Composition containing collagenase, calcium, histidine and glycine and method for stabilizing collagenase

Abstract

The present invention provides a composition comprising collagenase as an enzyme, calcium, histidine, and glycine as coenzymes, and a method for stabilizing an enzyme, comprising preparing the composition by adding histidine and glycine to collagenase. According to the enzyme composition and the method for stabilizing an enzyme of the present invention, it is possible to minimize or inhibit the aggregation of the enzyme, and it is possible to provide a dosage form in which the concentration of the lyophilized enzyme does not decrease even during long-term storage, and which has excellent reconstitution time and moisture content and excellent enzyme activity.

Description

[Technical field]

[0001] The present invention relates to a composition containing collagenase, calcium, histidine and glycine and a method for stabilizing an enzyme, and more specifically, to a composition having increased stability by containing collagenase, calcium, histidine and glycine, and a method for stabilizing collagenase using the same. [Background technology]

[0002] Collagen is the main structural component of mammalian organisms and constitutes a large proportion of the total protein content in the skin and other parts of the animal body. In humans, it is particularly important during the wound healing process and the natural aging process. Various skin injuries, such as burns, surgery, infections and accidents, are often characterized by irregular accumulations of collagen-rich fibrous material and increased proteoglycan content. In addition to replacing damaged or destroyed normal tissue, excessive and misshapen deposition of new tissue is often formed during the healing process. Excessive collagen deposition occurs because the balance between collagen synthesis and collagen degradation is disturbed.

[0003] Many diseases and disorders are associated with excessive collagen deposition and the irregular accumulation of collagen-rich fibrous tissue. Such diseases and disorders are collectively referred to as "collagen-mediated disorders." Collagenase has been used to treat a variety of collagen-mediated disorders. Collagenase is an enzyme that has the specific ability to digest collagen.

[0004] Collagenase for therapeutic use can be obtained from a variety of sources including mammalian (e.g., human), crustacean (e.g., crab, shrimp), fungi, and bacteria (e.g., from fermentation of Clostridium, Streptomyces, Pseudomonas, or Vibrio). Collagenase may also be genetically engineered. One common source of crude collagenase is obtained from bacterial fermentation processes, specifically, from the fermentation of C. histolyticum. Crude collagenase obtained from C. histolyticum can be purified using a number of chromatographic techniques.

[0005] On the other hand, proteins, including enzymes, can lose biological activity as a result of physiological instability (e.g., denaturation, aggregate formation, etc.) and chemical instability, such as hydrolysis, oxidation, and deamidation. Protein stability is additionally affected by factors such as pH, temperature, storage time, and number of freeze-thaw cycles.

[0006] To ensure stability, therapeutic protein formulations are generally supplied as protein solutions containing lyophilized protein that is dissolved immediately before use in a separately packaged water-soluble diluent (sometimes called "solvent") and additives to improve stability. For example, additives such as free amino acids (e.g., leucine, tryptophan, serine, arginine, and histidine) have been proposed as useful for formulating protein solutions. Some protein formulations currently available on the market contain proteins as stabilizers. For example, human serum albumin or purified gelatin are used to suppress chemical and physical changes in protein solutions. However, the addition of these proteins involves a complicated process to remove viral contamination.

[0007] Lyophilization is another method used to ensure stability, but this process increases production costs and involves increased risk of improper administration since the lyophilized protein must be dissolved immediately before use. The purpose of lyophilization is to remove water from the formulation, since harmful physical and chemical reactions often occur in the aqueous phase. That is, lyophilization is performed because stability is reduced in the aqueous liquid state, but even with lyophilization, there are problems with the above-mentioned problems and storage stability problems.

[0008] On the other hand, when preparing a liquid formulation using collagenase as an enzyme, there is a problem that water-soluble aggregates are generated due to acute stress. In addition, even if lyophilization is performed, there is a disadvantage that the concentration is low, the time required for reconstitution is increased, and the humidity content is high. In some cases, there is a disadvantage that the enzyme activity is reduced, so it is difficult to formulate it into a powder form. Furthermore, collagenase requires calcium as a coenzyme, and if collagenase lyophilization powder is produced by adding calcium, the frozen powder is not properly produced, and the desired collagenase specifications are not properly preserved, making the development of a powder form even more difficult.

[0009] Therefore, most collagenase formulations currently on the market are shipped in separate vials containing collagenase freeze-dried powder and calcium-containing buffer, which are then mixed together just before use to prepare a ready-to-use injection formulation. In other words, when freeze-dried powder is produced by incorporating calcium into collagenase, there is no need to separately prepare a calcium-containing buffer, and only water for injection is needed to prepare a ready-to-use injection formulation just before use, which has the advantage of significantly reducing production costs. However, the development of such technology is still insufficient.

[0010] Therefore, the present invention aims to develop and provide a production technique for "lyophilized powder of calcium-added collagenase" that does not have the above-mentioned problems.

[0011] A major related prior patent is PCT International Patent Publication No. WO2020-142701 (published on July 9, 2020). Summary of the Invention [Problem to be solved by the invention]

[0012] Although collagenase freeze-dried powder is prepared by adding calcium, the present invention aims to develop and provide a powder formulation in which aggregation of collagenase enzyme is minimized, the concentration of freeze-dried enzyme does not decrease even during long-term storage, and the liquid reconstitution time (dissolution time) and moisture content are excellent, and the enzyme activity can be maintained and restored to an excellent state by using various formulation additives focused on in the present invention. [Means for solving the problem]

[0013] The present invention provides a composition comprising collagenase as an enzyme; calcium as a coenzyme; histidine as a dosage form additive; and glycine as a dosage form additive.

[0014] In the composition of the present invention, the content of the collagenase enzyme is preferably 0.3% to 20.8% by weight based on the total weight of the composition.

[0015] In the composition of the present invention, the calcium is preferably contained in the form of a calcium salt.

[0016] In the composition of the present invention, the calcium or calcium salt is preferably present in an amount of 0.4% to 28% by weight based on the total weight of the composition.

[0017] In the composition of the present invention, the content of the histidine is preferably 0.4% to 29.9% by weight based on the total weight of the composition.

[0018] In the composition of the present invention, the content of the glycine is preferably 15% by weight to 94.6% by weight based on the total weight of the composition.

[0019] The composition of the present invention preferably further comprises trehalose as a dosage form additive.

[0020] It is preferable that the composition of the present invention further contains polysorbate 20 (PS20) as a surfactant.

[0021] In the composition of the present invention, the pH of the composition is preferably 6.0 to 7.0.

[0022] In the composition of the present invention, the composition is preferably in a lyophilized dosage form.

[0023] The present invention provides a solution prepared by mixing the lyophilized composition of the present invention with a diluent. Effect of the Invention

[0024] The present invention has an effect of minimizing or suppressing the generation of water-soluble aggregates even when a sudden stress is applied, even though a collagenase freeze-dried powder is prepared in a state where calcium is added, by using many dosage form additives developed in the present invention. In addition, it has an effect of not decreasing the concentration of freeze-dried collagenase even during long-term storage, and maintaining high enzyme activity. In addition, the composition of the present invention has an effect of minimizing the time required to dissolve a freeze-dried preparation in water for injection, and reducing the humidity content during freeze-drying. Furthermore, such a composition of the present invention can extend the period during which the activity is maintained at room temperature even after dissolving the freeze-dried preparation in water for injection, so that when a liquid preparation is used in portions, it is possible to maintain stability for a long period of time, and when a vial containing this composition is packaged, it is possible to improve the stability of the enzyme and reduce the amount of enzyme adsorbed to the vial.

[0025] Also, from an economic standpoint, the technology of the present invention has the advantage that collagenase formulations can be produced more economically since there is no need to prepare a separate solution containing calcium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Since the present invention can be modified in various ways and has various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to a specific embodiment, and should be understood as including all modifications, equivalents, and alternatives included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related publicly known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

[0027] The terms used in this application are merely used to describe specific embodiments and are not intended to limit the present invention. A singular expression includes a plural expression unless the context clearly indicates otherwise. In this application, the terms "include" or "have" are intended to specify the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and should be understood not to preclude the presence or additional possibility of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0028] Terms such as "first" and "second" may be used to describe various components, but the components should not be limited by the terms. The terms are used only to distinguish one component from another.

[0029] One embodiment of the present invention is a composition comprising: collagenase as an enzyme; calcium as a coenzyme; histidine as a dosage form additive; and glycine as a dosage form additive.

[0030] Collagenase binds calcium ions (Ca 2+ ) provides structural stability and increases substrate affinity, so calcium ions are required as a coenzyme. Calcium ions are generally supplied through calcium salts, with calcium chloride being the most commonly used calcium salt. However, calcium chloride is inherently deliquescent, and therefore has the property of hindering freezing when added to an aqueous solution. Therefore, when developing a freeze-dried formulation containing calcium chloride, the freezing process can be carried out smoothly only if a special formulation is developed to increase collagenase stability. In addition, only if a special formulation is used can the deliquescent nature of calcium chloride be overcome when drying is performed in a vacuum after freezing, and the moisture can be sufficiently removed to complete drying.

[0031] Since it is difficult to freeze-dry collagenase with calcium chloride added, a vial of freeze-dried collagenase and a vial of a diluent for dissolving the collagenase and supplying calcium ions are usually provided in separate forms. Providing two vials increases the manufacturing cost, so developing a collagenase formulation with calcium chloride added would have the advantages of convenience in manufacturing and reduced manufacturing costs, and this technology has been developed in the present invention.

[0032] In one embodiment, the collagenase used as an enzyme in the present invention is not necessarily limited to a specific one, and may be, for example, collagenase I, collagenase II, or a mixture thereof.

[0033] In the present invention, calcium used as a cofactor is preferably a calcium salt, and an example of such a cofactor may be calcium chloride. The calcium acts as a cofactor for the activity of the collagenase. That is, calcium can act as an activator or cofactor for the enzyme, and collagenase activity is increased by the number of calcium ions (Ca) per mole of enzyme. 2+ ) 0.1 mol. Also, four calcium ions can promote binding with collagen molecules.

[0034] The present invention is characterized in that it contains histidine as a dosage form additive and contains glycine as a dosage form additive.

[0035] In the present invention, the term dosage form additive refers to a compound that can form and maintain the dosage form of the present invention or solve problems in the dosage form that may occur in the absence of this additive.

[0036] In the present invention, histidine used as a dosage form additive serves as a buffer component, and L-histidine is preferably used. In conventional pharmaceutical compositions, various types of buffers have been used to adjust pH. As will be seen in the Examples below, the present inventors have conducted various experiments using sodium phosphate, sodium acetate, succinate, histidine, and Tris as buffers for dosage form buffering. As a result, when sodium phosphate was used as a buffer, precipitates such as calcium phosphate were observed, and it was confirmed that the purity and stability of the titer were the best when histidine was used compared to other buffers.

[0037] Glycine used as a dosage form additive in the present invention serves as a bulking agent. In conventional pharmaceutical compositions, various kinds of sugars, amino sugars, sugar alcohols, and amino acids are used as bulking agents, including monosaccharides such as glucose, mannose, galactose, fructose, and sorbose; disaccharides such as sucrose, lactose, maltose, and trehalose; trisaccharides such as rapinose; and sugar alcohols such as xylitol, sorbitol, mannitol, and inositol. As amino acids, basic amino acids such as glycine, arginine, lysine, histidine, and ornithine are mainly used. As can be seen in the examples below, the present inventors conducted various experiments using mannitol and glycine as bulking agents for buffering the dosage form, and found that when mannitol is used as a bulking agent, problems such as a decrease in concentration, a delay in reconstitution time, high humidity, an increase in HMWS (%), and an increase in fine particle concentration occur under temperature acceleration conditions, and that the use of glycine provides the best stability in purity and potency compared to other bulking agents. That is, the present invention is characterized by including glycine as a dosage form additive, and has the effect of minimizing the time required for reconstituting a lyophilized formulation in water for injection and reducing the humidity during lyophilization.

[0038] In one embodiment, the present invention may further include trehalose as a dosage form additive. Trehalose used as a dosage form additive serves as an osmolality regulator, and is preferably trehalose dihydrate. In general, an osmolality regulator refers to a compound or substance that functions as a protectant in a pharmaceutical composition and increases the stability of an active substance during or after the drying process, or is added for long-term storage stability of the resulting dry powder. Suitable protectants are generally easily dissolved in a solution and do not become viscous or polymerize upon contact with water.

[0039] For example, suitable protective agents include, but are not limited to, proteins such as human and bovine serum albumin, egg white, gelatin, immunoglobulins, whey protein, soy protein, caseinate, gelatin, immunoglobulins, carbohydrates such as monosaccharides (galactose, D-mannose, sorbose, etc.), disaccharides (lactose, trehalose, sucrose, etc.), monosodium glutamate, and the like. glutamate, lysine, glycine, alanine, arginine, histidine, and other amino acids, as well as hydrophobic amino acids (tryptophan, tyrosine, leucine, phenylalanine, and the like); methylamines such as betaine; excipient salts such as magnesium sulfate; polyols such as trihydric and higher sugar alcohols (e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol); propylene glycol; polyethylene glycol; pluronics; surfactants; and mixtures thereof.

[0040] As will be seen in the Examples below, the present inventors conducted various experiments using sodium chloride (NaCl), sorbitol, and trehalose as osmotic pressure regulators for buffering dosage forms, and confirmed that when sodium chloride or sorbitol is used as an osmotic pressure regulator, the concentration or content of the active ingredient is reduced or the active ingredient is decomposed, resulting in a decrease in stability, whereas when trehalose is used, stability is maintained even when stored at high temperatures for a long period of time. Trehalose is a non-reducing sugar formed by linking two glucose molecules with an α(1→1) α-glycosidic bond. Due to this bond, trehalose has very strong resistance to acid hydrolysis and is considered to be stable even at high temperatures and under acidic conditions.

[0041] In one embodiment, the composition according to the present invention may have a pH of 6.0 to 7.0. As described above, the present invention is characterized in that it contains histidine as a buffer, but the inventors have confirmed that the pH of the composition may vary depending on the type of buffer used. Therefore, when the composition according to the present invention has a pH of 6.0 to 7.0, the purity and titer stability are the most excellent.

[0042] According to this, in the composition of the present invention, the buffer is not particularly limited as long as it can have a pH value of about 6.5, similar to histidine, and other types of buffers can be used in place of or in addition to histidine. For example, buffers that can be used for pH 6.5 include MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, and Bis-Tris Propane.

[0043] In one embodiment, the composition according to the present invention may further include polysorbate 20 (PS20) as a surfactant.

[0044] In general, surfactants are commonly used in pharmaceutical compositions.Adding surfactants can reduce the tendency of protein to unfold by reducing the tension between interfaces, or reduce the amount of protein present at interfaces, so that protein can be dissolved.Adding surfactants can also protect protein from surface-induced denaturation and aggregation.

[0045] Thus, the composition according to the present invention may contain, as a surfactant, various surfactants known in the art, without any particular limitation. For example, polysorbate 20 or 80 (PS20 or PS80), poloxamer 188 (Pluronic® 68), polyoxyethylene-polyoxypropylene polymers such as Tween® 20, or non-ionic surfactants consisting of a hydrophilic polyoxyethylene head group and a hydrophobic fatty acid tail, etc. After testing many types of surfactants, the present inventors confirmed that polysorbate 20 (PS20) is the most effective.

[0046] In one embodiment, the composition of the present invention may be in a lyophilized dosage form. The composition may be for stabilizing the lyophilized dosage form.

[0047] Such a freeze-dried dosage form is a solid preparation obtained by freeze-drying a composition containing collagenase as an enzyme, calcium as a coenzyme, histidine as a dosage form additive, and glycine as a dosage form additive.

[0048] In addition, in the present invention, the composition may be in the form of a lyophilized solution or a solution prepared from a lyophilized composition and a diluent. That is, the composition may be a solution before lyophilization containing a solvent in addition to the enzyme, histidine, and glycine, or a liquid preparation prepared by dissolving a lyophilized solid preparation in a diluent. The diluent may be water for injection. In one embodiment, the concentration of collagenase enzyme in the solution may be various concentrations depending on the indication to be used, and may be 2.9 mg / mL, 2.3 mg / mL, or 0.225 mg / mL, for example.

[0049] When the composition containing the enzyme is a lyophilized solid preparation, it has excellent stability in terms of enzyme activity. When the composition containing the enzyme is a liquid preparation, the activity stability of the enzyme is reduced compared to a solid preparation, but the composition according to the present invention maintains the activity stability of the enzyme even in a liquid preparation. That is, according to the present invention, the period during which the activity of the lyophilized preparation is maintained at room temperature can be extended even after the lyophilized preparation is reconstituted in water for injection, so that when the liquid preparation is used in portions, the stability can be maintained for a long period of time, and when the vial containing the composition is packaged, the stability of the enzyme can be improved and the amount of the enzyme adsorbed to the vial can be reduced.

[0050] In one embodiment, the content of the collagenase enzyme in the present invention may be 0.3% to 20.8% by weight based on the total weight of the composition. Alternatively, the enzyme may be contained in an amount of 0.09 mg to 9 mg per 3 cc vial. The amount of collagenase enzyme to be added may vary depending on the indication. However, it must be assumed that the effect can be exerted, and it must be used within a range that can prevent the occurrence of side effects.

[0051] In one embodiment, the content of calcium or calcium salt may be 0.4% to 28% by weight based on the total weight of the composition. Alternatively, calcium or calcium salt may be contained in an amount of 0.132 mg to 13.2 mg per 3 cc vial. If the content of calcium or calcium salt is less than the above range, the coenzyme function may be insufficient and the enzyme activity may be reduced, while if the content is more than the above range, excessive precipitation or aggregation may occur.

[0052] In one embodiment, the histidine content may be 0.4% to 29.9% by weight based on the total weight of the composition. Alternatively, the histidine content may be 0.144 mg to 14.4 mg per 3 cc vial. If the histidine content is below this range, the pH of the composition may change and the potency stability may decrease, while if the histidine content exceeds this range, excessive precipitation or aggregation may occur.

[0053] In one embodiment, the content of glycine may be 15% to 94.6% by weight based on the total weight of the composition. Alternatively, glycine may be contained in an amount of 2.25 mg to 225 mg per 3 cc vial. If the content of glycine is less than this range, the stability may decrease when stored at high temperatures for a long period of time, while if the content of glycine is more than this range, excessive precipitation or aggregation may occur.

[0054] In one embodiment, the trehalose content may be 3.3 to 77.4% by weight based on the total weight of the composition. Alternatively, 0.90 to 90 mg of trehalose may be contained per 3 cc vial. If the trehalose content is below this range, the resistance to acid hydrolysis may be weakened and the stability may be reduced when stored at high temperatures for a long period of time, while if the trehalose content exceeds this range, excessive precipitation or aggregation may occur.

[0055] In one embodiment, the content of polysorbate 20 (PS20) may be 0.03% to 2.5% by weight based on the total weight of the composition, or 0.009 mg to 0.9 mg of polysorbate 20 may be included per 3 cc vial. If the content of polysorbate 20 is less than the above range, enzyme denaturation may occur quickly, whereas if the content of polysorbate 20 is more than the above range, excessive precipitation or aggregation may occur.

[0056] Another embodiment of the present invention is a method for stabilizing collagenase, comprising preparing a composition by adding histidine and glycine as dosage form additives to collagenase as an enzyme and calcium as a coenzyme, and freeze-drying the prepared composition.

[0057] First, a composition is prepared by adding histidine and glycine to collagenase as an enzyme, the collagenase enzyme being preferably added in an amount of 0.3% to 20.8% by weight based on the total weight of the composition, the histidine being preferably added in an amount of 0.4% to 29.9% by weight based on the total weight of the composition, and the glycine being preferably added in an amount of 15% to 94.6% by weight based on the total weight of the composition.

[0058] The enzyme, coenzyme, histidine, glycine, trehalose, and polysorbate 20 (PS20) used in the present invention are as described above. The addition of histidine and glycine as dosage form additives makes it possible to maintain the stability of the enzyme when the composition is in a solid or liquid phase.

[0059] The composition thus prepared is then freeze-dried. Freeze-drying can be accomplished by any method known in the art.

[0060] The lyophilized composition can then be dissolved in a diluent to produce a solution. When the composition of the present invention is formulated as a liquid formulation, the enzyme activity is still maintained.

[0061] The present invention provides a method for stabilizing an enzyme, comprising adding histidine and glycine to an enzyme to prepare a composition. The method for stabilizing an enzyme according to the present invention can minimize or inhibit enzyme aggregation, and can provide a formulation in which the concentration of a lyophilized enzyme does not decrease even during long-term storage, and which has excellent reconstitution time and moisture content and excellent enzyme activity.

[0062] The present invention can be better understood by the following examples, which are intended to be illustrative of the invention and are not intended to limit the scope of protection defined by the appended claims.

[0063] <Example 1: Preparation of materials>

[0064] In this example, collagenase was used as the pharmacologically active ingredient (API) enzyme. That is, recombinant collagenase I (CNXT1) and recombinant collagenase II (CNXT2) were prepared, and CNXT1 stock solution and CNXT2 stock solution were obtained, and then mixed to produce a collagenase mixed solution (CNT201).

[0065] Specifically, the recombinant collagenase I (CNXT1) and the recombinant collagenase II (CNXT2) were each produced as high-purity collagenase I and II solutions by obtaining transformants by introducing an expression vector carrying genes encoding foreign proteins (collagenase I and II) into an E. coli cell line, expressing the foreign proteins using the transformants, and then carrying out culture, recovery and purification steps. In the final step of the purification step, the recombinant collagenase I (CNXT1) and the recombinant collagenase II (CNXT2) obtained in the previous step were replaced with a buffer containing a mixture of histidine and calcium chloride to obtain the following CNXT1 stock solution and CNXT2 stock solution.

[0066] (1) CNXT1 stock solution: CNXT1 (recombinant collagenase I), 2.03 mg / mL, 10 mM L-histidine, 10 mM calcium chloride, pH 7.0

[0067] (2) CNXT2 stock solution: CNXT2 (recombinant collagenase II), 1.97 mg / mL, 10 mM L-histidine, 10 mM calcium chloride, pH 7.0

[0068] Then, the CNXT1 stock solution and the CNXT2 stock solution were mixed in the same amount based on the weight of protein to prepare a mixed solution (CNT201) with a collagenase concentration of 2.00 mg / mL.

[0069] In addition, the formulation of CNT201 and the chemical components and raw materials used in the analysis are as follows:

[0070] (1) L-Histidine: JT Baker, Cat# 2080-05, Batch# 0000181214

[0071] (2) Calcium Chloride Dihydrate: Sigma, Cat# 12022-1KG, Lot# BCCB9183

[0072] (3) Super Refined Polysorbate 20: Croda, Cat# SR40606, Batch# 0001204756

[0073] (4) Highly purified polysorbate 80: Croda, Cat# SR48833, Batch# 0001603779

[0074] (5) Poloxamer 188: Spectrum, Cat# P1169, Batch# 2EK0152

[0075] (6) Glycine: JT Baker, Cat# 0581-01, Batch# 0000226798

[0076] (7) Succinic Acid: Sigma, Cat# 398055-1KG, SLBZ5010

[0077] (8) Mannitol: JT Baker, Cat# 2553-01, Batch# 0000253262

[0078] (9) Sodium Succinate dibasic hexahydrate: Sigma, Cat# S2378-1KG, Lot# SLBX5905

[0079] (10) Trehalose Dihydrate: Pfanstiehl, Cat# T-104-4, Lot# 404441A

[0080] (11) Sodium Chloride: EMD Millipore, Cat# 1.06604.5000, Lot# K51865804002

[0081] (12) Glacial Acetic Acid: EMD Millipore, Cat# AX0073-9, Lot# 49287

[0082] (13) Tromethamine (Tris): JT Baker, Cat# 4102-01, Batch# 0000229888

[0083] (14) Imidazole: Sigma-Aldrich, Cat# I2399-100G, Lot# SLCG0769

[0084] (15)HEPES:VWR, Cat# 0511-250G, Lot# 18G1856652

[0085] (16) Sodium Azide: VWR, Cat# 0639-250G, Lot# 0386C067

[0086] (17) Tricine: Sigma-Aldrich, Cat# 5704-04-1, Lot# SLCC2910

[0087] (18) Calcium chloride: Sigma-Aldrich, Cat# 10043-52-4, Lot# SLBT0806

[0088] (19)FALGPA: Bachem AG, Cat# M1385.0100, Lot# 1075966

[0089] (20) Sodium Acetate Trihydrate: JT Baker, Cat# JT3461-1, Lot# 0000130474

[0090] (21) Sorbitol: EMD Millipore, Cat# 1.11597.1003, Lot# M835497840

[0091] (22) Sodium Phosphate Monobasic Monohydrate: JT Baker, Cat# 3802-01, Batch# 0000126400

[0092] (23) Sodium Phosphate Dibasic Dihydrate: Sigma-Aldrich, Cat# 04272-1KG, Lot# BCCB8057

[0093] Example 2: Preparation of liquid dosage form

[0094] The collagenase mixed solution (CNT201) prepared in Example 1 was used to prepare a liquid formulation for collagenase.

[0095] Specifically, eight dialysis cassettes (Slide-A-Lyzer Dialysis Cassettes, 3500 MWCO) were prepared by adding 7.8 mL of a mixed solution with a collagenase concentration of 2.0 mg / mL, and then the dialysis cassettes were placed one by one in the formulation buffers F1 to F8 shown in Table 1 below, and waited until they reached equilibrium. The mixed solution was exchanged for each formulation through the dialysis membrane of the dialysis cassette. After a sufficient time had passed to complete equilibrium, the collagenase solution was collected from each dialysis cassette and diluted to a collagenase concentration of 0.232 mg / mL by adding the corresponding formulation buffer. The formulation buffer consisted of 10 mM sodium acetate, histidine, or Tris as a buffer, 150 mM sodium chloride (NaCl) or 5% sorbitol as a tonicity modifier, and calcium chloride (CaCl) as a coenzyme. 2 ) 10 mM, 0.01% polysorbate 20 (PS20) was included as a surfactant, and no bulking agent was included.

[0096] The control group, F13, was prepared by diluting collagenase mixed solution (CNT201) with 10 mM L-histidine, 10 mM calcium chloride, pH 7.0 buffer to a collagenase concentration of 0.232 mg / mL, and then adding 10% PS20 stock solution to adjust the concentration to 0.01% PS20.

[0097] [Table 1]

[0098] Note 1) Form. is an abbreviation for formulation. Note 2) Dosage forms F5-F8 used sodium phosphate as a buffer, but it was observed that calcium phosphate precipitated, so they were formulated using histidine or tris as a buffer instead of sodium phosphate.

[0099] The osmolality, pH, and concentration of each formulation were then verified.

[0100] Each liquid formulation was then sterile filtered through a 0.2 μm PES membrane in a sterile BSC and then dispensed in 1.0 mL portions into 3 cc sterile glass vials, which were then sealed with crimp caps and labeled.

[0101] Example 3: Preparation of freeze-dried dosage form In this example, a lyophilized formulation for collagenase was prepared using the collagenase mixed solution (CNT201) prepared in Example 1. Before performing dialysis, 10% PS20 stock solution was added to make the concentration of PS20 0.01%.

[0102] Specifically, 7.8 mL of a mixed solution with a collagenase concentration of 2.0 mg / mL was added to four dialysis cassettes (Slide-A-Lyzer Dialysis Cassette, 3500 MWCO), and the dialysis cassettes were then placed one by one in the formulation buffers F9 to F12 shown in Table 2 below, and allowed to reach equilibrium. After the time had elapsed, the collagenase solution was collected from each dialysis cassette, and the corresponding formulation buffer was added to dilute the collagenase to a concentration of 0.232 mg / mL. The formulation buffer contained 10 mM succinate or histidine as a buffer, 1.0% trehalose as an osmotic pressure regulator, and calcium chloride (CaCl) as a coenzyme. 2 ) 10 mM, 4.0% mannitol or 2.5% glycine as a bulking agent, and 0.01% polysorbate 20 (PS20) as a surfactant.

[0103] [Table 2]

[0104] The osmolality, pH, and concentration of each formulation were then verified.

[0105] Each liquid formulation was then sterile filtered through a 0.2 μm PES membrane in a sterile BSC and then dispensed in 1.0 mL portions into 3 cc sterile glass vials.

[0106] Then, the vial was subjected to the following freeze-drying process: a portion of the vial was sealed with a freeze-drying vent stopper, and freeze-dried under the conditions shown in Table 3 below.

[0107] [Table 3]

[0108] After lyophilization, the chamber was backfilled with nitrogen and the vials were subjected to partial vacuum to approximately 580 Torr using a stopper. The lyophilized glass vials were then sealed with crimp caps and labeled. Additional vials were stored and frozen separately prior to the lyophilization process for pre-lyo analysis performed at T=0.

[0109] <Experimental Example 1: Analysis method and analysis items>

[0110] The liquid dosage form and the lyophilized dosage form prepared in Examples 2 and 3 were evaluated for their influence and effect on acute stress and accelerated stability using the following 11 analytical methods.

[0111] 1.1: Visual Inspection

[0112] Visual inspection was performed under a white light source (13 W fluorescent lamp) against black and white backgrounds. All dosage forms were digitally photographed at all time points.

[0113] Liquid and lyophilized dosage forms were visually inspected in their original state.

[0114] 1.2: Concentration Measurement

[0115] 1.315mg / mL -1 x cm -1 A was obtained using an Implen NanoPhotometer Pearl. 280 The concentration of CNT201 was analyzed.

[0116] The lyophilized formulations were measured after dissolving in Milli-Q water and reconstituting to a liquid state.

[0117] 1.3: pH

[0118] pH analysis was performed using a Symphony pH meter (VWR Scientific, catalog# SB70P) calibrated with three pH standard solutions (pH 4, 7, 10) with a correction slope of 95% or greater. Each dosage form sample was not temperature adjusted and was equilibrated to room temperature prior to pH measurement.

[0119] The lyophilized formulations were measured after dissolving in Milli-Q water and reconstituting to a liquid state.

[0120] 1.4: Osmolarity

[0121] Osmolality at T=0 was measured using a Model 2020 Freezing Point Osmometer (Advanced Instruments).

[0122] The lyophilized formulations were measured after dissolving in Milli-Q water and reconstituting to a liquid state.

[0123] 1.5:SE-HPLC(Size Exclusion-High Performance Liquid Chromatography)

[0124] SE-HPLC was carried out under the following conditions.

[0125] The lyophilized formulations were measured after dissolving in Milli-Q water and reconstituting to a liquid state.

[0126] [Table 4]

[0127] 1.6:Potency Assay

[0128] The potency assay was carried out under the following conditions.

[0129] The lyophilized formulations were measured after dissolving in Milli-Q water and reconstituting to a liquid state.

[0130] [Table 5]

[0131] 1.7:FlowCAM

[0132] The FlowCAM particle imaging system automated particle analysis by combining optics, electronics, and fluidics. An optical system was used to capture real-time images of particles in the fluid as they passed through a flow cell. Imaging software was used to assess particle size and morphology. All sample formulations were analyzed and degassed at 75 torr for 30 minutes prior to analysis. Lyophilized formulations were dissolved in Milli-Q water and reconstituted prior to analysis.

[0133] 1.8:Reconstitution Time

[0134] To the vial containing the lyophilized dosage form, 1.0 mL of sterile filtered Milli-Q water was added and a timer was started while the vial was gently agitated. Reconstitution, i.e., the point at which dissolution was complete, was considered to be the point at which the cake material completely disappeared when gently agitated.

[0135] 1.9:Moisture Content (Karl-Fischer titration)

[0136] A Karl Fisher Coulometer C20 (Mettler Toledo) was used to analyze moisture content in lyophilized dosage forms. AquaStar Water Oven Standard 1% or AquaStar Lactose Oven Standard 5% were used to determine system accuracy. Sample vials were stored at room temperature prior to removing the vial caps for analysis. Approximately 30 mg to 50 mg of material was used for each analysis.

[0137] 1.10:Sub-Ambient DSC

[0138] Approximately 10 μL of each sample was frozen at −60° C. using a Pyris Diamond DSC with Intercooler II. The samples were heated at a temperature ramp rate of 5° C. / min until thawed, and the heat flow during the thawing process was recorded.

[0139] 1.11: Fourier Transform Infrared Spectroscopy (FTIR)

[0140] FTIR is a technique used to obtain infrared absorption spectra of gases, liquids, or solids. IBI uses an FTIR-660 Plus spectrometer to measure the infrared absorption and wavenumber [cm] of sample materials. -1 The data provide information on the secondary structure of the polypeptide. The liquid formulation before lyophilization and the lyophilized powder were dissolved in Milli-Q water to reconstitute the liquid formulation, and the lyophilized powder itself were analyzed by FTIR spectroscopy.

[0141] Using the above 11 types of analysis methods, the influence and effect on acute stress and accelerated stability were evaluated, and the specific analysis items are as shown in Table 6 below. Accelerated stability monitors the effect of temperature over time (changes over time), and the temperature used for accelerated stability is set higher than the appropriate storage temperature (accelerated condition, Temperature Stress), and by using such a high temperature, changes over time can be observed more quickly.

[0142] [Table 6]

[0143] Note: "X" means done.

[0144] According to the above analysis, in the case of the liquid dosage forms F1 to F8 and F13 in Example 2, all the sample dosage forms were not stable due to the reduction in the content of the active ingredient and decomposition during long-term storage. Therefore, the present invention is aimed at developing a lyophilized powder dosage form, and is intended to confirm under what conditions a product of normal quality can be produced by producing lyophilized powder using compositions under various conditions, and whether the product produced in this way can maintain normal quality for a long time.

[0145] Accordingly, the evaluation results of the acute stress and accelerated stability of the samples having the lyophilized dosage forms F9 to F12 according to Example 3 will be described below.

[0146] <Experimental Example 2: Acute Stress Evaluation Results> Representative environmental factors that act as stress in the laboratory (the term "stress" is sometimes expressed as "stress") are stirring, ultraviolet light, and reconstitution to a liquid state. In actual usage environments, these would be physical impact, exposure to sunlight or indoor fluorescent lights, and dissolution of diluents. In Experimental Example 2, since it is generally difficult to obtain results quickly when evaluating something that changes slowly over time during storage, a method was used to select superior dosage forms by applying stress suddenly.

[0147] In Experimental Example 2, the lyophilized dosage form prepared in Example 3 of the present invention was subjected to rapid stress and evaluated using the method of Experimental Example 1. The rapid stress was evaluated in three aspects, including the effect of agitation, the effect of ultraviolet (UV) exposure, and the effect of liquid reconstitution (Recon.).

[0148] <Experimental Example 2-1: Effect of stirring> Agitation is one method of acute stress used to replicate physical shocks in a real-life application environment.

[0149] A sample vial having the dosage form according to Example 3 was dissolved in Milli-Q water, reconstituted into a liquid state, and then continuously stirred for 4 hours at room temperature with an orbital shaker set at 1,000 RPM. In addition, another sample having the same dosage form as a control sample was incubated for the same time in a static environment at room temperature without stirring.

[0150] The results are shown in Table 7 below.

[0151] [Table 7]

[0152] As shown in Table 7, the visual analysis showed that all samples were transparent and colorless regardless of the stress conditions. Visual analysis, which is evaluated by the naked eye, is qualitative in that it cannot directly confirm the size of particles, so it is only meaningful to determine whether or not large particles are generated. Visual analysis could not be used to determine the superiority or inferiority of the dosage forms.

[0153] SE-HPLC is a common method for quantitatively analyzing aggregates that are difficult to see with the naked eye. SE-HPLC analysis showed a slightly higher level of HMWS (high-molecular-weight species) aggregation in the F9 sample.

[0154] That is, when comparing the F9 and F10 samples, under the same dosage form conditions with a succinate buffer of pH 5.5, when mannitol was used as a bulking agent (F9 sample), slightly higher HMWS aggregation was observed due to shear force caused by stirring, but when glycine was used (F10 sample), such aggregation was not observed.

[0155] Also, comparing the F9 and F11 samples, under the same formulation conditions with mannitol as a bulking agent, slightly higher HMWS aggregation was observed in the succinate buffer and pH 5.5 formulation, but no such aggregation was observed in the histidine buffered pH 6.5 formulation.

[0156] Additionally, collagenase activity assays revealed collagenase activity of 3.71–4.23 FALGPA Units / mg protein in all formulations, which was also observed in the static control samples (3.53–3.86 FALGPA Units / mg protein).FlowCAM analysis also showed low changes in microparticle concentration after 4 hours in all formulations, independent of agitation stress.

[0157] This indicates that glycine is a more suitable bulking agent than mannitol, and histidine is a more suitable buffer than succinate.

[0158] <Experimental Example 2-2: Effects of ultraviolet (UV) exposure> The dosage form according to Example 3 and a formulation reconstituted with water immediately before the experiment were placed in a photostability chamber at 25° C. and exposed to ultraviolet (UV) light to simulate everyday UV exposure.

[0159] In addition, other samples (lyophilized dosage forms and their liquid reconstitution solutions) with the same dosage forms as the control samples were prepared, wrapped in foil, and placed in sample boxes protected from light. These protected dosage forms were incubated in the same light stability chamber for the same period of time as the exposed dosage forms.

[0160] The results are shown in Table 8 below.

[0161] [Table 8]

[0162] As shown in Table 8, visual analysis showed that all samples were clear and colorless both before and after UV exposure, with a small number of visually visible particles. For visual analysis, the freeze-dried powder samples were dissolved in water and observed.

[0163] SE-HPLC analysis also showed similar chromatographic profiles for all samples after the end of incubation, regardless of the stress conditions, and no significant differences emerged between UV-shielded and exposed samples, regardless of the freeze-drying conditions.

[0164] Similarly, collagenase activity assays showed that after UV exposure, all UV-exposed lyophilized formulations had collagenase activity of 3.83-4.12 FALGPA Units / mg protein, similar to the UV-blocked controls (3.79-4.12 FALGPA Units / mg protein).

[0165] <Experimental Example 2-3: Effect of liquid restoration> Each sample for each dosage form in Example 3 was dissolved in 1 mL of Milli-Q water (reconstituted in liquid form) and stored for 24 hours at 25° C. Other samples were dissolved in parallel (reconstituted in liquid form) and stored for the same period at 5° C.

[0166] The results are shown in Table 9 below.

[0167] [Table 9]

[0168] If aggregation or decomposition occurs due to dissolution caused by liquid restoration, the collagenase concentration will change. Therefore, by measuring the concentration, the magnitude of change due to dissolution can be confirmed. Similarly, changes in pH and the presence or absence of an increase in particulates using FlowCAM are also methods of measurement that can predict the effects of dissolution.

[0169] As shown in Table 9 above, visual analysis showed that all samples were clear and colorless after one day, regardless of storage temperature, with a small number of particles visible.

[0170] Concentration analysis showed that after dissolution and 1-day temperature stress exposure, concentrations similar to the target of 221 mg / mL to 0.240 mg / mL were observed in all dosage forms, regardless of storage temperature.

[0171] pH analysis showed that after dissolution and 1 day of temperature stress exposure, all dosage forms exhibited a pH identical or similar to the target pH (with small deviations of ±0.1), regardless of storage temperature.

[0172] Similar chromatographic profiles were observed for all formulations after dissolution and 1 day of temperature stress, regardless of storage temperature, with all formulations at 25°C showing similar peak percentages as the 5°C samples.

[0173] Collagenase activity assays showed that after dissolution and 1 day of temperature stress, similar collagenase activities of 4.00-4.25 FALGPA Units / mg protein were observed in all formulations, regardless of storage temperature.

[0174] FlowCAM analysis showed that after dissolution and 1 day of temperature stress, low changes in fine particle concentration were observed for all formulations, regardless of storage temperature.

[0175] <Experimental Example 3: Acceleration Stability (Temperature Stress) Evaluation Results>

[0176] Using the method of Experimental Example 1, the accelerated stability (Temperature Stress Time Points) of the dosage forms of the present invention was evaluated.

[0177] Sample vials having the dosage forms according to Examples 2 and 3 were placed at 5° C., 25° C., and 40° C., respectively, starting from T=0 week, and then removed and analyzed after 8 weeks.

[0178] <Experimental Example 3-1: Visual analysis under accelerated temperature conditions> At T=0 (Time zero), all pre-lyo dosage forms were clear and colorless, with no visible particles.

[0179] [Table 10]

[0180] At the start of the experiment, the lyophilized dosage forms containing glycine (F10, F12) were in good condition, while the lyophilized dosage forms containing mannitol (F9, F11) were in a slightly shrunk state. At the 8th week, there was no significant change in the lyophilized cake. For all dosage forms, the lyophilized dosage forms dissolved before and immediately after lyophilization were clear and colorless, with no particles observed. At the 8th week, the lyophilized dosage forms dissolved were clear and colorless, with a few particles visually observed.

[0181] <Experimental Example 3-2: Concentration analysis under temperature acceleration conditions> When aggregation or decomposition occurs due to the effect of temperature, the concentration of collagenase changes, so the degree of change due to the effect of temperature can be confirmed by measuring the concentration.

[0182] At T=0, all formulations showed concentrations similar to the target of 0.257mg / mL to 0.264mg / mL. Up until the 8th week after the start of the experiment, most formulations showed concentrations similar to the target at 5°C and 25°C, but at the 8th week, a decrease in concentration was observed for F9 to F12. At 40°C, most formulations showed concentrations lower than the target, with F10, F11, and F12 formulations showing the highest concentrations.

[0183] [Table 11]

[0184] In particular, up until the 8th week after the start of the experiment, the F9 formulation showed lower concentrations than at the initial stage at 5°C, 25°C, and 40°C.

[0185] <Experimental Example 3-3: pH analysis under temperature acceleration conditions> pH analysis showed that after 8 weeks all formulations showed a pH similar to that at T=0 within small deviations (±0.1), regardless of storage temperature.

[0186] [Table 12]

[0187] <Experimental Example 3-4: Osmotic pressure analysis under temperature acceleration conditions> Osmolality is an index to check whether the dosage form is manufactured correctly. If the difference from the theoretical value is larger than the experimental error, it indicates that the dosage form is manufactured incorrectly. Since osmolality is not a test method that can detect the effect of temperature, it was measured only at T=0 in the solution before lyophilization and immediately after lyophilization.

[0188] Osmolality analysis showed that at T = 0, all lyophilized formulations (except for the control group) had osmolalities ranging from 292 mOsm / kg to 398 mOsm / kg after reconstitution in water, which were similar to the values ​​of the pre-lyo samples.

[0189] [Table 13]

[0190] <Experimental Example 3-5: Analysis of liquid recovery time under temperature acceleration conditions> Measuring reconstitution time (i.e., dissolution time) is one way to determine how a dosage form changes over time under accelerated temperature conditions. For example, lyophilized dosage forms with relatively high moisture content will harden over time as the lyophilized cake hardens and clumps together, which will be manifested as an increase in reconstitution time (i.e., dissolution time).

[0191] Dissolution time analysis showed that all dosage forms showed fast dissolution times (≦10 seconds) from week 8 onwards, with the glycine-containing dosage forms (F10 and F12) showing much faster dissolution times than the mannitol dosage forms (F9 and F11) at T=0. Nevertheless, since the dissolution times of all dosage forms were less than 10 seconds at week 8, it is not possible to distinguish between the dosage forms in terms of dissolution time alone, and all can be judged to be superior.

[0192] [Table 14]

[0193] <Experimental Example 3-6: Humidity analysis under temperature acceleration conditions> Moisture content is an important factor in the formation and maintenance of a lyophilized dosage form. The better the combination of the formulation components, the lower the measured moisture content. In the industry, the standard for moisture content is often set at 3.0% or less.

[0194] Moisture content analysis showed that at T=0, all lyophilized dosage forms showed low moisture content (<1.5%), with the glycine dosage forms (F10 and F12) showing lower moisture content compared to the mannitol dosage forms (F9 and F11).

[0195] [Table 15]

[0196] <Experimental Example 3-7: FTIR analysis under accelerated temperature conditions> FTIR analysis showed that at T=0, all dissolved lyophilized formulations showed FTIR spectra similar to that of the pre-lyo samples, indicating that no denaturation of collagenase occurred during the lyophilization process.

[0197] <Experimental Example 3-8: SE-HPLC analysis under temperature acceleration conditions> By SE-HPLC analysis, at T=0, all formulations showed similar chromatographic profiles.

[0198] [Table 16]

[0199] However, after 8 weeks of incubation, a slight increase in HMWS was observed in the lyophilized succinate formulations (F9 and F10). The proportion of HMWS increased proportionally at 25°C and 40°C, where the temperature effect is greatest, and since the same buffer components showed similar HMWS, it can be concluded that the histidine formulation is more advantageous under temperature accelerated conditions than the succinate formulation. Although the pH to be maintained may differ depending on the buffer components, it can also be concluded that the histidine formulation at pH 6.5 is superior to the succinate formulation at pH 5.5.

[0200] <Experimental Example 3-9: Collagenase activity analysis under temperature acceleration conditions> Collagenase activity assay showed that at T=0, all formulations showed similar collagenase activity ranging from 2.77 to 3.33 FALGPA Units / mg protein, which was slightly higher than the assay control (2.37 FALGPA Units / mg protein).

[0201] [Table 17]

[0202] After 8 weeks of incubation at 25°C, all formulations except F13 showed collagenase activity similar to or slightly lower than that at T=0 (2.49-3.28 FALGPA Units / mg protein).

[0203] However, after 8 weeks of incubation at 40°C, the histidine-containing lyophilized formulations (F11, F12) showed the highest collagenase activity (3.24-3.29 FALGPA Units / mg protein) compared to all other formulations.

[0204] <Experimental Example 3-10: FlowCAM analysis under temperature acceleration conditions> At T=0, all lyophilized dosage form samples showed low microparticle concentration variation according to FlowCAM analysis.

[0205] After 8 weeks, most of the dosage forms showed low particle concentration changes at 5°C and 25°C, and at 40°C, the lyophilized dosage forms (F10-F12) except for F9 showed relatively low particle concentration changes. The F12 dosage form showed relatively high particle concentration in the pre-lyo form stored at -70°C. Meanwhile, the F12 dosage form showed relatively superior low particle concentration changes than the F9-F11 dosage forms under temperature accelerated conditions when it was a lyophilized dosage form.

[0206] [Table 18]

[0207] Comparing the F9 and F10 samples in the accelerated stability evaluations of Experimental Examples 3-1 to 3-10, when mannitol was used as a bulking agent under the same conditions of a dosage form having a pH of 5.5 as succinate buffer, a decrease in concentration, a delay in dissolution time, high humidity, an increase in HMWS (%), and an increase in fine particle concentration occurred under temperature accelerated conditions. This shows that glycine is more suitable as a bulking agent than mannitol.

[0208] In addition, when comparing samples F9 and F11, under the same conditions of formulations with mannitol as a bulking agent, in the case of a pH 5.5 formulation with succinate buffer, there was a decrease in concentration, a decrease in collagenase activity, an increase in HMWS (%), and an increase in microparticle concentration under temperature acceleration conditions, indicating that histidine is more suitable as a buffer than succinate.

[0209] In addition, when comparing samples F10 and F12, under the same conditions of formulations containing glycine as a bulking agent, in the case of a pH 5.5 formulation adjusted to succinate buffer, collagenase activity decreased and HMWS (%) increased under temperature acceleration conditions, indicating that histidine is more suitable as a buffer than succinate.

[0210] Although the present invention has been illustrated and described in connection with certain preferred embodiments, it will be apparent to those skilled in the art that the present invention can be modified and changed in various ways without departing from the technical characteristics and field of the present invention as defined by the following claims.

Claims

1. A composition comprising: collagenase as an enzyme; calcium as a coenzyme; histidine as a dosage form additive; and glycine as a dosage form additive.

2. 2. The composition of claim 1, wherein the collagenase enzyme content is from 0.3% to 20.8% by weight based on the total weight of the composition.

3. The composition of claim 1 , wherein the calcium is included in the form of a calcium salt.

4. 4. The composition of claim 1 or claim 3, wherein the calcium or calcium salt is present in an amount of from 0.4% to 28% by weight based on the total weight of the composition.

5. The composition according to claim 1 , wherein the histidine content is from 0.4% to 29.9% by weight based on the total weight of the composition.

6. The composition according to claim 1 , wherein the glycine content is from 15% to 94.6% by weight based on the total weight of the composition.

7. 10. The composition of claim 1, further comprising trehalose as a dosage form additive.

8. The composition according to claim 1 , further comprising polysorbate 20 (PS20) as a surfactant.

9. The composition of claim 1 , wherein the pH of the composition is from 6.0 to 7.

0.

10. The composition of claim 1 , wherein the composition is in a lyophilized form.

11. A solution produced by mixing the lyophilized composition of claim 10 with a diluent.

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