lot U16H016; Glycine USP Baker 0583-1 lot 198055; and Calcium Chloride Dihydrate ACS Fisher C79-3 lot 142537. The formulations produced from these materials included: #01 - 6% Collagenase, 1% Calcium Chloride, 12% DEAE Dextran, 81% Dextran; #02 - 6% Collagenase, 1% Calcium Chloride, 12% DEAE Dextran, 81% Glycine; and #03 - 6% Collagenase, 1% Calcium Chloride, 12% DEAE Dextran, 41% Glycine, 40% Dextran

[0139] The bulk solids were dissolved in water and the pH adjusted to 7.4 with sodium hydroxide. Liquid collagenase was added, and the formulations diluted to 50 milligrams solids / milliliter. An aliquot was dispensed into a dish and the remainder into bottles. One bottle was kept at +2-8°C and the remainder frozen at -20°C. One bottle was kept frozen, and the remaining bottles and the dish sample lyophilized. Lyophilized samples of all three formulations were kept in an incubator at +37°C for 4 weeks. These samples were then rehydrated and analyzed using Mono Q ion Exchange Chromatography, Wunsch enzyme assay, and Superdex- 200 size exclusion chromatography. Collagen degrading activity assay was performed on the +37°C samples.

[0140] Results - All three formulations were found to be stable to freezing and lyophilization by the three analytical methods. The three dish formulations powdered easily and experienced no change upon exposure to room temperature air. As shown in Table 4, all three formulations kept at +37°C showed no change in the Wunsch enzyme assay, the Superdex-200 size exclusion Chromatography or collagen degrading assay.Table 4. Formulation 220412 Activity recovery for 4 weeks at 37°C3710527945:v2

[0141] Example 11 - Debridement Formulation Experiment 220425

[0142] This experiment was performed to evaluate formulations with higher concentrations of DEAE Dextran and change to Dextran 40KDa as this material was most used in pharmaceutical preparations. The materials used included: Collagenase Gold Plus bulk liquid Lot 210312; Dextran 40 KDa Alfa Aesar J63690 lot W09G052; DEAE Dextran Alfa Aesar J63781 lot U16H016; and Glycine LISP Baker 0583-1 lot 198055Calcium Chloride Dihydrate ACS Fisher C79-3 lot 142537. The formulations tested were: #01 - 6% Collagenase, 1% Calcium Chloride, 31% DEAE Dextran, 31% Dextran, 31% Glycine; #02 - 6% Collagenase, 1% Calcium Chloride, 21.5% DEAE Dextran, 21.5% Dextran, 50% Glycine; and #03 - 6% Collagenase, 1% Calcium Chloride, 20% DEAE Dextran, 63% Glycine, 10% Dextran.

[0143] During the experiments, the bulk solids were dissolved in water and the pH adjusted to 7.4 with sodium hydroxide. Liquid collagenase was added, and the formulations diluted to 50 milligrams solids / milliliter. An aliquot was dispensed into a dish and the remainder into bottles. One bottle was kept at +2-8°C and the remainder frozen at -20°C. One bottle was kept frozen, and the remaining bottles and the dish sample lyophilized. Lyophilized samples of each formulation were kept at +37°C for four weeks. These samples were rehydrated and analyzed using Mono Q ion Exchange Chromatography, Wunsch enzyme assay, Superdex-200 size exclusion chromatography. Collagen degrading activity assay was performed on the +37°C samples.

[0144] Results - All three lyophilized dish samples had a uniform cake with no obvious water absorption when exposed to ambient atmosphere. Formulation #01 had a significantly harder cake which did not powder easily. Formulations #02 and #03 were easily powdered. The physical and enzymatic properties of these formulations were unchanged upon freezing and lyophilization. As shown in Table 5, all three formulations kept at +37°C showed no change in3810527945:v2the Wunsch enzyme assay, and collagen degrading assay in comparison to the starting Collagenase Gold Plus starting material. In short, the Dextran 40 KDa product concentrations up to 30% solids appeared to be acceptable for stability testing. DEAE Dextran Concentrations up to 30% solids appear to be acceptable for stability testing.Table 5. Formulation 220425 Activity recovery for 4 weeks at 37°C

[0145] Example 12 - Debridement Formulation Experiment 230726

[0146] For this experiments, several debridement formulations were prepared with high concentrations of DEAE dextran along with a variety of dextrans. The materials used included: Collagenase Gold Plus bulk liquid Lot 210312; Dextran 40 KDa Alfa Aesar J63690 lot W09G052; Dextran 20 KDa Alfa Aesar J61217 lot X01H007; Dextran 86KDa Acros 40627 LotA0386491; Maltodextrin Thermo Fisher 466680 Lot A0437342; DEAE Dextran Alfa Aesar J63781 lot U16H016; Glycine USP Baker 0583-1 lot 198055; Calcium Chloride Dihydrate ACS Fisher C79-3 lot 142537.; and Mannitol Sigma Aldrich M9647 Lot SLBF7222V. The formulations tested are shown in Table 6 below.Table 6. Exemplary Debridement Formulations.3910527945:v2

[0147] During the experiments, a total of 50 milliliters of 50 milligram solids / milliliter of solution was prepared for each formulation. The excipients were first dissolved in water and the pH adjusted to 7.4 with sodium hydroxide. The collagenase Gold plus solution was then added and the volume brought to 50 milliliters. A total of 20 milliliters of each formulation was dispensed into petri dishes and the remainder was dispensed into bottles (2 milliliters / bottle). One bottle was kept at +2-+8°C. The petri dishes and remaining bottles were frozen at -20°C. One bottle of each formulation was kept at -20°C for later analysis and the remaining bottles and the petri dishes were lyophilized. After lyophilization the petri dish samples were left exposed to ambient conditions for two weeks. Samples of each formulation were placed in a 45°C incubator for two and four weeks. Samples were evaluated by Mono Q chromatography and Wunsch activity assay. The assessed Wunsch Activity is shown in Table 7 below:Table 7. Assessed Wunsch Activity.4010527945:v2ND = Not Determined

[0148] Based upon the Mono Q results only selected formulations were analyzed for Wunsch activity. When assessing the petri dish lyophilized formulations exposed to ambient conditions for two weeks, the following observations were made:Formulation #01 - Uniform cake which powders nicely.Formulation #02 - Some evidence of phase separation during freezing. The cake collapsed. Formulation #03 - Uniform cake which powders nicely. Cake is harder than Formulation #01. Formulation #04 - Some evidence of phase separation during freezing. The cake collapsed.Formulation #05 - Uniform cake which can be powdered. Cake is harder than Formulation #03. Formulation #06 - Some evidence of phase separation during freezing. The cake collapsed. Formulation #07 - Uniform cake with plate like crystal structure. Does not powder well.Formulation #08 - Similar to Formulation #07 but with a denser tighter cake.Formulation #09 - Similar to Formulation #08 but with a denser tighter cake.Formulation #10 - Uniform soft cake with a fine crystal structure.Formulation #11 - A uniform soft cake which powders nicely.

[0149] All plates had a small weight increase of about 5 to 8% except formulation #11 with an increase of only 2%. All of the lyophilized Formulations had Mono Q profiles consistent4110527945:v2with previous formulations. After two weeks at +45°C, formulations #02, #04, #06, #10, #11 all showed broadening of the collagenase I & II peaks. Formulations #01, #03, #05 had some front shouldering on the Collagenase I & II peaks. Formulations #07, #08, #09 had almost perfect peak overlays. After four weeks at +45°C, all formulations showed very high levels of enzyme deterioration. From these studies, it was thus determined that formulations with high concentrations of DEAE Dextran had undesirable lyophilizate properties, and it was thought that the DEAE Dextran percentage should be kept below 30%. Addition of Glycine to the formulations appeared to improve the stability and chromatographic integrity of the Collagenase enzymes. It also produced lyophilized cakes with better powdering properties. The higher the molecular weight of the dextran the harder the cake. High percentage of dextran in the formulations yields lyophilized formulations with undesirable plate like crystalline lyophilizates. Lower concentrations of the dextran may be advantageous. Maltodextrin and mannitol appeared to be inferior excipients.

[0150] Example 13 - Debridement Experiment 231002

[0151] Using the data from the previous experiments a design of experiment stability study was set up. The objective was to identify the best ratios of components to yield a formulation with the best stability at elevated temperatures. A second objective was to identify the formulation which is least expensive to manufacture with the desired stability. Glycine, Dextran 40 KDa, DEAE Dextran, and Calcium Chloride were used as the excipients in this experimental design. The materials included: Collagenase Gold Plus bulk liquid Lot 230918 - 30.6 mg / mL; Dextran 40 KDa Alfa Aesar J63690 lot W09G052; DEAE Dextran Alfa Aesar J63781 lot U16H016; Glycine USP Baker 0583-1 lot 198055; and Calcium Chloride Dihydrate ACS Fisher C79-3 lot 142537. All formulations contained 10% Collagenase Gold Plus and 1% Calcium Chloride dry weight. Dextran 40 KDa was chosen for this experimental design because of its multiple use pharmaceutical preparations and its lyophilization properties. The formulations were as follows:Table 8. Additional Exemplary Debridement Formulations.4210527945:v2**Formulation #01 was inserted into the design to identify the contribution of the glycine to the stabilization and the collagenase gold plus sample was added as an excipient free control.

[0152] A total of 35 milliliters of each formulation was prepared. The Glycine, DEAE Dextran, Dextran 40KDa and calcium chloride were weighed and transferred into 50 milliliter tubes. The solids were dissolved in 25 milliliters of RO / DI water and the pH adjusted to 7.4-7.5 using IN sodium hydroxide. A total of 175 milligrams (5.73 milliliters) of Collagenase Gold Plus was added and the final volume brought to 35 milliliters. For each formulation two milliliters of formulation were dispensed into twelve ten milliliter lyophilization vials and 10 milliliters into a petri dish and the samples frozen at -20°C. The petri dishes and eleven vials were freeze-dried with the twelfth kept as a frozen control. Into ten jars was placed one vial of each formulation and some drying agent. Five of the jars were placed in a 37°C oven and five jars in a 45°C oven. The petri dish samples were weighed and placed on a table at ambient conditions. The samples were analyzed at 4, 8, 13, 17, and 22 weeks. For analysis, the +37°C4310527945:v2and +45°C stability samples were taken out of the incubators and the +37°C samples placed in a - 20°C Freezer for future later analysis if needed. The +45°C samples were rehydrated with 2.0 milliliters of RO / Di water and analyzed by Mono Q chromatography, and Wunsch and collagen degrading assays.

[0153] The Wunsch activity for 45°C Stability Samples and the Mono Q Chromatographic Results are provided in Tables 9 and 10 below.Table 9. Wunsch activity for 45°C Stability Samples.4410527945:v2* Collegenase II control used.Table 10. Mono Q Chromatographic Results.4510527945:v24610527945:v2

[0154] Results of the Mono Q chromatographic studies of the above formulations are further illustrated in FIGS. 1-8. Elution of Collagenase Gold (see FIG. 1) resulted in a large peak at about 11 minutes which was intact Collagenase II enzyme. The shoulder at about 12 to 13 minutes is proteolyzed Collagenase I & II related substances which have had their collagen binding domain removed. The peak at about 15 minutes is a Collagenase I related substance which has lost both collagen binding domains. It was determined to be a fully active gelatinase but with little collagen degrading activity. The large peak at about 18 minutes is intact Collagenase I enzyme. The shoulder eluting on the backside of the Collagenase peak at 19 to 20 minutes is composed of two collagenase I related proteins which have had one of the collagen binding domains proteolytically removed. They are both active gelatinase and collagenases.The peak at 25 minutes contains aggregated forms of the collagenase enzymes. FIG. 2 shows the elution profde of a collagenase Gold sample prepared, lyophilized, and maintained at -20°C.

[0155] FIG. 3 shows elution profdes of the lyophilized and stored collagenase Gold sample of FIG. 2 against Formulation #01. FIG. 4 shows elution profiles of the lyophilized and stored collagenase Gold sample of FIG. 2 against Formulation #02, both maintained at 45°C for 22 weeks. The large peak at 2-3 minutes is DEAE Dextran.

[0156] FIG. 5 shows elution profiles of the lyophilized and stored collagenase Gold sample of FIG. 2 against Formulation #06, both maintained at 45°C for 22 weeks. The large peak at 2-3 minutes is DEAE Dextran. FIG. 6 shows elution profiles of the lyophilized and stored collagenase Gold sample of FIG. 2 against Formulation #09, both maintained at 45°C for 22 weeks. The large peak at 2-3 minutes is DEAE Dextran.

[0157] FIG. 7 shows elution profiles of the lyophilized and stored collagenase Gold sample of FIG. 2 against Formulation #12, both maintained at 45°C for 22 weeks. FIG. 8 shows elution profiles of the lyophilized and stored collagenase Gold sample of Figure 2 against Formulation #15, both maintained at 45°C for 22 weeks. The large peak at 2-3 minutes in each chromatograph is DEAE Dextran.

[0158] As is clear from the figures, each formulation evaluated showed good stability, similar to the collagenase Gold standard.

[0159] Because of the deterioration seen in the Mono Q chromatograms of Formulations #05 to #16, only formulations #01 to #04 and the Collagenase gold stability samples were4710527945:v2analyzed as part of a soluble collagen assay. The results of the soluble collagen assay are shown in Table 11 below:Table 11. Soluble Collagen Assay.

[0160] Moisture absorption experiments were also performed and the results of the moisture absorption experiments are shown in Table 12 below.Table 12. Moisture absorption Results.4810527945:v2

[0161] Upon analysis of the results from the above-described experiments, it was observed that there was no major trend within the Wunsch activity data to corelate with the enzyme stability. This was not surprising as only the catalytic domain of the molecule was required for full enzymatic activity so any deterioration to the collagen binding regions will have no effect on this assay. For the moisture absorption experiment, there were no upward trends in the weights of any of the formulations, which indicated that none of the formulations have any significant hydroscopic property. Also, for the Mono Q analyses, it was observed that keeping the samples dry in the desiccator jars eliminated previous observations where the formulations containing DEAE Dextran had complete breakdown of the collagenase enzymes. Those results appear now to have been the result of moisture getting into the sample vials during the incubation.

[0162] From these analyses the following observations were identified: 1) Addition of glycine to the collagenase prior to the lyophilization (Formulation #01) improves the collagenase thermal stability; 2) Formulations #02, #03, and #04 containing DEAE Dextran showed no significant changes in the chromatograms when compared to the starting preparations; 3) Formulations #05, #06, and #07 containing plain dextran showed deterioration of the Collagenase I & II peaks, and these formulations showed no improvement in thermal stability over the Collagenase Gold stability sample; and 4) the remaining Formulations #08 to #16 containing both DEAE Dextran and plain Dextran 40KDa showed a variety of deteriorations4910527945:v2indicating that the presence of DEAE Dextran did not overcome the destabilizing effects of the Dextran 40 KDa but instead seems to have further decreased the stability of the collagenase enzymes.

[0163] For the soluble collagen assay, and based upon the Mono Q chromatographic profiles, only formulations #01 to #04 were analyzed along with the Collagenase Gold Plus stability along with collagenase gold plus control material.

[0164] In summary, and from the analysis of the data, the following conclusions could be drawn: 1) the lyophilized Collagenase Gold Plus experiences a loss of activity during the stability study; 2) lyophilization of the Collagenase Gold Plus with Glycine reduces the deterioration of collagen degrading activity by about 50%; 3) inclusion of DEAE Dextran into the collagenase Glycine formulation results in total thermal stability of the collagenase activity for the experimental duration; and 4) Collagenase to DEAE Dextran ratios of 1 : 1, 1 :2, and 1 :3 all have the same thermal stability.

[0165] Example 14 - Debridement Formulation Experiment 240409

[0166] In this experiment, experiment 231002 was repeated with Formulations #02, #03, #04 but with replacing the Glycine with Alanine, Serine, and Threonine. The materials utilized included: Collagenase Gold Plus bulk liquid Lot 230918 - 30.6 mg / mL; L-Alanine Thermo Fisher J60279.32 lot Y07J005; L-Serine Beantown Biochemicals 128350 Lot 50058942; L- Threonine Beantown Biochemicals 211050 Lot 50036745; DEAE Dextran Alfa Aesar J63781.22 lot V16H016; and Calcium Chloride Dihydrate ACS Fisher C79-3 lot 142537. The following formulations were produced and incubated at 45 °C and tested for recovery of collagen degrading activity:Table 13. Formulations for Debridement Formulation Experiment 240409.5010527945:v2

[0167] A total of 25 milliliters of a 50 milligram solids / milliliter was prepared for each formulation. The DEAE Dextran and the appropriate amino acid were dissolved in water and the pH adjusted to 7.4 using IN sodium hydroxide solution. The Calcium Chloride and Collagenase Gold solution were then added, and the final volume brought to 25 milliliters. A total of 2.0 milliliters of each formulation was dispensed into bottles. One sample was kept at refrigerator temperature and the remaining samples were frozen. The remaining 5 milliliters of formulations were dispensed into Petri Dishes. A total of eight of the frozen samples were lyophilized along with the samples in the Petri dishes. A total of five samples of each formulation were placed in a +45°C incubator for stability testing. Because the Wunsch assay has not demonstrated any value for characterizing the status of the collagenase stability, its use was discontinued for this experiment. Only the Mono Q Chromatography and soluble collagen assay were used to characterize the enzyme stability.

[0168] Because the 50 mg / mL trinitrobenzene sulfonic acid in methanol product was discontinued it was replaced with 1.2-Napthoquinone-4-Sulfonic Acid Sodium salt (Acros Organics Cat # 415400100 Lot A20343901). The crystalline powder was dissolved in color development buffer at a concentration of 2 mg / mL immediately prior to incubation with the assay samples. All other operations were unchanged. The absorbance of the colored product was monitored at 301 nm.

[0169] Results - For the Mono Q Analysis of the starting formulations, except for minor differences in collagenase peak areas due to small variations in formulation preparations there were no obvious differences between the pre and post lyophilized samples. After thirteen weeks peak broadening was observed for all samples especially for the alanine and threonine formulations. The collagen degrading activities are shown in the Table below for the samples maintained at 45°C.Table 14. Collagen Degrading Activities.5110527945:v2*The apparent high activity values for the time zero- and four-week samples are an artifact of the low specific activity seen for the collagenase gold control material in these assays.

[0170] While no formulation met the criteria for collagen degrading activity stability failure, the three serine formulations appear to have the best stabilization of activity. It is possible that the less hydrophobic serine molecule provides enhanced protein stability.

[0171] Example 15 - Debridement Formulation Experiment 241014

[0172] In this experiment, experiment 231002 was repeated with Formulations #02, but with replacing the Collagenase Gold with individually purified Collagenase II, and Collagenase I proteins. This formulation was chosen because previous experiments indicated this ratio of Collagenase to DEAE dextran (1 : 1) might have lower stability than higher ratios of DEAE Dextran to Collagenase (1 :2 or 1 :3). The materials utilized included: Collagenase II bulk liquid Lot 241010 - 21.98 mg / mL; Collagenase I bulk lot 241007; USP Glycine JT Baker 0582-01 lot 198055; DEAE Dextran Alfa Aesar J63781.22 lot V16H016; and Calcium Chloride Dihydrate ACS Fisher C79-3 lot 142537. Only one formulation was produced and tested for each enzyme: Collagenase 10% (375 milligrams), DEAE Dextran 10% (375 milligrams), Calcium Chloride dihydrate 1% (37.5 milligrams), Glycine 79% (2,962.5 milligrams).

[0173] A total of 75 milliliters of a 50 milligram solids / milliliter was prepared for each formulation. The DEAE Dextran and the glycine were dissolved in water and the pH adjusted to 7.4 using IN sodium hydroxide solution. The Calcium Chloride and Collagenase solution were5210527945:v2then added, and the final volume brought to 75 milliliters. A total of 2.0 milliliters of each formulation was dispensed into 37 bottles, frozen at -20°C and lyophilized. A total of 12 samples of each lyophilized formulation were placed in a -20°C freezer or a +45°C or +55°C incubator for stability testing. Because the Wunsch assay has not demonstrated any value for characterizing the status of the collagenase stability, its use was discontinued for this experiment. Because of aggregation at the higher temperatures the Mono Q Chromatography was less informative about the enzyme stability and was replaced with a Superdex 200 chromatography and soluble collagen assay.

[0174] Results. A Mono Q overlay of the purified Collagenase I and II proteins can be seen in FIG. 9 and the Superdex 200 overlay is shown in FIG. 10. It should be noted that while both enzymes have an almost identical molecular weight the Collagenase I enzyme elutes significantly ahead of the Collagenase II protein indicating that the Collagenase II protein appears to have a more compact structure than the Collagenase I protein under these conditions. The recovery of the soluble collagen degrading activity of the high temperature samples is recorded as a percentage of the -20°C control samples.Table 15. Collagen Degrading Activity.

[0175] Superdex 200 Overlay for the 46-week Collagenase II samples at -20°C, +45°C or +55°C is shown in FIG. 11 while the overlay for the Collagenase I can be seen in FIG.12. There appears to be little breakdown of either enzyme but there is a steady increase in the formation of dimers and oligomers for both enzymes. On average the Collagenase II enzyme5310527945:v2appears to have slightly enhanced stability when compared to the Collagenase I enzyme at a higher temperature.

[0176] Example 16 - Debridement Formulation Experiment 250327

[0177] In this experiment, experiment 241014 was repeated but with purified Collagenase II. The same formulation was use as in the previous experiment with the DEAE dextran being replaced with poly epsilon - L - Lysine hydrochloride. The poly epsilon - L - lysine was selected for several reasons for analysis. First, it has a more compact molecular structure and may bind more tightly to the collagenase and possibly enhance the thermal stability to denaturation. Secondly, this molecule has known antibiotic properties with no reported antigenic properties. The materials utilized included: Collagenase II bulk liquid Lot 241010 - 21.98 mg / mL; USP Glycine JT Baker 0582-01 lot 198055; Poly epsilon - L - Lysine Biosynth FP 14985 lot 213602; and Calcium Chloride Dihydrate ACS Fisher C79-3 lot 142537. Collagenase 10% (375 milligrams), Poly epsilon - L - Lysine (375 milligrams), Calcium Chloride dihydrate 1% (37.5 milligrams), Glycine 79% (2,962.5 milligrams).

[0178] A total of 75 milliliters of a 50 milligram solids / milliliter was prepared. The poly epsilon - L - lysine and the glycine were dissolved in water and the pH adjusted to 7.4 using IN sodium hydroxide solution. The Calcium Chloride and Collagenase solution were then added, and the final volume brought to 75 milliliters. A total of 2.0 milliliters of each formulation was dispensed into 37 bottles, frozen at -20°C and lyophilized. A total of 12 samples of each lyophilized formulation were placed in a -20°C freezer or a +45°C or +55°C incubator for stability testing. Because the Wunsch assay has not demonstrated any value for characterizing the status of the collagenase stability, its use was discontinued for this experiment. Because of aggregation at the higher temperatures the Mono Q Chromatography was less informative about the enzyme stability and was replaced with a Superdex 200 chromatography and soluble collagen assay.

[0179] Results. A table recording the recovery of collagen degrading activity of the temperature stability samples is shown below. The recovery of the soluble collagen degrading activity of the high temperature samples is recorded as a percentage of the -20°C control samples. An overlay of the Superdex 200 chromatographies at seventeen weeks, twenty-two, and twenty-six weeks is seen in FIGS. 13-15.5410527945:v2

[0180] The recovery of the soluble collagen degrading activity of the high temperature samples is recorded as a percentage of the -20°C control samples.Table 16. Collagen Degrading Activity.

[0181] *The Collagenase II control specific activity of the 17-week analysis was about 10% higher than the previous Collagenase II control samples.

[0182] The poly epsilon - L - lysine appears to have enhanced stabilizing of the Collagenase II enzyme activity when compared to the DEAE Dextran. At 45°C approximately 95% of the collagen degrading activity and at 55 °C approximately 90% of the collagen degrading activity is retained. For both temperatures these remaining activities appear to have reached a plateau early in the testing. This indicates that perhaps a slightly higher ratio of poly epsilon - L-Lysine ratio above the 1 : 1 ratio might improve the stability even more. From the Superdex 200 overlay there is almost no formation of dimer or oligomers at elevated temperatures. This is superior to the DEAE Dextran. The possible reasons for this observation are that the molecule has four methylene groups in each lysine subunit which could be covering any hydrophobic patches on the collagenase surface reducing the probability of interacting with other hydrophobic patches on other collagenase molecules, the polymer has amide bonds which can potentially hydrogen bond to the surface increasing the energy of activation needed to dissociate the complex, and lastly the repeating alpha amino groups are directly linked to the poly lysine chain providing more rigidity to the interaction. This data suggests that other poly5510527945:v2epsilon - L - lysine analogs with less than four methylene groups might also have similar stabilizing properties. An example is poly delta -L- ornithine.

[0183] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0184] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.5610527945:v2

Claims

CLAIMSWhat is claimed is:

1. An enzymatic composition having enhanced thermal stability, comprising: an enzyme; a polymeric charged molecule having a charge opposite a charge of the enzyme; and a zwitterionic stabilization agent.

2. The composition of claim 1, wherein the composition has a pH greater than an isoionic point of the enzyme, and wherein the polymeric charged molecule has a positive charge.

3. The composition of claim 1, wherein the composition has a pH less than an isoionic point of the enzyme, and wherein the polymeric charged molecule has a negative charge.

4. The composition of claim 1, wherein the enzyme is a protease.

5. The composition of claim 4, wherein the protease is a collagenase.

6. The composition of claim 5, wherein the collagenase is derived from Hcithewaya histolytica by natural or recombinant fermentation.

7. The composition of claim 6, wherein the collagenase comprises collagenase Type I, Type II, or combinations thereof.

8. The composition of claim 5, wherein the collagenase is a natural or a recombinant collagenase Type I and / or Type II.

9. The composition of claim 6, wherein the collagenase is enriched prior to inclusion in the composition.5710527945:v210. The composition of claim 8, wherein the composition comprises collagenase Type II, and wherein the collagenase Type II is provided in an amount of from about 10 to about 60% of the total weight of the collagenase in the composition.

11. The composition of claim 5, wherein the collagenase and the polymeric charged molecule are provided in the composition at a mass ratio of from about 1 : 1 to about 1 : 3.

12. The composition of claim 1, wherein the polymeric charged molecule is an amine containing molecule or a resin-based amine derived synthetic polymer.

13. The composition of claim 12, wherein the amine containing molecule is selected from the group consisting of: a chemically derivatized cellulose; a linear, branched, or cross-linked dextran; a derivatized polyacrylate or polymethacrylate having charged groups selected from primary, secondary or tertiary amines; diethyl amino ethyl (DEAE); DEAE Dextran; poly- epsilon-lysine; poly-lysine; poly-arginine; poly-ornithine; poly delta ornithine; poly 2,3 -diamino propionic acid; poly beta 2,3 diamino propionic acid; poly 2,4-diamino butyric acid; poly gamma 2,4 diamino butyric acid; poly 2,5 diamino valeric acid; poly delta 2,5 diamino valeric acid; DEAE cellulose; DEAE Sephadex; DEAE Sepharose; QAE Sephadex; and Q Sepharose.

14. The composition of claim 13, wherein the amine containing molecule is DEAE Dextran or poly-epsilon-lysine.

15. The composition of claim 1, further comprising an enzyme-stabilizing metal salt.

16. The composition of claim 15, wherein the enzyme-stabilizing metal salt is a non-chelated calcium salt.

17. The composition of claim 16, wherein the non-chelated calcium salt is selected from the group consisting of calcium chloride, calcium bromide, and calcium acetate.5810527945:v218. The composition of claim 16, wherein the non-chelated calcium salt is provided in an amount of from about 0.1% to about 2% of the total weight of the composition.

19. The composition of claim 1, wherein the zwitterionic stabilization agent is an amino acid or an amino acid-peptide blend.

20. The composition of claim 19, wherein the amino acid is selected from the group consisting of glycine, alanine, serine, threonine, and combinations thereof.

21. The composition of claim 20, wherein the amino acid is glycine.

22. The composition of claim 1, wherein the composition has a pH of from about 6.0 to about8.0.

23. The composition of claim 1, wherein the composition is spray dried or lyophilized.

24. The composition of claim 23, wherein the spray dried or lyophilized composition is admixed with a suitable carrier or bulking agent.

25. An enzymatic composition having enhanced thermal stability and formulated for use in wound healing and debridement, comprising: from about 5% w / w to about 20% w / w enriched collagenase; from about 5% w / w to about 30% w / w DEAE dextran or poly-epsilon-lysine; from about 0.1% w / w to about 2% w / w calcium salt; and from about 48% w / w to about 89.9% w / w free amino acids.

26. The enzymatic composition of claim 25, comprising: about 10% w / w enriched collagenase; about 10% w / w DEAE dextran or poly-epsilon4ysine; from about 0.5% w / w to about 2% w / w calcium salt; and from about 78% w / w to about 79.5% w / w free amino acids.5910527945:v227. The enzymatic composition of claim 25, comprising: about 10% w / w enriched collagenase; about 10% w / w DEAE dextran or poly-epsilon-lysine; about 1% w / w calcium salt; and about 79% w / w free amino acids.

28. The enzymatic composition of any one of claims 25-27, wherein the free amino acids are selected from the group consisting of glycine, alanine, serine, and threonine.

29. The enzymatic composition of claim 28, wherein the free amino acid is glycine.

30. The enzymatic composition of claim 25, wherein the enriched collagenase comprises collagenase Type I and Type II, and wherein the collagenase Type II comprises from about 10 wt% to about 60 wt% of the collagenase in the composition.

31. The enzymatic composition of claim 30, wherein the enriched collagenase is a native or recombinant collagenase Type I and Type II.

32. The enzymatic composition of claim 31, wherein the recombinant collagenase has a sequence identity of at least 95% to native Hathewaya histolytica collagenase.

33. A method for enhancing thermal stability of an enzyme, comprising: providing an enzyme in need of enhanced thermal stabilization; and mixing the enzyme with a polymeric charged molecule having a charge opposite a charge of the enzyme and with a zwitterionic stabilization agent to produce an enhanced thermally- stable composition.

34. The method of claim 33, wherein the enhanced thermally-stable composition has a pH greater than an isoionic point of the enzyme, and wherein the polymeric charged molecule has a positive charge.6010527945:v235. The method of claim 33, wherein the enhanced thermally-stable composition has a pH less than an isoionic point of the enzyme, and wherein the polymeric charged molecule has a negative charge.

36. The method of claim 33, wherein the polymeric charged molecule is an amine containing molecule or a resin-based amine derived synthetic polymer.

37. The method of claim 36, wherein the amine containing molecule is selected from the group consisting of: a chemically derivatized cellulose; a linear, branched, or cross-linked dextran; a derivatized polyacrylate or polymethacrylate having charged groups selected from primary, secondary or tertiary amines; diethyl amino ethyl (DEAE); DEAE Dextran; poly- epsilon-lysine; poly-lysine; poly-arginine; poly-ornithine; poly delta ornithine; poly 2,3 -diamino propionic acid; poly beta 2,3 diamino propionic acid; poly 2,4-diamino butyric acid; poly gamma 2,4 diamino butyric acid; poly 2,5 diamino valeric acid; poly delta 2,5 diamino valeric acid; DEAE cellulose; DEAE Sephadex; DEAE Sepharose; QAE Sephadex; and Q Sepharose.

38. The method of claim 37, wherein the amine containing molecule is DEAE Dextran or poly-epsilon-lysine.

39. The method of claim 33, further comprising mixing the enzyme, the polymeric charged molecule having a charge opposite a charge of the enzyme, and the zwitterionic stabilization agent with an enzyme-stabilizing metal salt to form the enhanced thermally-stable composition.

40. The method of claim 39, wherein the metal salt is a non-chelated calcium salt.

41. The method of claim 40, wherein the non-chelated calcium salt is selected from the group consisting of calcium chloride, calcium bromide, and calcium acetate.

42. The method of claim 40, wherein the non-chelated calcium salt is provided in an amount of from about 0.1% to about 2% of the total weight of the composition.6110527945:v243. The method of claim 33, wherein the zwitterionic stabilization agent is an amino acid or an amino acid-peptide blend.

44. The method of claim 43, wherein the amino acid is selected from the group consisting of glycine, alanine, serine, threonine, and combinations thereof.

45. The method of claim 44, wherein the amino acid is glycine.

46. The method of claim 33, wherein the enzyme is a protease.

47. The method of claim 36, wherein the protease is selected from the group consisting of a collagenase, an elastase, trypsin, bromelain, and papain.

48. The method of claim 47, wherein the collagenase is derived from Hathewaya histolytica by natural or recombinant fermentation.

49. The method of claim 47, wherein the collagenase comprises collagenase Type I, Type II, or combinations thereof.

50. The method of claim 49, wherein the collagenase is a natural or a recombinant collagenase Type I and / or Type II.

51. The method of claim 48, wherein the collagenase is enriched prior to mixing the collagenase with the polymeric charged molecule having a charge opposite a charge of the enzyme and with the zwitterionic stabilization agent.

52. The method of claim 50, wherein the collagenase Type II is provided in an amount of from about 10% to about 60% of the total weight of the collagenase.6210527945:v253. The method of claim 47, wherein the protease is collagenase, and wherein the collagenase and the polymeric positively charged molecule are included in the composition at a mass ratio of from about 1 : 1 to about 1 :3.

54. The method of claim 33, further comprising dissolving the enhanced thermally-stable composition at a pH of from about 6.0 to about 8.0.

55. The method of claim 54, further comprising drying the dissolved enhanced thermally- stable composition by spray drying or lyophilization.

56. The method of claim 55, further comprising admixing the enhanced thermally-stable composition with a suitable carrier or bulking agent.

57. A method for treating a wound, comprising administering to a subject an effective amount of a composition of claim 1 or claims 25-27.

58. The method of claim 57, wherein administering the composition comprises administering an amount of the composition sufficient for wound healing and / or debridement.

59. The method of claim 58, wherein the composition is in a spray dried or lyophilized form, and wherein the composition is applied directly to a wound or is placed in a water permeable pouch prior to being applied to a wound.

60. The method of claim 58, wherein the composition is applied to a substrate.

61. The method of claim 60, wherein the substrate is a solid support.

62. The method of claim 61, wherein the solid support is a medical gauze.6310527945:v2

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