Collagenase preparation and method for manufacturing the same

Abstract

To provide collagenase-containing formulations that are improved for shelf life and enzyme stability, and methods of preparing the same.SOLUTION: Collagenase-containing formulations comprise a collagenase, about 30 mM to about 240 mM of a disaccharide, about 50 mM to about 800 mM of mannitol, and about 6 mM to about 10 mM of a Tris-HCl. Lyophilized and reconstituted formulations are also provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 788,916, filed on January 6, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] This application includes a sequence listing electronically submitted on January 3, 2020, as a text file titled "117326_000001_Sequence_Listing.txt" with a size of 17,447 bytes. This sequence listing is incorporated herein by reference.

[0003] This specification discloses collagenase-containing formulations with improved stability and shelf life. [Background technology]

[0004] XIAFLEX® (collagenase derived from Clostridium histolyticum (CCH)) is currently approved for the treatment of Dupuytren's contracture (DC) and Peyronie's disease (PD). The currently approved XIAFLEX® formulation is supplied as a lyophilized cake containing 0.9 mg of CCH in a 3 CC vial with a diluent. The current XIAFLEX® formulation (before lyophilization) has a lyophilization cycle time of approximately 72 hours in the vial. Efficient lyophilization is necessary for shelf life and enzyme stability. The following are prior art documents related to the invention of this application (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries): (Prior art document) (Patent Document) (Patent Document 1) International Publication No. 2013 / 059619 (Patent Document 2) International Publication No. 2007 / 100675 (Patent Document 3) International Publication No. 2018 / 160905 (Patent Document 4) U.S. Patent Application Publication No. 2011 / 033464 (Patent Document 5) U.S. Patent Application Publication No. 2018 / 099049 (Patent Document 6) U.S. Patent Application Publication No. 2006 / 263347 [Overview of the Initiative] [Means for solving the problem]

[0005] This specification discloses a formulation comprising collagenase, a disaccharide of about 30 mM to about 240 mM; mannitol of about 50 mM to about 800 mM, and Tris-HCl of about 6 mM to about 10 mM.

[0006] Also provided in this specification is a lyophilized formulation comprising collagenase, a disaccharide, mannitol, and Tris-HCl.

[0007] A reconstituted formulation comprising collagenase, a disaccharide, mannitol, Tris-HCl, calcium chloride, and sodium chloride is also disclosed.

[0008] A kit is also provided, which kit comprises a container containing any of the disclosed lyophilized formulations and a container containing a sterile diluent comprising calcium chloride and sodium chloride.

Brief Description of the Drawings

[0009] This summary, and the following detailed description, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the disclosed formulations, exemplary embodiments of the formulations are shown in the drawings, but the formulations are not limited to the specific embodiments disclosed.

[0010] [Figure 1] FIG. 1 shows the effect of pH on protein interaction in an exemplary formulation containing trehalose, mannitol, and various collagenases. [Figure 2A] FIGS. 2A and 2B show an exemplary hydrogen peroxide challenge analyzing the effect of pH and excipients on turbidity. NTU - Nephelometric Turbidity Units, PS - Polysorbate, T - Trehalose, S - Sucrose, M - Mannitol, H2O2 - Hydrogen peroxide, 7.5, 8.0, and 8.5 indicate the pH of the formulation. [Figure 2B] Figures 2A and 2B show an exemplary hydrogen peroxide problem analyzing the effects of pH and excipients on turbidity. NTU (Nephelometric Turbidity Units), PS (Polysorbate), T (Trehalose), S (Sucrose), M (Mannitol), H2O2 (Hydrogen peroxide), 7.5, 8.0, and 8.5 represent the pH of the formulations. [Figure 3A-3D] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show various lyophilized formulations (Va: 0.93 mg / ml CCH; 60 mM sucrose; 112.5 mM mannitol; 10 mM Tris / HCl buffer pH 8.5; Vb: 0.93 mg / ml CCH; 60 mM sucrose; 225 mM mannitol; 10 mM Tris / HCl buffer pH 8.5; and Vc: 0.93 mg / ml CCH; 60 mM sucrose; 337.5 mM mannitol; 10 mM Tris / HCl buffer pH 8.5). [Figure 3E-3H] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show various lyophilized formulations (Va: 0.93 mg / ml CCH; 60 mM sucrose; 112.5 mM mannitol; 10 mM Tris / HCl buffer pH 8.5; Vb: 0.93 mg / ml CCH; 60 mM sucrose; 225 mM mannitol; 10 mM Tris / HCl buffer pH 8.5; and Vc: 0.93 mg / ml CCH; 60 mM sucrose; 337.5 mM mannitol; 10 mM Tris / HCl buffer pH 8.5). [Figure 3I-3L]Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show various lyophilized formulations (Va: 0.93 mg / ml CCH; 60 mM sucrose; 112.5 mM mannitol; 10 mM Tris / HCl buffer pH 8.5; Vb: 0.93 mg / ml CCH; 60 mM sucrose; 225 mM mannitol; 10 mM Tris / HCl buffer pH 8.5; and Vc: 0.93 mg / ml CCH; 60 mM sucrose; 337.5 mM mannitol; 10 mM Tris / HCl buffer pH 8.5). [Figure 3M-3P] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show various lyophilized formulations (Va: 0.93 mg / ml CCH; 60 mM sucrose; 112.5 mM mannitol; 10 mM Tris / HCl buffer pH 8.5; Vb: 0.93 mg / ml CCH; 60 mM sucrose; 225 mM mannitol; 10 mM Tris / HCl buffer pH 8.5; and Vc: 0.93 mg / ml CCH; 60 mM sucrose; 337.5 mM mannitol; 10 mM Tris / HCl buffer pH 8.5). [Figure 3Q-3R] Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show various lyophilized formulations (Va: 0.93 mg / ml CCH; 60 mM sucrose; 112.5 mM mannitol; 10 mM Tris / HCl buffer pH 8.5; Vb: 0.93 mg / ml CCH; 60 mM sucrose; 225 mM mannitol; 10 mM Tris / HCl buffer pH 8.5; and Vc: 0.93 mg / ml CCH; 60 mM sucrose; 337.5 mM mannitol; 10 mM Tris / HCl buffer pH 8.5). [Figure 4]Figures 4A, 4B, and 4C show images of cakes from various lyophilized formulations under pressures of 128 μbar (Figure 4A), 380 μbar (Figure 4B), and 1030 μbar (Figure 4C). [Figure 5] Figures 5A and 5B show images of cakes from various lyophilized formulations under pressures of 128 μbar (Figure 5A) and 4000 μbar (Figure 5B). [Figure 6] Figure 6 shows the moisture content of various freeze-dried formulations over time. [Modes for carrying out the invention]

[0011] The disclosed formulations can be more readily understood by referring to the following detailed description relating to the accompanying figures which form part of this disclosure. It should be understood that the disclosed formulations are not limited to the specific formulations described and / or shown herein, and that the terms used herein are for illustrative purposes to describe specific embodiments and are not intended to limit the claimed formulations.

[0012] Unless otherwise specified, descriptions of possible mechanisms of action, modes of operation, and reasons for improvement are for illustrative purposes only, and the disclosed formulations are not limited by the correctness or incorrectness of the suggested mechanisms of action, modes of operation, or reasons for improvement.

[0013] This document describes formulations and methods for preparing such formulations. Where this disclosure describes or claims features or embodiments relating to a formulation, such features or embodiments also apply to methods for forming the formulation. Similarly, where this disclosure describes or claims features or embodiments relating to methods for forming a formulation, such features or embodiments also apply to the formulation.

[0014] Where a numerical range is described or established herein, that range includes its endpoints and all individual integers and fractions within those endpoints, and further includes each of the narrower ranges within it formed by all possible combinations of those endpoints and the integers and fractions within them, each of which forms a subgroup of the larger group of values ​​within the described range, to the same extent as when it is explicitly described. Where a numerical range is described herein as being greater than a described value, the range is nevertheless finite, and its upper limit is bounded by values ​​that are operable within the context of the invention as described herein. Where a numerical range is described herein as being less than a described value, the range is nevertheless bounded by non-zero values. The scope of this disclosure is not intended to be limited to any specific values ​​described in defining a range. All ranges are inclusive and combinable.

[0015] When a value is expressed as an approximation, the use of the antecedent "approximately" indicates that the particular value forms another embodiment. Referring to a specific number means including at least that particular number unless the context clearly indicates otherwise.

[0016] For clarification, it should be understood that certain features of the disclosed formulation described herein in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for simplicity, various features of the disclosed formulation described in the context of a single embodiment may also be provided separately or in any subcombination.

[0017] In this specification, the singular forms "a," "an," and "the" include the plural forms.

[0018] This specification and claims use various terms relating to aspects thereof. Unless otherwise specified, such terms shall have their common meanings in the art. Other specifically defined terms shall be construed in accordance with the definitions set forth herein.

[0019] The term "comprising" is intended to include examples that are encompassed by the terms "consisting essentially of" and "consisting of," and similarly, the term "consisting essentially of" is intended to include examples that are encompassed by the term "consisting of."

[0020] Abbreviations such as collagenase (CCH), United States Pharmacopeia (USP), Nephelometric Turbidity Units (NTU), polysorbate (PS), and hydrogen peroxide (H2O2) are used.

[0021] This specification provides formulations comprising, or comprising: Collagenase and Disaccharides ranging from approximately 30 mM to approximately 240 mM, Mannitol in concentrations of approximately 50 mM to 800 mM, and Tris-HCl at approximately 6 mM to 10 mM.

[0022] The formulations may contain approximately 0.2 mg / ml to approximately 50 mg / ml of collagenase. For example, lyophilized formulations may contain approximately 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.6 mg / ml, 1.8 mg / ml, 2 mg / ml, 2.5 mg / ml, 3 mg / ml, 3.5 mg / ml, 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or approximately 50 mg / ml of collagenase.

[0023] In this specification, “collagenase” means any of the following: (a) collagenase (including variants) having activity as defined in EC3.4.24.3 (www.brenda-enzymes.org / enzyme.php?ecno=3.4.24.3 (accessed July 3, 2019)), (b) collagenase produced by fermentation of Clostridium histolyticum (also known as Hathewaya histolytica), (c) CCH (as described herein), (d) collagenase having at least 50% sequence consistency with collagenase I (also known as class I collagenase) as determined by BLAST, (e) collagenase having at least 50% sequence alignment with collagenase II (also known as class II collagenase) as determined by BLAST, (f) collagenase produced by fermentation of organisms from other sources (e.g., non-Clostridium (g) collagenases from mammals, crustaceans, fungi, bacteria or microorganisms (h) histolyticum, (i) collagenases from mammals, crustaceans, fungi, bacteria or microorganisms, (h) collagenases obtained by recombinant technology, (i) collagenases having a molecular weight of approximately 65 kDa to approximately 130 kDa, (j) collagenases designated as collagenase I or collagenase II, (k) mixtures of collagenase I and II, (l) collagenases derived from JCM 1403 strain (ATCC 19401) or its derivatives, (m) collagenases derived from ATCC 21000 strain or its derivatives, (n) collagenases derived from ATCC 69334 or its derivatives, (o) collagenases derived from C. perfringens, (p) collagenases derived from Vibrio alginolyticus, (q) collagenases derived from Streptomyces, (r) collagenases derived from Pseudomonas, (r) Achromobacter Collagenases derived from iophagus, (s)collagens described by Worthington Biochemical Corp. (www.Worthington-biochem.com; “Product Highlights”), (t)collagens described by Sigma-Aldrich (www.sigma-aldrich.com), (u)collagens having one or more of the following characteristics: ·Vmax (min -1) is about 0.08 - 7.70 (SRC assay) or about 0.3 - 30.5 (GPA assay). ·K M is about 4.1 - 410 nM (SRC assay) or about 0.03 - 3.1 mM (GPA assay) ·K cat (sec -1 ) is about 1.1 - 107 (SRC assay) or about 93 - 9,179 (GPA assay) ·1 / K cat is about 376 - 37,222 microseconds (SRC assay) or about 4 - 428 (GPA assay), or ·K cat / K M 、mM -1 sec -1 is about 5,140 - 508,814 (SRC assay) or about 60 - 5,934 (GPA assay), (v) collagenase described in Nordmark Arzneimittel GmbH & Co. KG, (w) collagenase derived from stock 004, or (x) any equivalents thereof or combinations of some of them. Non-limiting examples of collagenase that can be used in the disclosure of this specification are described in U.S. Patent No. 7,811,560, U.S. Patent No. 9,757,435, U.S. Patent No. 9,744,138, and International Publication No. WO2012 / 125948.

[0024] In some embodiments, the collagenase can include collagenase I. Suitable collagenase I includes, for example, collagenase I having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In some aspects, the collagenase I includes the amino acid sequence of SEQ ID NO: 1.

[0025] In some embodiments, the collagenase may include collagenase II. Preferred collagenase IIs include, for example, those containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 2. In some embodiments, collagenase II consists of the amino acid sequence of sequence ID number: 2. [Table 1] TIFF0007855655000002.tif65141

[0026] In some embodiments, the collagenase may include a mixture of collagenase I and collagenase II. For example, the collagenase may include a mixture of collagenase I containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 1, and collagenase II containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 2. In some aspects, the collagenase includes a mixture of collagenase I containing the amino acid sequence of sequence ID number: 1 and collagenase II containing the amino acid sequence of sequence ID number: 2. Suitable mixtures of collagenase I and collagenase II include, for example, collagenase I:collagenase II mass ratios of 0.1:1, 0.25:1, 0.5:1, 0.75:1, 1:1, 1.1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, 1:0.1, 1:0.25, 1:0.5; 1:0.75, 1:1.1, 1:1.25, 1:1.5, 1:1.75, or 1:2. Each of collagenase I and collagenase II may have a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, as measured, for example, by reverse-phase HPLC.

[0027] In some embodiments, the collagenase may include collagenase Clostridium histolyticum (CCH). As used herein, "CCH" refers to collagenase Clostridium histolithium, which contains a mixture of collagenase I (sequence ID number: 1) and collagenase II (sequence ID number: 2) in a mass ratio of about 1:1. CCH is obtained by fermenting collagenase Clostridium histolyticum (also known as Hathewaya histolytica).

[0028] Suitable disaccharides include the following: • Stabilizes proteins, • Protects proteins during both freezing and dehydration. • Suppresses unfolding due to freeze-drying. • Non-reducing, and / or • Tends to become amorphous during freeze-drying.

[0029] In some embodiments, the disaccharide comprises sucrose or trehalose. In some aspects, the formulation comprises: collagenase, about 30 mM to about 240 mM sucrose, about 50 mM to about 800 mM mannitol, and about 6 mM to about 10 mM Tris-HCl. In some aspects, the formulation comprises collagenase, about 30 mM to about 240 mM trehalose, about 50 mM to about 800 mM mannitol, and about 6 mM to about 10 mM Tris-HCl.

[0030] Disaccharides may be present at concentrations of approximately 30 mM to 240 mM, 60 mM to 240 mM, 90 mM to 240 mM, 120 mM to 240 mM, 150 mM to 240 mM, 180 mM to 240 mM, 210 mM to 240 mM, 30 mM to 210 mM, 30 mM to 180 mM, 30 mM to 150 mM, 30 mM to 120 mM, 30 mM to 90 mM, or 30 mM to 60 mM. Disaccharides may be present at concentrations of approximately 30 mM, 60 mM, 90 mM, 120 mM, 150 mM, 180 mM, 210 mM, or 240 mM.

[0031] Mannitol is found in the following concentrations: approximately 50mM to 800mM, 100mM to 800mM, 150mM to 800mM, 200mM to 800mM, 250mM to 800mM, 300mM to 800mM, 350mM to 800mM, 400mM to 800mM, 450mM to 800mM, 500mM to 800mM, 550mM to 800mM, 600mM to 800mM, 650mM to 800mM, and 700mM to 800mM. These are approximately 750mM to 800mM, 50mM to 750mM, 50mM to 700mM, 50mM to 650mM, 50mM to 600mM, 50mM to 550mM, 50mM to 500mM, 50mM to 450mM, 50mM to 400mM, 50mM to 350mM, 50mM to 300mM, 50mM to 250mM, 50mM to 200mM, 50mM to 150mM, or 50mM to 100mM. Mannitol may be present at concentrations of approximately 50 mM, 100 mM, 150 mM, 200 mM, 225 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, or 800 mM.

[0032] The pH of the formulation can be approximately 7.8 to 8.8. The pH can be approximately 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.

[0033] The formulation contains the following: CCH, approximately 60 mM sucrose, approximately 225 mM mannitol, and approximately 10 mM Tris-HCl, and the formulation has a pH of approximately 8.5. The formulation contains the following: approximately 0.9 mg CCH / ml, approximately 60 mM sucrose, approximately 225 mM mannitol, and approximately 10 mM Tris-HCl, and the formulation has a pH of approximately 8.5.

[0034] The formulation consists of the following: CCH, approximately 60 mM sucrose, approximately 225 mM mannitol, and approximately 10 mM Tris-HCl, and the formulation has a pH of approximately 8.5. The formulation consists of the following: approximately 0.9 mg CCH / ml, approximately 60 mM sucrose, approximately 225 mM mannitol, and approximately 10 mM Tris-HCl, and the formulation has a pH of approximately 8.5.

[0035] The disclosed formulations may further contain surfactants. Suitable surfactants include, for example, polysorbate 20, polysorbate 80, or poloxamer 188. The surfactants are present in concentrations of about 0.01% to 2%, about 0.05% to 2%, about 0.1% to 2%, about 0.15% to 2%, about 0.2% to 2%, about 0.25% to 2%, about 0.3% to 2%, about 0.4% to 2%, about 0.5% to 2%, about 1% to 2%, about 1.5% to 2%, about 0.01% to 1.5%, about 0.01% to 1%, about 0.01% to 0.5%, about 0.01% to 0.1%, or about 0.01% to 0.05%. The surfactant may be present in concentrations of about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about 1%, about 1.5%, or about 2%. In some embodiments, the formulation further comprises polysorbate 20 at a concentration of about 0.02%. In some embodiments, the formulation further comprises polysorbate 80 at a concentration of about 0.02%. In some embodiments, the formulation further comprises poloxamer 80 at a concentration of about 0.02%.

[0036] The above formulation may also be in liquid form.

[0037] The disclosed formulations can be lyophilized under more aggressive conditions compared to conventional collagenase-containing formulations such as XIAFLEX®. For example, the disclosed formulations can be lyophilized in a shorter time, at higher pressure, and / or with fewer drying steps (e.g., drying at a single temperature), resulting in a lyophilized formulation with improved stability and maintained acceptable collagenase activity upon reconstitution. As described in the specification, pH and mannitol lead to the formation of a more robust formulation that can subsequently undergo more aggressive lyophilization.

[0038] Furthermore, lyophilized formulations are provided herein. Lyophilized formulations can be formed by lyophilizing any of the above formulations. In some embodiments, the lyophilized formulation is as follows: Collagenase and Disaccharides and, Mannitol and, It contains tris-HCl or consists of the following:

[0039] Lyophilized preparations may contain approximately 0.2 mg to approximately 50 mg of collagenase. For example, lyophilized preparations may contain approximately 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 0.9 mg, 1 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg of collagenase.

[0040] Collagenase may include collagenase I. A preferred collagenase I is, for example, one containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 1. In some embodiments, collagenase I contains the amino acid sequence of sequence ID number: 1.

[0041] Collagenase may include collagenase II. Preferred collagenase II includes, for example, collagenase II containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 2. In some embodiments, collagenase II contains the amino acid sequence of sequence ID number: 2.

[0042] Collagenase may comprise a mixture of collagenase I and collagenase II. For example, collagenase may comprise a mixture of collagenase I containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 1, and collagenase II containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 2. In some embodiments, collagenase comprises a mixture of collagenase I containing the amino acid sequence of sequence ID number: 1 and collagenase II containing the amino acid sequence of sequence ID number: 2. Suitable mixtures of collagenase I and collagenase II include, for example, collagenase I:collagenase II mass ratios of 0.1:1, 0.25:1, 0.5:1, 0.75:1, 1:1, 1.1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, 1:0.1, 1:0.25, 1:0.5, 1:0.75, 1:1.1, 1:1.25, 1:1.5, 1:1.75, or 1:2. In some embodiments, the collagenase is collagenase Clostridium histolyticum (CCH).

[0043] Collagenase I and collagenase II may each have a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, as measured, for example, by reverse-phase HPLC.

[0044] Suitable disaccharides include, for example, sucrose or trehalose. In some embodiments, the lyophilized formulation comprises or consists of collagenase, sucrose, mannitol, and Tris-HCl.

[0045] Lyophilized formulations can be placed in unit-dose vials, multi-dose vials, cartridges, or syringes. Lyophilized formulations may contain approximately 0.2 mg to approximately 50 mg of collagenase. For example, lyophilized formulations may contain approximately 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 0.9 mg, 1 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg of collagenase. Vials, cartridges, or syringes may have capacities ranging from 2 mL to 50 mL, such as 5 mL, 7.5 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, or 50 mL. A vial, cartridge, or syringe may contain approximately 0.2 mg to approximately 50 mg of collagenase. For example, a vial, cartridge, or syringe may contain approximately 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 1 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg of collagenase. A vial, cartridge, or syringe may contain approximately 0.2 mg to approximately 50 mg of lyophilized preparation. For example, vials, cartridges, or syringes may contain lyophilized formulations of approximately 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 1 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg.

[0046] The formulation before lyophilization contains, or comprises, collagenase, approximately 30 mM to 240 mM disaccharides, approximately 50 mM to 800 mM mannitol, and approximately 6 mM to 10 mM Tris-HCl. The formulation before lyophilization contains, or comprises, approximately 0.9 mg collagenase / ml, approximately 30 mM to 240 mM disaccharides, approximately 50 mM to 800 mM mannitol, and approximately 6 mM to 10 mM Tris-HCl. Before lyophilization, the formulation contains, or comprises, CCH, 60 mM sucrose, 225 mM mannitol, 10 mM Tris-HCl, and a pH of approximately 8.5. In some embodiments, prior to lyophilization, the formulation may contain or consist of approximately 0.9 mg of CCH / ml, 60 mM sucrose, 225 mM mannitol, and 10 mM Tris-HCl, and have a pH of approximately 8.5.

[0047] The disclosed lyophilized formulation exhibits improved stability compared to conventional collagenase-containing formulations such as XIAFLEX®. For example, the disclosed lyophilized formulation has a stable 380 μbar. exceed , 400 μbar exceed , 450 μbar exceed , 500 μbar exceed , 550 μbar exceed , 600 μbar exceed , 650 μbar exceed 700 μbar exceed 750 μbar exceed , 800 μbar exceed 850 μbar exceed 900 μbar exceed 950 μbar exceed , 1000 μbar exceed , 1500 μbar exceed , 2000 μbar exceed , 2500 μbar exceed , 3000 μbar exceed , 3500 μbar exceed , or 4000 μbar exceed It stabilizes under pressure. In some embodiments, the lyophilized formulation stabilizes at a pressure of approximately 4000 μbar.

[0048] The disclosed lyophilized formulation exhibits improved shelf life and storage conditions compared to conventional collagenase-containing formulations. For example, the disclosed lyophilized formulation shows an extended shelf life at low temperatures such as 2-8°C and at high temperatures such as room temperature (40°C / 75% relative humidity). The disclosed lyophilized formulation can be stabilized in the following locations, for example: (a) At 2-8°C for at least 36 months, (b) At 25°C / 60% relative humidity for at least 36 months, (c) At least 6 months at 40℃ / 75% relative humidity, (d) Any combination of (a) to (c).

[0049] The disclosed lyophilized formulation can be formed by a method comprising the steps of freezing the formulation at a temperature between approximately -25°C and -55°C to form a lyophilized formulation, and drying the lyophilized formulation at a temperature between approximately 25°C and approximately 50°C to form a lyophilized formulation. Suitable temperatures for the freezing step include approximately -25°C, approximately -30°C, approximately -35°C, approximately -40°C, approximately -45°C, approximately -50°C, or approximately -55°C. Suitable temperatures for the drying step are approximately 25°C, approximately 30°C, approximately 35°C, approximately 40°C, approximately 45°C, or approximately 50°C.

[0050] It is shown that lyophilized formulations can be formed using a single-temperature freezing step and a single-temperature drying step. For example, a lyophilized formulation can be formed by a method comprising the steps of freezing the formulation at a single temperature between approximately -25°C and -55°C to form a lyophilized formulation, and drying the lyophilized formulation at a single temperature between approximately 25°C and approximately 50°C to form a lyophilized formulation. The single-temperature freezing step can be carried out at a temperature between approximately -25°C and approximately -55°C. For example, the single-temperature freezing step can be carried out at approximately -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, or -55°C. The single-temperature drying step can be carried out at a temperature between approximately 25°C and approximately 50°C. For example, the single-temperature drying step can be carried out at approximately 25°C, approximately 30°C, approximately 35°C, approximately 40°C, approximately 45°C, or approximately 50°C. When this method is performed in a single-temperature freezing step and a single-temperature drying step, the method may further include a "ramp-up" step between freezing and drying to allow the freeze dryer to reach an appropriate drying temperature.

[0051] The disclosed lyophilized formulations can be formed by a lyophilization method that is much faster than the lyophilization methods used to form other collagenase-containing lyophilized formulations. The lyophilized formulations can be formed by a lyophilization method carried out in less than 72 hours. In some embodiments, the method can be carried out in less than 30 hours. In some embodiments, the method can be carried out in less than 18 hours. In some embodiments, the method can be carried out for about 15 to about 25 hours.

[0052] The disclosed lyophilized formulation can be formed by a lyophilized method that uses much higher pressure than the lyophilized methods used to form other collagenase-containing lyophilized formulations. The lyophilized formulation can be formed by a lyophilized method carried out at a pressure between approximately 380 μbar and approximately 4000 μbar. The disclosed method can be carried out at pressures between approximately 500 μbar and approximately 4000 μbar, between approximately 750 μbar and approximately 4000 μbar, and between approximately 1000 μbar and approximately 4000 μbar. The disclosed method can be carried out at 380 μbar, 500 μbar, 750 μbar, 1000 μbar, 1500 μbar, 2000 μbar, 2500 μbar, 3000 μbar, 3500 μbar, or 4000 μbar.

[0053] The disclosed lyophilized formulation, once reconstituted, can be used to treat or alleviate collagen-mediated conditions such as severe cellulite (also known as edematous fibrosclerosing subcutaneous lipopathy (EFP)), Dupuytren's contracture (DC) with palpable cords, or Peyronie's disease (PD) with palpable plaques and curvature deformities of 30 degrees or more.

[0054] Furthermore, this specification provides a reconstituted formulation comprising, or consisting of, collagenase, disaccharides, mannitol, Tris-HCl, calcium chloride, and sodium chloride.

[0055] Collagenase may include collagenase I, and a preferred collagenase I is, for example, a collagenase I containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 1. In some embodiments, collagenase I contains the amino acid sequence of sequence ID number: 1. Collagenase may include collagenase II. A preferred collagenase II includes, for example, a collagenase II containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 2. In some embodiments, collagenase II contains the amino acid sequence of sequence ID number: 2. Collagenase may include a mixture of collagenase I and collagenase II. The collagenase may include, for example, a mixture of collagenase I containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 1, and collagenase II containing an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of sequence ID number: 2. In some embodiments, the collagenase includes a mixture of collagenase I containing the amino acid sequence of sequence ID number: 1 and collagenase II containing the amino acid sequence of sequence ID number: 2. Suitable mixtures of collagenase I and collagenase II include, for example, collagenase I:collagenase II mass ratios of 0.1:1, 0.25:1, 0.5:1, 0.75:1, 1:1, 1.1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, 1:0.1, 1:0.25, 1:0.5; 1:0.75, 1:1.1, 1:1.25, 1:1.5, 1:1.75, or 1:2. In some embodiments, the collagenase is collagenase Clostridium histolyticum (CCH).

[0056] Collagenase I and collagenase II may each have a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, as measured, for example, by reverse-phase HPLC.

[0057] Suitable disaccharides include, for example, sucrose or trehalose. In some embodiments, the reconstituted formulation comprises or consists of collagenase, sucrose, mannitol, Tris-HCl, calcium chloride, and sodium chloride. In some embodiments, the lyophilized formulation comprises or consists of collagenase, trehalose, mannitol, Tris-HCl, calcium chloride, and sodium chloride.

[0058] Appropriate amounts of calcium chloride and sodium chloride include those that allow the reconstituted preparation to be isotonic to human blood. In some embodiments, the reconstituted preparation contains about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or greater than 0.1% calcium chloride. In some embodiments, the reconstituted preparation contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or greater than 1% sodium chloride.

[0059] Furthermore, the reconstituted formulation may contain water for injection (WFI).

[0060] The disclosed reconstituted formulations can be used to treat or alleviate collagen-mediated conditions, including severe cellulite (also known as edematous fibrosclerosing subcutaneous lipopathy (EFP)), Dupuytren's contracture (DC) with palpable cords, or Peyronie's disease (PD) with palpable plaques and curvature deformities of 30 degrees or more.

[0061] The reconstituted formulation can contain approximately 0.01 mg to 50 mg of collagenase in a single dose or divided dose. For example, the reconstituted formulation can contain approximately 0.05 mg to 15 mg, 0.10 mg to 10 mg, 0.15 mg to 5 mg, 0.20 mg to 3 mg, or 0.25 mg to 2 mg of collagenase in a single dose or divided dose. Reconstituted formulations include, for example, approximately 0.05 mg, 0.10 mg, 0.15 mg, 0.20 mg, 0.25 mg, 0.30 mg, 0.35 mg, 0.40 mg, 0.45 mg, 0.50 mg, 0.55 mg, 0.60 mg, 0.65 mg, 0.70 mg, 0.75 mg, 0.80 mg, 0.85 mg, 0.90 mg, and 0.95 mg. mg, about 1.00mg, 1.05mg, about 1.10mg, about 1.15mg, about 1.20mg, about 1.25mg, about 1.30mg, about 1.35mg, about 1.40mg, about 1.45m g, about 1.50mg, about 1.55mg, about 1.60mg, about 1.65mg, about 1.70mg, about 1.75mg, about 1.80mg, about 1.85mg, about 1.90mg, about 1.95mg , about 2.00mg, about 2.05mg, about 2.10mg, about 2.15mg, about 2.20mg, about 2.25mg, about 2.30mg, about 2.35mg, about 2.40mg, about 2.45mg , about 2.50mg, about 2.55mg, about 2.60mg, about 2.65mg, about 2.70mg, about 2.75mg, about 2.80mg, about 2.85mg, about 2.90mg, about 2.95mg , about 3.00mg, about 3.05mg, about 3.10mg, about 3.15mg, about 3.20mg, about 3.25mg, about 3.30mg, about 3.35mg, about 3.40mg, about 3.45mg , about 3.50mg, about 3.55mg, about 3.60mg, about 3.65mg, about 3.70mg, about 3.75mg, about 3.80mg, about 3.85mg, about 3.90mg, about 3.95mg.95mg, about 4.00mg, about 4.05mg, about 4.10mg, about 4.15mg, about 4.20mg, about 4.25mg, about 4.30mg, about 4.35mg, about 4.40mg, about 4.45mg, about 4.50mg, about 4.55 mg, about 4.60mg, about 4.65mg, about 4.70mg, about 4.75mg, about 4.85mg, about 4.90mg, about 4.95mg, about 5.00mg, about 5.05mg, about 5.10mg, about 5.15mg, about 5.20mg, Each product contains approximately 5.25 mg, 5.30 mg, 5.35 mg, 5.40 mg, 5.45 mg, 5.50 mg, 5.55 mg, 5.60 mg, 5.65 mg, 5.70 mg, 5.75 mg, 5.80 mg, 5.85 mg, 5.90 mg, 5.95 mg, 6.00 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg of collagenase.

[0062] The reconstituted preparation may have a total volume of approximately 0.1 mL to approximately 50 mL. For example, the reconstituted preparation may have a total volume of approximately 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, or 50 mL.

[0063] A kit containing the disclosed lyophilized formulations and sterile diluents is also provided. This kit may include a container containing one of the disclosed lyophilized formulations and a container containing sterile diluents containing calcium chloride and sodium chloride.

[0064] Suitable containers for lyophilized preparations and / or sterile diluents include, for example, vials, cartridges, or syringes. Vials can be unit-dose vials or multi-dose vials. Suitable container sizes include, for example, containers ranging from 2 mL to 50 mL, such as 5 mL, 7.5 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, or 50 mL.

[0065] A container containing the disclosed lyophilized formulation may contain approximately 0.2 mg to approximately 50 mg of collagenase. For example, a container may contain approximately 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 1 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg of collagenase. A container containing the disclosed lyophilized formulation may contain approximately 0.2 mg to approximately 50 mg of the lyophilized formulation. For example, a container may contain lyophilized preparations of approximately 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 1 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg.

[0066] A container containing a sterile diluent may contain an amount of sterile diluent that, when the lyophilized preparation is reconstituted, will be an isotonic solution with human blood. In some embodiments, the sterile diluent contains about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or greater than 0.1% calcium chloride. In some embodiments, the sterile diluent contains about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or greater than 1% sodium chloride.

[0067] The amount of sterile diluent can range from approximately 0.1 mL to approximately 50 mL. For example, it can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, or 50 mL.

[0068] This specification provides a method for lyophilizing any of the disclosed formulations, the method comprising the steps of freezing the formulation at a temperature between approximately -25°C and -55°C to form a frozen formulation, and drying the frozen formulation at a temperature between approximately 25°C and approximately 50°C to form a lyophilized formulation.

[0069] In some embodiments, freezing is performed at a single temperature, and drying is performed at a single temperature. In such embodiments, the method may further include a “ramp-up” step between freezing and drying to allow the freeze dryer to reach a suitable drying temperature. The single-temperature freezing step can be performed at temperatures between approximately -25°C and approximately -55°C. For example, the single-temperature freezing step can be performed at approximately -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, or -55°C. The single-temperature drying step can be performed at temperatures between approximately 25°C and approximately 50°C. For example, the single-temperature drying step can be performed at approximately 25°C, approximately 30°C, approximately 35°C, approximately 40°C, approximately 45°C, or approximately 50°C.

[0070] The disclosed method can be carried out in less than 72 hours. In some embodiments, the method can be carried out in less than 30 hours. In some embodiments, the method can be carried out in less than 18 hours. In some embodiments, the method can be carried out in about 15 to about 25 hours.

[0071] The disclosed method can be performed at pressures between approximately 128 μbar and approximately 4000 μbar, between approximately 380 μbar and approximately 4000 μbar, between approximately 500 μbar and approximately 4000 μbar, between approximately 750 μbar and approximately 4000 μbar, and between approximately 1000 μbar and approximately 4000 μbar. The disclosed method can be performed at 380 μbar, 500 μbar, 750 μbar, 1000 μbar, 1500 μbar, 2000 μbar, 2500 μbar, 3000 μbar, 3500 μbar, or 4000 μbar. [Examples]

[0072] The following examples are provided to further illustrate some of the embodiments disclosed herein. The examples are intended to illustrate, not limit, the embodiments disclosed herein.

[0073] The XIAFLEX® formulation (CCH, 10 mM Tris / HCl pH 8.0, 60 mM sucrose) has a relatively long lyophilization cycle time of 72 hours. The objective of the following study was to achieve a more efficient lyophilization process that would shorten the cycle time and improve yield.

[0074] The following criteria were considered in optimizing the formulation and freeze-drying process. formulation: • pH to optimize the thermodynamic stability of proteins. • Trehalose or sucrose as a cryoprotectant / freeze-proofing agent to stabilize proteins. • Addition of bulking agents / isotonic agents (e.g., mannitol), • If necessary, use nonionic surfactants to suppress protein aggregation. Freeze-drying treatment: • Suppresses protein unfolding during freezing and drying. • The glass transition temperature of the product exceeds the planned storage temperature. • The ability to maintain a relatively low moisture content, and • Elegant cake structure.

[0075] As part of the process of identifying the optimal formulation composition for freeze-drying, several solution formulations ("pre-freeze-drying") were evaluated, each containing various combinations of excipients such as sucrose, trehalose, and mannitol, and different levels of surfactants. Furthermore, both untreated and silicone-treated (baked) vials were evaluated.

[0076] Research on the robustness of pharmaceutical formulations - Study 1 The objective of these studies was to challenge variants of the two formulations against the original XIAFLEX® formulation by subjecting them to freeze / thaw testing, shear stress, thermal stress, peroxide stress, and hydrophobic surface contact. The following formulations were subjected to thermal / shear stress and freeze / thaw stress testing. Variant A(V A ): CCH, 10 mM Tris / HCl pH 8.5, 60 mM Trehalose, 225 mM Mannitol Variant B(V B ): CCH, 10 mM Tris / HCl pH 8.0, 60 mM Trehalose, 225 mM Mannitol Original XIAFLEX® active pharmaceutical ingredient (OR): CCH, 10 mM Tris / HCl pH 8.0, 60 mM Sucrose

[0077] To investigate the effect of hydrophobic surfaces on protein stability, standard glass vials and siliconized glass vials were used in the tests. Two surfactants (polysorbate 20 and poloxamer 188) were added to the formulations, and the effect of the surfactants on the stability of the formulations during the challenge tests was examined. In addition, tests were conducted in the presence of a strong oxidizing agent (hydrogen peroxide) to induce oxidative stress. A total of 21 different matrices were used in the challenge tests.

[0078] Experimental Design CCH was formulated with three different formulation variants. To challenge the candidate formulations, the formulation variants were subjected to thermal stress and freeze / thaw stress with agitation. Hydrogen peroxide was added to the samples to induce oxidative stress on the proteins. To monitor aggregate formation, each sample after stress exposure was analyzed by turbidity measurement.

[0079] Disposal of glass vials and stoppers The vials were washed with pure water using a laboratory dishwasher. Afterward, the vials were dried and heat-treated at 300°C for 2 hours to remove pyrogenic substances and sterilize them. The stoppers were placed in a sterile bag and autoclaved at 2 bar and 121°C for 20 minutes, then dried at 80°C for 8 hours.

[0080] Sample preparation To achieve quantitative buffer exchange, dialysis was performed in three independent dialysis cycles. 60 ml of XIAFLEX® API was dialyzed against formulation variant A, and 40 ml of XIAFLEX® API was dialyzed against formulation variant B. The XIAFLEX® API was transferred to two pre-prepared Slide-A-Lyzer® cassettes (Thermo Scientific, Rockford, USA). After incubating the filled Slide-A-Lyzer® cassettes in 2000 ml or 1000 ml of target buffer for 2 hours, the first buffer exchange (2000 ml / 1000 ml) was performed. After another 2 hours of dialysis, the second buffer exchange (2000 ml / 1000 ml) was performed, and dialysis was completed overnight. Protein samples were removed from the Slide-A-Lyzer® cassettes and diluted to a concentration of 1 mg / ml.

[0081] Addition of detergent or hydrogen peroxide Post-dialysis samples were supplemented with stock solutions (10% w / w polysorbate 20, 10% w / w poloxamer 188, 30% w / w H2O2) containing polysorbate 20, poloxamer 188, hydrogen peroxide, and combinations thereof. Variants of the original XIAFLEX® formulation were prepared by directly adding detergents or hydrogen peroxide to the bulk solution. The composition of each variant is shown in Table 2. [Table 2]

[0082] Each formulation was aseptically filtered under laminar flow and subjected to heat, agitation, and freeze / thaw stress.

[0083] Freeze / thaw test The freeze / thaw stability of the formulations was tested by performing a total of three freeze / thaw cycles. 1.0 ml of each formulation was filled into siliconized 6R glass vials or 2R standard glass vials. Three vials were prepared for each variant. Liquid samples were frozen to 25°C to -30°C within 55 minutes, with controlled freezing rate, and then heated back to room temperature within 55 minutes, with controlled heating rate. For proper temperature control, the samples were placed in a pilot freeze-dryer. After each freeze / thaw cycle, the turbidity of the samples was measured. Turbidity was measured by placing 1 ml of each sample into a single-use turbidity cuvette for analysis. After analysis, the liquid was returned to the glass vials, and the experiment was continued.

[0084] Thermal stress test Test samples of all formulations were placed in 2R vials (filled with 1 ml) and subjected to stress at 40°C for 4 days under agitation (200 rpm). The turbidity of the stressed samples was then analyzed.

[0085] Turbidity measurement The turbidity of the samples was measured using a 2100AN turbidimeter (Hach Lange, Dusseldorf, Germany) in accordance with the European Pharmacopoeia. The system was calibrated as follows: • Hydrazine sulfate solution: 1.0 g of hydrazine sulfate was dissolved in purified water and diluted to 100.0 ml with the same solvent. It was left to stand for 4-6 hours. • Hexamethylenetetramine solution: 2.5 g of hexamethylenetetramine was dissolved in 25.0 ml of distilled water and placed in a 100 ml glass volumetric flask. • Primary opal-colored suspension: 25.0 ml of hydrazine sulfate solution was added to hexamethylenetetramine solution in a volumetric flask. Mixed and left to stand for 24 hours. • Standard for milky white color: A 15.0 ml primary milky white suspension was diluted with purified water to 1000.0 ml. This suspension was freshly prepared (stored for up to 24 hours). • Reference suspensions were prepared according to Table 3. [Table 3]

[0086] Results and Discussion Table 4 shows the results of the freeze / thaw test. None of the variants showed an increase in turbidity with increasing freeze / thaw cycles. The enzyme appears to be stable against freeze-thaw stress. Addition of surfactants or hydrogen peroxide did not affect the turbidity of the samples.

[0087] Table 5 shows the results of thermal stress at 40°C and stirring at 200 rpm. A clear increase in turbidity was observed after thermal stress in the variant containing hydrogen peroxide (indicated by an increase in nephelometric turbidity units (NTU)). Variation V A and Variation V B In the case where both formulations contained mannitol, the resulting turbidity was significantly lower than that of the modified version of the original XIAFLEX® formulation ("Or" - without mannitol) (reduced NTU). The beneficial effect of mannitol is thought to be due to its radical scavenging properties. [Table 4] [Table 5]

[0088] conclusion The increase in turbidity is V A and V B The results were significantly different. This is likely because the intermolecular repulsive force is higher at pH 8.5 compared to pH 8.0, thus suppressing the tendency for aggregate formation due to oxidative stress.

[0089] The addition of surfactants had neither a positive nor negative effect on the turbidity of the samples. The surface of the hydrophobic glass vials did not affect the turbidity of the samples after thermal stress.

[0090] To monitor aggregate formation, samples were analyzed by turbidity measurement. Sample turbidity did not increase with freeze / thaw cycles. This formulation appeared stable against aggregate formation even under freeze-thaw stress (up to 3 cycles). Hydrophobic surface contact and the addition of hydrogen peroxide did not affect sample turbidity under freeze / thaw stress, regardless of the presence or absence of surfactants. Under thermal stress, a clear increase in turbidity (measured by NTU) was observed under forced oxidative stress (addition of hydrogen peroxide). In variants containing mannitol and with high intermolecular repulsion, the increase in turbidity under forced oxidative stress was not as pronounced. The stability of the formulations against oxidative stress was highest in the order of Variant A > Variant B > Original XIAFLEX® formulation. The presence of detergents under forced oxidative stress did not affect sample turbidity. In the absence of oxidative stress, all samples maintained transparency even under thermal / agitation stress. Hydrophobic surface contact did not affect sample turbidity under thermal / agitation stress, regardless of the presence or absence of detergents.

[0091] Research on the robustness of pharmaceutical formulations - Study 2 Twelve formulations with different pH values ​​and mannitol concentrations were investigated using CG-MALS and nanoDSC. Furthermore, each variant was subjected to thermal stress by agitation and freeze-thaw stress tests. In addition, 0.1% H2O2 was added to reduce oxidative stress. The objectives of this study are as follows: Using CG-MALS and nanoDSC, we will elucidate the intermolecular interactions between collagenase I and collagenase II in various pharmaceutical formulations. • Test the stability of XIAFLEX® and additional formulation variants against freeze / thaw and heat stress.

[0092] The following CCH preparations were prepared and tested. [Table 6]

[0093] Collagenase I and Collagenase II were dialyzed against their corresponding formulation buffers. After dialyzing, the sample solutions were further diluted. The colloidal and thermodynamic stability of Collagenase I, Collagenase II, and the mixture were measured by CG-MALS and nanoDSC, respectively. Furthermore, the protein stability of each formulation was examined by stress tests (freeze / thaw and heat / shear stress). To induce oxidative stress during stability testing, each formulation was examined in the presence and absence of 0.1% H2O2 (variants without hydrogen peroxide are indicated by a, and variants with hydrogen peroxide are indicated by b).

[0094] dialysis Collagenase I and Collagenase II were dialysisd in three independent dialysis steps to achieve quantitative buffer exchange. 14 ml intermediate samples of Collagenase I and Collagenase II were transferred to two pre-prepared Slide-A-Lyzer® cassettes (Thermo Scientific, Rockford, USA) (in dialysis buffer). The filled Slide-A-Lyzer® cassettes were incubated in 1000 ml of target buffer for 2 hours before the first buffer exchange (1000 ml). After another 2 hours of dialysis, a second buffer exchange (1000 ml) was performed, and dialysis was finally completed overnight. Protein samples were removed from the Slide-A-Lyzer® cassettes and further processed. Each dialysis step represented a 1 / 35 buffer exchange, resulting in a total calculated buffer exchange factor of approximately 2 × 10⁻⁶. 5 This is the result.

[0095] Nano Differential Scanning Calorimetry Differential scanning calorimetry (DSC) is a technique used to evaluate the stability of proteins in their native form by measuring the thermal changes associated with molecular thermal denaturation when heated at a constant rate. Proteins in solution maintain equilibrium between their original structure (folded state) and a denatured structure (unfolded state). Native proteins undergo unfolding (thermal denaturation) at a characteristic temperature (T onset) when heated. The more intrinsically stable the biopolymer, the higher the onset temperature for unfolding transition. DSC measures the enthalpy of unfolding resulting from thermal denaturation, allowing us to elucidate factors contributing to the folding and stability of native biomolecules. These include hydrophobic interactions, hydrogen bonding, conformational entropy, and the physical environment. The following experimental methods were used: Mode: Scan Temperature parameter: Lower limit 20℃ Upper limit: 100℃ Ratio: 1°C / min Heating / Cooling Equilibration: 600s Pressure parameter: Manual, 3.0 bar Data interval: 1s

[0096] A buffer scan was performed before each sample was run to create a baseline. Sample preparation

[0097] Dialysis samples were diluted to 1 mg / ml with the corresponding formulation buffer. The corresponding dialysis buffer was used as a buffer scan and buffer reference.

[0098] CG-MALS Interactions between protein molecules in solution were characterized by analyzing changes in light scattering behavior at different concentrations by calculating the second virial coefficient (A2). A2 is an indicator of protein-protein interactions in solution. A negative A2 value indicates attractive protein-protein interactions, while a positive value indicates repulsive protein interactions. When A2 is negative, the protein solution is "colloidally unstable." A larger A2 value indicates greater repulsion, less protein interaction, less likelihood of protein aggregation, and higher stability. A2 values ​​were measured for various formulations using CG-MALS and used as an indicator of nonspecific protein-protein interactions.

[0099] Apparent weight-average molecular weight (Mw app ) is determined at each step of the concentration gradient by analyzing light scattering and concentration data. Significant interactions between polymers are determined by Mw app This appears as a change in relative concentration. The calculation of A2 was performed via Zimm plot analysis by extrapolating to a concentration of 0 mg / ml according to the following equation I.

number

[0100] Sample preparation For CG-MALS measurements, the dialyzed samples were used in their stock form. In the CG-MALS experiments, the corresponding dialyzing buffer was used as the diluent. Both the sample and buffer were filtered through a 0.1 μm filter. Before loading the sample into the CG-MALS system, the exact concentration of the sample was determined by UV absorption measurement. This concentration was used to calculate the concentration at each gradient step.

[0101] Sample measurement Using the Calypso II CG-MALS system, a concentration gradient of the analyte was supplied to the MALS detector. The sample was placed in syringe pumps 1 and 2 of the system, and the dialysis buffer was placed in syringe pump 3. The CG-MALS measurement consisted of three steps. In the first step, the concentration of sample 1 was varied from 10% to 100%, and the autoviral coefficient of sample 1 in the formulation was measured. In the second step, a crossover gradient was performed by decreasing the concentration of sample 1 from 90% to 10% and increasing the concentration of sample 2 from 10% to 90%. This step was performed to determine the cross-viral coefficient. In the third step, a concentration gradient of sample 2 was performed from 100% to 10%, and the autoviral coefficient of sample 2 in the formulation was measured. In each gradient step, 0.7 ml of sample was injected into the MALS detector. The resulting light scattering signals were recorded over a period of 180 seconds. A multi-component Zimm plot analysis with fixed molecular weight was performed using Calypso software version 2.1.5.

[0102] UV measurement The concentrations of collagenase I and collagenase II in solution were measured using an 8452A UV spectrometer (Agilent Technologies, Santa Clara, USA). Samples were measured at a concentration of approximately 3 mg / ml using a plastic cuvette with a light path thickness of 0.2 cm. The concentration for collagenase I was 1.52 ml / (mg). * (cm), collagenase II is 1.48 ml / (mg *The extinction coefficient was used (in cm) and calculated according to the Lambert-Beer law.

[0103] Freeze / thaw test The freeze-thaw stability of the formulation was tested by performing a total of three freeze-thaw cycles. Dialyzed collagenase I and dialyzed collagenase II were mixed to produce a solution containing 0.5 mg / ml of collagenase I and 0.5 mg / ml of collagenase II.

[0104] Each formulation was filled with 1.0 ml into a 2R glass vial. Three vials were prepared for each variant. The liquid samples were frozen at 25°C to -30°C within 55 minutes, with controlled freezing rate, and then reheated to room temperature within 55 minutes, with controlled heating rate. For proper temperature control, the samples were placed in a pilot freeze-dryer. After each freeze / thaw cycle, the turbidity of the samples was measured. Turbidity was measured by placing 1 ml of each sample into a single-use turbidity cuvette for analysis. After analysis, the liquid was returned to the glass vial, and the experiment was continued.

[0105] Thermal stress test Dialyzed collagenase I and dialyzed collagenase II were mixed to produce a solution containing 0.5 mg / ml of collagenase I and 0.5 mg / ml of collagenase II. Test samples of all formulations were placed in 2R vials (filled with 1 ml) and stressed at 40°C for 4 days under agitation (200 rpm). The turbidity of the stressed samples was later analyzed.

[0106] Turbidity measurement The turbidity of the sample was measured using the method described above.

[0107] Results and Discussion Table 7 shows the measurement results for CG-MALS and nanoDSC for each formulation.

[0108] Colloidal StabilityThe pH value of the formulation strongly affected the colloidal stability of collagenase I, collagenase II, and mixtures thereof. The strongest repulsive interaction was observed at pH 8.5. At pH 7.5, the repulsive interaction was low. At pH 7.5, collagenase I showed an attractive interaction when mannitol was not present in the formulation, and the repulsive interaction became stronger with increasing mannitol concentration. At more basic pH values, the effect of mannitol became subordinate.

[0109] thermodynamic stability Collagenase I showed a slight pH dependence on the unfolding initiation temperature. It exhibited a higher T value at pH 7.5 than at pH 8.5. Mannitol showed a slight concentration-dependent positive effect on the thermodynamic stability of collagenase I. The thermodynamic stability of collagenase II did not depend on the pH value investigated. The presence of mannitol was considered beneficial to the thermodynamic stability of collagenase II. [Table 7]

[0110] Table 8 shows the turbidity values ​​of the samples during the subsequent freeze-thaw cycle. [Table 8]

[0111] None of the formulations showed an increase in turbidity after the freeze / thaw test. The presence of hydrogen peroxide was well tolerable in each formulation under freeze / thaw stress.

[0112] Table 9 shows the turbidity values ​​of each formulation before and after heat stress. [Table 9]

[0113] In the absence of oxygen radicals, turbidity remained constant across all formulation variants examined. In the presence of hydrogen peroxide, sample turbidity increased significantly, indicating that oxidative stress induces aggregate formation. A basic solution at pH 8.5 stabilized the proteins against oxidative stress-induced aggregation. Mannitol showed an even more beneficial effect on sample turbidity after thermal stress in the presence of hydrogen peroxide. Mannitol is thought to act as a radical scavenger, improving protein stability against oxidative stress.

[0114] Research on the robustness of pharmaceutical formulations - Study 3 In the following studies, sucrose and trehalose were used at a concentration of 60 mM, and mannitol at a concentration of 225 mM. Several concentrations of surfactants were evaluated, and the concentrations used for specific samples are listed in the data table below. As part of formulation optimization, the effect of pH before lyophilization on various formulations was evaluated.

[0115] The robustness of the proposed formulation as a solution was investigated by subjecting it to the following stresses: • Three freeze-thaw cycles (-30°C to 25°C), and / or Shake at 200 rpm and 40°C for 4 days.

[0116] Subsequently, the turbidity of the stressed samples was examined, and the main quality characteristics of the product were evaluated using the following method: UV A280 Protein concentration • Mass and ratio of collagenase I and collagenase II obtained by reverse-phase high-performance liquid chromatography (as described herein) • Specific soluble rat tail collagen (SRC) enzyme activity assay (collagenase I; described herein) • Glycyl-L-prolyl-L-alanine (GPA) enzyme activity assay method (collagenase II: as described herein)

[0117] result TurbidityThe data obtained from the freeze / thaw cycle is shown in Table 10. [Table 10]

[0118] These results indicate that multiple freeze-thaw cycles do not affect protein aggregation, and turbidity values ​​were consistent across all formulation / container combinations tested. These results suggest that the tested excipients are suitable for further evaluation.

[0119] Table 11 shows the data under conditions of shaking and exposure to high temperatures. [Table 11]

[0120] These results, showing consistent turbidity values ​​across all tested formulation / container combinations, indicate that heat / shaking does not affect protein aggregation. This suggests that the tested excipients are suitable for further evaluation.

[0121] In the second test, the effects of various concentrations of polysorbate 20 and a high pH (8.8) on the turbidity of the product under stress conditions were evaluated. The results of this test are shown in Table 12. [Table 12]

[0122] These data indicate that the surfactant concentrations (0.02% and 0.1%) and pH values ​​(8.5 and 8.8) examined did not adversely affect protein aggregation, and consistent turbidity values ​​were obtained during freeze / thaw cycles and heat / shaking. These results suggest that thermodynamically stable formulations are possible, and that polysorbate 20 can be used as a surfactant within the investigated concentration range. Furthermore, it was shown that the use of trehalose or mannitol, either alone or in combination, did not adversely affect protein aggregation as measured by sample turbidity.

[0123] Main product quality attribute indicators Tables 13 and 14 show the data created to evaluate the effects of three freeze-thaw cycles from -30°C to 25°C and four days of thermal stress / shaking at 40°C and 200 rpm, respectively. [Table 13] [Table 14]

[0124] These data confirm that modifying the formulation at various pH levels using the three tested excipients did not adversely affect protein stability / concentration or enzyme activity, and that all of these components are suitable for further evaluation in lyophilized formulations. Furthermore, the pH range tested did not adversely affect product quality.

[0125] Research on the robustness of pharmaceutical formulations - Study 4 Furthermore, the properties of various formulations were evaluated using composition gradient multi-angle light scattering (CG-MALS) to assess self and heteroprotein interactions, and nano-differential scanning calorimetry (DSC) to assess protein unfolding. CG-MALS and nanoDSC were performed using the methods described above.

[0126] Furthermore, a study was conducted to investigate whether hydrogen peroxide, as an oxidizing agent, affects the target formulation (solution before freeze-drying) based on its turbidity level.

[0127] result CG-MALS -The results are shown in Table 15. [Table 15]

[0128] In the case of formulations containing trehalose, the most repulsive interactions were observed at pH 8.5, both within and between proteins (collagenase I and collagenase II), as shown in Figure 1.

[0129] Furthermore, trehalose, sucrose, and mannitol were also considered suitable formulation ingredients because they do not adversely affect the rebound force.

[0130] Nano-DSC -Table 16 shows the variables (pH, type and concentration of excipients) investigated using Nano-DSC to evaluate protein unfolding. [Table 16]

[0131] As a result, the various formulation components examined were found to have a protein separation initiation temperature (T onset ) and the thawing temperature of the protein (T m It was shown that it does not affect T. onsetの The data showed that a temperature of 40°C can be effectively utilized in the secondary drying step of the freeze-drying process without affecting protein stability.

[0132] Hydrogen Peroxide Challenge-The formulations were challenged in short-term tests using hydrogen peroxide (Figures 2A and 2B). Trehalose-containing formulations (with or without mannitol) and sucrose-containing formulations (with or without mannitol) showed significantly lower turbidity at pH 8.5 compared to the same formulations at pH 8.0 and pH 7.5 after exposure to hydrogen peroxide. Lower turbidity values ​​indicate a more stable formulation with less protein aggregation. These results are consistent with the high A2 value (high repulsive interaction) observed in the pH 8.5 formulation, suggesting the possibility of improved stability. Furthermore, exposure to hydrogen peroxide showed that formulations containing trehalose and mannitol (with or without polysorbate) (pH 8.0 or 8.5) showed significantly lower turbidity compared to a sucrose-containing formulation without mannitol (pH 8.0). The presence of surfactants did not have a positive or negative effect on turbidity.

[0133] conclusion Based on the results of formulation optimization work that identified the qualitatively and quantitatively optimal excipients, the following formulations (before lyophilization) were proceeded to evaluation of the lyophilization cycle. Mix CCH, sucrose, and mannitol (with or without polysorbate 20) at pH 8.5. Mix CCH, trehalose, and mannitol (with or without polysorbate 20) at pH 8.5.

[0134] Furthermore, in future analyses, increasing the concentration of polysorbate 20 did not prove effective, so the lowest concentration of polysorbate 20 (0.02%) was used.

[0135] The lyophilization cycles for both formulations were developed using 5 mL vials. Table 17 shows the details of the evaluated formulations. [Table 17]

[0136] Optimization of the freeze-drying cycle - Study 1: Changes in mannitol concentration The purpose of this study was to investigate the effects of varying the ratio of mannitol to a constant concentration of sucrose on the freeze-drying process and cycle time in the absence of surfactants. The following experimental variants were prepared along with a placebo Lyo sample. [Table 18]

[0137] An intermediate freeze-drying cycle ("Lyo cycle") was used for freeze-drying the product (with a target total cycle time of approximately 36 hours). The information on the pilot freeze-dryer used in this experiment is as follows: ·Manufacturer: Hof Sonderanlagenbau(Lohra,Germany) • Shelf area: 0.5m² 2 • Ice storage capacity: 10kg • Adjustable ice condenser temperature • Temperature record in vial • Differential pressure measurement • Connectable radiation cages

[0138] Preparation of packaging materials The freeze-dried stopper was autoclaved at 121°C for 15 minutes and dried at 105°C for 8 hours. The vial was washed with pure water and dehydrogenated at 300°C for 2 hours.

[0139] The formulation variants were prepared by dialysis. Dialysis of the XIAFLEX® active pharmaceutical ingredient was performed in three independent dialysis steps to achieve quantitative buffer exchange. 125 ml of XIAFLEX® active pharmaceutical ingredient was dialyzed against variants a, b, and c. The XIAFLEX® active pharmaceutical ingredient was transferred to two pre-prepared dialysis tubes (in dialysis buffer). The filled dialysis tubes were incubated in 1 L of target buffer for 2 hours, followed by the first buffer exchange (1 L). After another 2 hours of dialysis, a second buffer exchange (2 L) was performed, and dialysis was finally completed overnight. Protein samples were taken from the dialysis tubes and diluted to a concentration of 0.93 mg / ml (±10%) (concentration confirmed by UV 280 nm). As a control, XIAFLEX® active pharmaceutical ingredient was used without dialysis.

[0140] Filtration and filling The lyophilized solution was passed through a 0.22 μm filter before being filled. 1 ml of the corresponding lyophilized solution was then filled into the vial.

[0141] Freeze drying Vials filled with freeze-drying stoppers labeled "Lyo-position" were loaded into a pilot freeze-dryer. Approximately 125 vials of each formulation and 100 placebo vials of Vb were added. The freeze-drying cycle was performed with the following parameters. ·Freezing temperature (shelf): -50℃ ·Primary drying temperature (shelf): -10℃ ·Secondary drying temperature (shelf): 40℃ • Pressure: 0.25 mbar • Total time: Approximately 41 hours

[0142] A thermocouple was inserted into the product vial to control the product temperature during freeze-drying. A Pirani pressure sensor was used for pressure control during freeze-drying. Pressure regulation was managed using vacuum and a dosing valve (nitrogen injection). The vial was closed at a pressure of 750 mbar under a nitrogen atmosphere.

[0143] Karl Fischer titration The contents of one vial of the corresponding lyophilized material were weighed into a glass vial sealed with a crimped cap. This sample was transferred to a Karl Fischer coulometer (756 / 774; Metrohm) oven heated to 100°C. The cap's partition was punctured with a hypodermic needle, and the resulting steam was directly introduced into the titration chamber of the Karl Fischer coulometer via dry nitrogen. The measurement was repeated once. An empty glass vial was used for blank correction.

[0144] Analysis of crystal water The Oven Sample Processor 774 enables a unique temperature rise method in Karl Fischer titration. The sample is heated at a defined heating rate, and the released water is directly transferred to the titration chamber of the Karl Fischer titrator. By recording the water generated and the water drift due to the oven temperature (μg water / min), specific events in which water is released (such as the release of hydrated water) can be detected. 50-100 mg of the lyophilized material was weighed into an empty 6R Type 1 glass vial and sealed with an Alcrimp cab. This sample was transferred to the oven of the sample processor, where the sample was heated from 50°C to 140°C over 45 minutes by a defined temperature ramp (2°C / min). To avoid undesirable Maillard reactions, the temperature rise was terminated at 140°C.

[0145] exterior The freeze-dried material was carefully removed from the glass vial by breaking it, and the freeze-dried cake was vertically cut to screen for any disintegration zones in its inner layer.

[0146] Scanning electron microscope The freeze-dried samples were analyzed using scanning electron microscopy (SEM) to evaluate their microstructure. The freeze-dried samples were cut, and the longitudinal section and top and bottom surfaces were analyzed using SEM at 50x and 150x magnification.

[0147] Reconstruction The vial was reconstituted with a 4 ml solution containing 0.03% calcium chloride and 0.66% sodium chloride. The time until the lyophilized material was completely dissolved was recorded.

[0148] Nano Differential Scanning Calorimetry NanoDSC was performed using the method described above.

[0149] Sample preparation The lyophilized solution and reconstituted lyophilized product were analyzed at a concentration of 0.93 mg CCH / ml. The corresponding buffer was used as a buffer scan and buffer reference.

[0150] result The acquisition of digital data proved that the freeze-drying process was carried out as intended. As indicated by the difference between the Pirani and conductive pressure sensors, the sublimation of the batch was completed approximately 15 hours after the total freeze-drying time. The completion of sublimation in each sublot was indicated by a sharp rise in product temperature during the primary drying.

[0151] The primary drying process was completed approximately 17 hours after the total freeze-drying time. After 18 hours of secondary drying, one vial of each variant was removed from the freeze-dryer, and the residual moisture level was measured by Karl Fischer titration while extending the secondary drying process for the remaining batch. The Karl Fischer analysis showed that the residual moisture level was already below the desired value. Based on these results, the remaining batch was immediately unloaded. The total secondary drying time was 19 hours.

[0152] exterior - All samples show excellent macroscopic appearance without any cake defects (data not shown).

[0153] Reconstruction behavior - All sublots of lyophilized products dissolved quickly and spontaneously within seconds (<30 seconds).

[0154] Residual moisture-Table 19 shows the residual water content of the samples measured by Karl Fischer titration. [Table 19]

[0155] Scanning electron microscope (SEM) SEM analysis of the Lyo cake showed no signs of collapse or other cake defects (see Figures 3A-3R). The internal structure of all formulation variants was equivalent. The top surface of variant Va (lowest sucrose content) had a more open, porous structure and less skin formation compared to variants Vb and Vc. The bottom surface of variant Vc appeared denser than the bottom surfaces of the other formulations.

[0156] Nano DSC -The results obtained are summarized in Table 20. [Table 20]

[0157] To demonstrate the accuracy of unfolding temperature and denaturation enthalpy, variants of Vc (before lyophilization) were analyzed six times. Samples were obtained in a single preparation, eliminating concentration uncertainties. The difference between the T onset and Tm results was less than 1%, but the enthalpy variation was greater than 7%, which is in good agreement with the instrument specifications (TA Instruments assumes a 5% uncertainty in lysozyme unfolding enthalpy) (data not presented). With respect to unfolding temperature and transition enthalpy, all formulations were equivalent within the margin of error of this method before and after lyophilization, and it can be concluded that the conditions of the lyophilization process used do not adversely affect CCH. Increasing the amount of mannitol appears to increase thermodynamic stability, as indicated by slightly higher values ​​of Tm and T onset.

[0158] conclusion Based on the observations and all the data, it was found that lyophilized CCH formulations can be produced using various concentrations of mannitol in the presence of sucrose in Tris buffer. This study also identified more moderate lyophilization conditions that resulted in a cycle time of approximately 40 hours.

[0159] Optimization of the freeze-drying cycle - Study 2: Fixed mannitol concentration The objective of this work was to update the freeze-drying cycle in order to reduce process time and achieve a more efficient and robust process.

[0160] Experimental Design Lab-scale lyophilization tests were conducted using 5cc vials with four experimentally developed formulations and the XIAFLEX® formulation as a control. These variations are shown in Table 21 below. [Table 21]

[0161] To prepare experimental formulations #2 to #5, formulation #1 was subjected to buffer exchange by dialysis, and new excipients (trehalose, mannitol, and polysorbate 20) were introduced into the formulation in Tris buffer (pH 8.5).

[0162] The initial freeze-drying cycle development work was carried out using slightly more aggressive conditions in terms of drying temperature, pressure, and time than those used for the XIAFLEX® formulation. Secondary drying was performed at 40°C to efficiently dry the product and reduce the target moisture content to 0.5% or less. A detailed comparison of the XIAFLEX® process and the experimental freeze-drying process is summarized in Table 22 below. [Table 22]

[0163] Results and conclusions The lyophilized cakes of all formulations dissolved rapidly in the diluent, with a reconstitution time of 10 seconds or less and a moisture content (KF) of less than 0.4%.

[0164] When the experimental cycle time was shortened, the XIAFLEX® cake appeared to shrink compared to the variants of the experimental formulations, but all of these formulations showed a firm, robust cake. All experimental formulations were tested for various properties, including protein concentration, collagenase I and collagenase II mass composition, and biological activity. The test results for all formulations were consistent with those of the XIAFLEX® formulation.

[0165] Experimental formulations #3 and #5 were selected and further tested. These formulations were used for informal short-term stability testing under accelerated storage conditions of 5°C and 25°C / 60%RH. Samples were analyzed according to approved testing methods. The results for both formulations were consistent with past data for XIAFLEX® formulations, indicating that this formulation change will not adversely affect product quality (data not presented). As previously stated, currently approved limits may be revised based on statistical analysis of experimentally obtained data.

[0166] Based on the development work and data collected to date for optimizing the formulations, it has been found that the addition of the two protein stabilizers (sucrose and trehalose), the bulking agent (mannitol), and the surfactant (polysorbate 20) does not adversely affect the quality of the product and is suitable for further refinement of these formulations.

[0167] Optimization of the freeze-drying cycle - Study 3: Pressure analysis Research purpose Pressure tests were conducted to facilitate the identification of optimal freeze-drying process conditions. The aim of these tests was to determine the maximum allowable pressure and other reliable process parameters for freezing, primary drying, and secondary drying under process-appropriate conditions, in order to achieve a fast and robust freeze-drying cycle.

[0168] Experimental Design As shown in Table 23, three different formulations in 5cc vials were used. [Table 23]

[0169] To determine the pressure during the sublimation phase, two pressure tests were performed at different shelf temperatures and pressure settings. A sample vial filled with the experimental formulation was placed on the shelf (center position) of a freeze-dryer. After freezing, the chamber pressure was set to the initial value of 128 μbar, and the shelf temperature was raised to the initial value (-10°C in Test 1, +10°C in Test 2). Freeze-drying was performed for a set period of time to generate a small amount of freeze-dried material on the ice interface. Subsequently, the chamber pressure was gradually increased (e.g., 380 μbar, 1030 μbar, etc.), and the sample vial was monitored by video to check for cake collapse or other visual adverse effects. The shelf temperature was set to -10°C, and a moderate energy input was applied.

[0170] Photographs of the vials at the end of each pressure step are shown in Figures 4A to 4C. As shown in these figures, the cake of the control (#1, XIAFLEX® in the vial shown on the far right) collapsed with increasing pressure. Therefore, the maximum allowable pressure for the XIAFLEX® formulation was 128 μbar to 380 μbar, corresponding to an ice interface temperature of -40°C to -30°C. Experimental formulations #3 and #5 remained intact throughout the entire pressure range investigated.

[0171] In the second pressure test, experimental formulations #3 and #5 were further investigated. The pressure range was extended up to 4 mbar. The shelf temperature during the test was set to +10°C to provide sufficient energy input to enable efficient sublimation in this pressure range.

[0172] The results are shown in FIGS. 5A and 5B. No cake collapse or other defects were observed in all the pressure ranges investigated. Based on these findings, it was concluded that formulations containing mannitol can be lyophilized at much higher chamber pressures, resulting in a more efficient and robust lyophilization process with shorter lyophilization cycle times.

[0173] The development work for this first lyophilization cycle supported the use of mannitol to pursue a more efficient and robust lyophilization cycle.

[0174] Optimization of the lyophilization cycle - Study 4 Further optimization Based on the results of the above pressurization tests, the lyophilization parameters were optimized for experimental formulations #3 and #5 (data not shown).

[0175] As a result of the pressure test, it was found that formulations containing mannitol can be lyophilized at chamber pressures up to 4 mbar without the risk of structural collapse during sublimation. Therefore, in the optimization test, recycling was carried out at a chamber pressure of 1 mbar to utilize a high sublimation rate.

[0176] Two freezing cycle experiments were conducted to identify the basic parameters of a robust and efficient lyophilization cycle. The samples after lyophilization were analyzed by appearance, residual moisture, physical stability, and scanning electron microscopy (SEM).

[0177] Experiment 1 : Only experimental formulation #3 was used. From experiment the results, it was found that to reach a residual moisture level of 0.5% or less, it is necessary to increase the temperature during secondary drying. The results of other physical property tests were within the acceptable range (data not shown). An overview of the lyophilization parameters used is shown below. · Freezing temperature (shelf): -50 °C · Drying temperature (sublimation · secondary drying) (shelf): 35 °C · Pressure: 1 mbar · Accumulated time: approximately

[0178] Experiment 2 : Only use experimental formulation #3. This experiment confirmed the conditions suitable for drying at 40°C. All results were within the acceptable range (data not shown). An overview of the lyophilization parameters used is shown below. · Freezing temperature (shelf): -50°C · Drying temperature (sublimation·secondary drying) (shelf): 40°C · Pressure: 1 mbar · Integration time: about 21.5 hours

[0179] Conclusion In the optimization study of the lyophilization cycle, the basic parameters for an efficient lyophilization cycle were identified. All tests on the physical attributes of the lyophilized cake were accepted.

[0180] Stability test - Long-term stability test Six process validation lots of the CCH formulation (a mixture of collagenase I and collagenase II at a concentration of 1 mg / mL in 10 mM Tris, 60 mM sucrose, 225 mM mannitol, pH 8.5 in a 1:1 ratio) were manufactured and their stability was confirmed. One initial formulation development lot using the same formulation but with a lower fill volume and an equivalent container closure system was manufactured and its stability was confirmed.

[0181] For the six process validation lots, long-term stability monitoring was performed. The storage conditions included long-term stability at various storage conditions (2 - 8°C; 25°C / 60% relative humidity (RH); and 40°C / 75% RH) to support the product's storage period, and short-term stability tests at various storage conditions to support the evaluation of potential temperature changes during transportation or storage. Data from the initial formulation development lot with the same formulation but a lower fill volume (0.46 mg) and a comparable container closure system were also included.

[0182] Table 24 shows an overview of these studies and the available stability data. [Table 24]

[0183] Stability batches are measured by appearance (before and after reconstitution), reconstitution time, osmotic pressure, pH, and UV. A280 The following tests were performed: concentration, quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reverse-phase high-performance liquid chromatography (RPHPLC) [purity], compositional mass and ratio by RP-HPLC, size exclusion high-performance liquid chromatography (SEC-HPLC), efficacy of collagenase I by soluble rat tail collagen (SRC) assay, efficacy of collagenase II by glycyl-L-prolyl-L-alanine (GPA) assay, moisture, particulate matter, endotoxin, and helium leak due to container airtightness.

[0184] Results and Analysis The following shows the evaluation and trend analysis of the stability results for each test under various storage conditions (2-8°C, 25°C / 60% relative humidity (RH), 40°C / 75% RH).

[0185] Appearance (before and after replacement) )-The results for appearance (before and after re-substitution) under the tested storage conditions did not show any significant changes or unexpected trends (data not presented).

[0186] Reconfiguration time - No significant changes or unexpected trends were observed in the reconstitution time under the tested storage conditions (data not presented).

[0187] Osmorality - No significant changes or unexpected trends were observed in the osmorality results under the tested storage conditions (data not presented).

[0188] pH - No significant changes or unexpected trends were observed in the pH results under the tested storage conditions (data not presented).

[0189] UV A280 Concentration by- Results of concentration by UV under the tested storage conditions showed no significant changes or unexpected trends (data not shown). A280

[0190] Quantitative SDS-PAGE - Results of quantitative SDS-PAGE under the tested storage conditions showed no significant changes or unexpected trends (data not shown).

[0191] RP-HPLC (purity - Results of RP-HPLC (purity) under the tested storage conditions showed no significant changes or unexpected trends (data not shown).

[0192] Composition by mass and ratio determined by RP-HPLC - Results of mass and ratio by compositional RP-HPLC under the tested storage conditions showed no significant changes or unexpected trends (data not shown).

[0193] SEC-HPLC - Results of SEC-HPLC under the tested storage conditions showed no significant changes or unexpected trends (data not shown).

[0194] SRC assay for the efficacy of collagenase I - Results of the SRC assay for the potency of collagenase I showed no significant changes or unexpected trends under the tested storage conditions (data not shown).

[0195] GPA assay of collagenase II efficacy - Results of the GPA assay for the potency of collagenase II showed no significant changes or unexpected trends under the tested storage conditions (data not shown).

[0196] moisture - As shown in Figure 6, at 25°C / 60% relative humidity, the mannitol-containing formulation had less moisture than the XIAFLEX® formulation, indicating improved stability of the mannitol-containing formulation.

[0197] fine particles - Results of the microparticles under the tested storage conditions showed no significant changes or unexpected trends (data not shown).<​

[0198] Container airtightness / helium leakage - No significant changes or unexpected trends were observed in the container airtightness / helium leakage results (data not provided).

[0199] Lightstable test After irradiating with 1.2 million lux of cold white light, 200 watt-hours / square meter (W / m 2 A test was conducted to investigate the photostability of CCH formulations by irradiating them with ultraviolet light. The samples were irradiated with 8.00 kilolux of cold white light for 150 hours, and then subjected to 10.00 W / m². 2 The subjects were exposed to UV light for 20 hours. All results after this exposure were equivalent to those of the unexposed control group (data not presented).

[0200] Reconstitution stability test Several reconstitution stability tests were conducted to demonstrate the compatibility between the lyophilized CCH formulation and the sterile diluent used for reconstitution, and to create stability data for the reconstituted product under potential usage conditions. The lyophilized CCH formulation was reconstituted with a sterile diluent, stored at a specific temperature, and then analyzed at specific time points. Table 25 summarizes the reconstitution stability tests performed. [Table 25]

[0201] These tests measured efficacy using the SRC and GPA methods (data not presented). Microplate versions of the SRC and GPA methods were used to measure potency. These methods offer higher throughput and faster results compared to cuvette-based methods. These results demonstrate the compatibility of lyophilized CCH formulations with sterile diluents and the stability of reconstituted CCH formulations under potential usage conditions.

[0202] conclusion Stability data from six process validation lots of the CCH formulation demonstrated stability to a pull point of at least 18 months under storage conditions of 5°C and 25°C / 60%RH, and 6 months under storage conditions of 40°C / 75%RH. Furthermore, data from formulation development lots of the CCH formulation in smaller vials confirmed stability to a pull point of 24 months under storage conditions of 5°C and 25°C / 60%RH, and 6 months under storage conditions of 40°C / 75%RH.

[0203] The CCH formulation showed acceptable photostability, with all results obtained after exposure being equivalent to those of the control (unexposed) sample.

[0204] The CCH preparation showed acceptable stability after reconstitution with sterile diluent, even when stored at 25°C / 60%RH for up to 24 hours and at 5°C for up to 120 hours. Furthermore, the reconstituted CCH preparation showed acceptable stability after storage at 25°C / 60%RH for 24 hours, followed by storage at 2°C-8°C for 96 hours, and then again at 25°C / 60%RH for another 24 hours.

[0205] In the future, we plan to conduct annual stability tests, including storage under conditions of 5°C±3°C and 25°C±2°C / 60%±5%RH. The material under both storage conditions will be evaluated over the proposed 36-month storage period.

[0206] Those skilled in the art will understand that numerous changes and modifications can be made to preferred embodiments of the present invention, and that such changes and modifications can be made without departing from the spirit of the invention. Accordingly, the appended claims are intended to cover all such equivalent variations that fall within the true spirit and scope of the invention.

[0207] Any patents, patent applications, and publications cited or referenced in this document are incorporated into this Agreement in their entirety by reference.

[0208] Embodiment The following list of embodiments is intended to complement, rather than replace, the preceding description. Embodiment 1 A pharmaceutical product, Collagenase and Disaccharides ranging from approximately 30 mM to approximately 240 mM, Mannitol in concentrations of approximately 50 mM to 800 mM, and Approximately 6 mM to approximately 10 mM Tris-HCl, A formulation containing the above. Embodiment 2 The formulation according to Embodiment 1, wherein the collagenase comprises collagenase I. Embodiment 3 The formulation according to Embodiment 2, wherein the collagenase I comprises the amino acid sequence with sequence ID number: 1. Embodiment 4 The formulation according to Embodiment 1, wherein the collagenase comprises collagenase II. Embodiment 5 The formulation according to Embodiment 4, wherein the collagenase II comprises the amino acid sequence with sequence ID number: 2. Embodiment 6 The formulation according to Embodiment 1, wherein the collagenase comprises a mixture of collagenase I and collagenase II. Embodiment 7 A formulation according to Embodiment 6, wherein collagenase I comprises the amino acid sequence of sequence ID number: 1, and collagenase II comprises the amino acid sequence of sequence ID number: 2. Embodiment 8 A formulation according to Embodiment 6 or 7, wherein the collagenase is collagenase Clostridium histolyticum (CCH). Embodiment 9 A formulation according to any one of Embodiments 1 to 8, wherein the disaccharide comprises sucrose or trehalose. Embodiment 10 A formulation according to any one of Embodiments 1 to 9, wherein the pH of the formulation is approximately 7.8 to approximately 8.8. Embodiment 11 A formulation according to any one of Embodiments 1 to 10, wherein the formulation is CCH and, Approximately 60 mM sucrose, Approximately 225 mM mannitol, and It contains approximately 10 mM Tris-HCl, The aforementioned formulation has a pH of approximately 8.5. Embodiment 12 A formulation according to any one of Embodiments 1 to 11, further comprising a surfactant containing polysorbate 20, polysorbate 80, or poloxamer 188. Embodiment 13 A formulation according to Embodiment 12, comprising about 0.01% to about 2% of the surfactant. Embodiment 14 A formulation according to Embodiment 13, comprising approximately 0.02% of the surfactant. Embodiment 15 A formulation according to any one of Embodiments 1 to 14, wherein the formulation is a liquid formulation. Embodiment 16 It is a freeze-dried preparation, Collagenase and Disaccharides and, Mannitol and, Tris-HCl and, A lyophilized preparation containing the above. Embodiment 17 The lyophilized preparation according to Embodiment 16, wherein the collagenase comprises collagenase I. Embodiment 18 In the lyophilized preparation described in Embodiment 17, the collagenase I comprises the amino acid sequence with sequence ID number: 1. Embodiment 19 A lyophilized preparation according to Embodiment 16, wherein the collagenase comprises collagenase II. Embodiment 20 In the lyophilized formulation described in Embodiment 19, the collagenase II comprises the amino acid sequence with sequence ID number: 2. Embodiment 21 A lyophilized preparation according to Embodiment 16, wherein the collagenase comprises a mixture of collagenase I and collagenase II. Embodiment 22 A lyophilized preparation according to Embodiment 21, wherein collagenase I contains the amino acid sequence of sequence ID number: 1, and collagenase II contains the amino acid sequence of sequence ID number: 2. Embodiment 23 A lyophilized preparation according to Embodiment 21 or 22, wherein the collagenase is collagenase Clostridium histolyticum (CCH). Embodiment 24 A lyophilized preparation according to any one of Embodiments 16 to 23, wherein the disaccharide comprises sucrose or trehalose. Embodiment 25 In the lyophilized formulation described in any one of Embodiments 16 to 24, before lyophilization, the formulation is CCH and, 60 mM sucrose and, 225 mM mannitol, and It contains 10 mM Tris-HCl and, A lyophilized formulation with a pH of approximately 8.5. Embodiment 26 A lyophilized preparation according to any one of Embodiments 16 to 25, wherein the lyophilized preparation is stable at a pressure of 380 μbar or higher. Embodiment 27 In the lyophilized formulation described in Embodiment 26, the lyophilized formulation is a lyophilized formulation that is stable at a pressure of approximately 4000 μbar. Embodiment 28 In the lyophilized preparation described in any one of Embodiments 16 to 27, the lyophilized preparation is: (i) At 2-8°C for at least 36 months, (j) At 25℃ / 60% relative humidity for at least 36 months, (k) At 40℃ / 75% relative humidity for at least 6 months, (l) A lyophilized formulation that is stable in any combination of (a) to (c). Embodiment 29 In the lyophilized preparation described in any one of Embodiments 16 to 28, the lyophilized preparation is: A step of freezing the aforementioned formulation at a temperature between approximately -25°C and -55°C to form a frozen formulation, and A lyophilized preparation formed by a method comprising the step of drying the aforementioned frozen preparation at a temperature between approximately 25°C and approximately 50°C to form the lyophilized preparation. Embodiment 30 In the lyophilized preparation described in Embodiment 29, the lyophilized preparation is A step of freezing the aforementioned formulation at a single temperature between approximately -25°C and -55°C to form a frozen formulation, and A lyophilized preparation formed by a method comprising the step of drying the aforementioned frozen preparation at a single temperature between approximately 25°C and approximately 50°C to form the lyophilized preparation. Embodiment 31 A lyophilized preparation according to any one of embodiments 16 to 30, wherein the lyophilized preparation is formed by a lyophilization method carried out in less than 72 hours. Embodiment 32 A lyophilized preparation according to any one of embodiments 16 to 31, wherein the lyophilized preparation is formed by a lyophilization method carried out at a pressure between approximately 380 μbar and approximately 4000 μbar. Embodiment 33 A lyophilized preparation according to any one of Embodiments 16 to 32, wherein the lyophilized preparation is contained in a unit-dose vial, a multi-dose vial, a cartridge, or a syringe. Embodiment 34 A reconstituted formulation, Collagenase and Disaccharides and, Mannitol and, Tris-HCl and, Calcium chloride and, Sodium chloride and A reconstituted formulation containing the above. Embodiment 35 In the reconstituted formulation according to Embodiment 34, the collagenase is collagenase Clostridium histolyticum (CCH). Embodiment 36 A reconstituted formulation according to Embodiment 34 or 35, wherein the disaccharide comprises sucrose or trehalose. Embodiment 37 A reconstituted preparation according to any one of embodiments 34 to 36, wherein the reconstituted preparation is isotonic with respect to human blood. Embodiment 38 It's a kit, A container containing a lyophilized preparation according to any one of embodiments 16 to 32, and A container containing a sterile diluent containing calcium chloride and sodium chloride, A kit that includes this.

Claims

1. A method for producing a freeze-dried collagenase preparation, wherein the method is To form a frozen collagenase preparation, the process involves freezing a composition containing collagenase, disaccharides, mannitol, and Tris-HCl, and To form the freeze-dried collagenase preparation, the process involves drying the freeze-dried collagenase preparation at a pressure of 380 μbar to 4000 μbar, Methods that include...

2. The method according to claim 1, wherein the method is To form the frozen collagenase preparation, the steps include freezing the composition in a temperature range of -25°C to -55°C, and / or A step of drying the frozen collagenase preparation at a temperature range of 25°C to 50°C. Methods that include...

3. The method according to claim 2, wherein the method is To form the aforementioned frozen collagenase preparation, the process involves freezing the composition at a single temperature between -25°C and -55°C, and The process involves drying the frozen collagenase preparation at a single temperature between 25°C and 50°C. Methods that include...

4. A method according to any one of claims 1 to 3, wherein the freezing step is carried out at -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, or -55°C.

5. A method according to any one of claims 1 to 4, wherein the drying step is carried out at 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C.

6. A method according to any one of claims 1 to 5, wherein the drying step is carried out at a pressure of 500 μbar to 4000 μbar.

7. A method according to any one of claims 1 to 5, wherein the drying step is performed at 380 μbar, 500 μbar, 750 μbar, 1000 μbar, 1500 μbar, 2000 μbar, 2500 μbar, 3000 μbar, 3500 μbar, or 4000 μbar.

8. A method according to any one of claims 1 to 7, wherein the disaccharide is sucrose or trehalose.

9. A method according to any one of claims 1 to 8, wherein the collagenase comprises collagenase I.

10. The method according to claim 9, wherein the collagenase I comprises the amino acid sequence of sequence ID number:

1.

11. A method according to any one of claims 1 to 8, wherein the collagenase comprises collagenase II.

12. The method according to claim 11, wherein the collagenase II comprises the amino acid sequence with sequence ID number:

2.

13. A method according to any one of claims 1 to 8, wherein the collagenase comprises a mixture of collagenase I and collagenase II.

14. The method according to claim 13, wherein collagenase I comprises the amino acid sequence of sequence ID number: 1, and collagenase II comprises the amino acid sequence of sequence ID number:

2.

15. A method according to claim 13 or 14, wherein the collagenase is collagenase Clostridium histolyticum (CCH).

16. A method according to any one of claims 1 to 15, wherein the collagenase is produced by recombinant technology.

17. In the method according to any one of claims 1 to 16, the composition is: The aforementioned disaccharides in concentrations of 30 mM to 240 mM, The mannitol in a concentration of 50 mM to 800 mM, and The Tris-HCl in a concentration of 6 mM to 10 mM, Methods that include...

18. The method according to claim 17, wherein the pH of the composition is 7.8 to 8.

8.

19. In the method according to claim 17 or 18, the composition is 60 mM sucrose and 225 mM mannitol, and It contains 10 mM Tris-HCl, The composition has a pH of 8.

5. method.

20. A method according to any one of claims 17 to 19, wherein the composition comprises a surfactant selected from polysorbate 20, polysorbate 80, and poloxamer 188.

21. The method according to claim 20, wherein the surfactant is present in an amount of 0.01% to 2%.

22. The method according to claim 21, wherein the surfactant is present in an amount of 0.02%.

23. A method according to any one of claims 1 to 22, wherein the composition is a liquid.

24. A method according to any one of claims 1 to 23, wherein the lyophilized collagenase preparation does not contain mannitol and has higher stability at a pressure exceeding 380 μbar compared to a control lyophilized preparation produced by the same method.

25. A method according to any one of claims 1 to 24, wherein the lyophilized collagenase preparation does not contain mannitol and has higher stability at a pressure greater than 1030 μbar compared to a control lyophilized preparation produced by the same method.

26. A method according to any one of claims 1 to 25, wherein the lyophilized collagenase preparation does not contain mannitol and has higher stability at a pressure exceeding 4000 μbar compared to a control lyophilized preparation produced by the same method.

27. In the method according to any one of claims 1 to 26, the lyophilized collagenase preparation is (a) at 2–8°C for at least 36 months, (b) At 25°C and 60% relative humidity for at least 36 months, (c) At 40°C and 75% relative humidity for at least 6 months, (d) Any combination of (a) to (c), A method to achieve stability.

Images