Collagenase formulations and methods of producing 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 January 6, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This application contains a Sequence Listing that has been submitted electronically as a text file titled "117326_000001_Sequence_Listing.txt" and is 17,447 bytes in size, created on January 3, 2020. This Sequence Listing is incorporated herein by reference.

[0003] Disclosed herein are collagenase-containing formulations that have improved stability and storage. [Background technology]

[0004] XIAFLEX® (collagenase from Clostridium histolyticum (CCH)) is currently approved for the treatment of Dupuytren's contracture (DC) and Peyronie's disease (PD). The currently approved formulation of XIAFLEX® is provided as a lyophilized cake containing 0.9 mg of CCH in 3 CC vials with diluent. The current formulation of XIAFLEX® (pre-lyophilized) has a lyophilization cycle time of approximately 72 hours in the vial. Efficient lyophilization is necessary for shelf life and enzyme stability. Summary of the Invention [Means for solving the problem]

[0005] Disclosed herein is a formulation comprising collagenase, about 30 mM to about 240 mM disaccharide; about 50 mM to about 800 mM mannitol, and about 6 mM to about 10 mM Tris-HCl.

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

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

[0008] Kits are also provided, comprising 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, as well as the following detailed description, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed formulations, there are shown exemplary embodiments of the formulations, but the formulations are not limited to the particular embodiments disclosed.

[0010] [Figure 1] FIG. 1 shows the effect of pH on protein interactions in an exemplary formulation containing trehalose, mannitol, and various collagenases. [Figure 2A] 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]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 3A-3D] 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show the different 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] 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show the different 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). [Fig. 3I-3L]3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show the different 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] 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show the different 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). [Fig. 3Q-3R] 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, 3Q, and 3R show the different 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). [Diagram 5] 5A and 5B show images of cakes from various lyophilized formulations under pressures of 128 μbar (FIG. 5A) and 4000 μbar (FIG. 5B). [Figure 6] FIG. 6 shows the moisture content over time of various lyophilized formulations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The disclosed formulations may be more readily understood by reference to the following detailed description in conjunction with the accompanying figures, which form a part of this disclosure: It is to be understood that the disclosed formulations are not limited to the specific formulations described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example, and is not intended to limit the claimed formulations.

[0012] Unless otherwise expressly stated, any statements regarding possible mechanisms, modes of action, or reasons for improvement are for illustrative purposes only, and the disclosed formulations are not limited to the correctness or incorrectness of any such suggested mechanisms, modes of action, or reasons for improvement.

[0013] In the present text, formulations and methods for preparing formulations are described.When the present disclosure describes or claims features or embodiments related to formulations, such features or embodiments also apply to the methods for forming formulations.Similarly, when the present disclosure describes or claims features or embodiments related to the methods for forming formulations, such features or embodiments also apply to formulations.

[0014] When a range of numerical values ​​is described or established herein, the range includes its endpoints and all individual integers and fractions within the range, and also includes each of the smaller ranges formed by all possible combinations of the endpoints and the integers and fractions within, forming a subgroup of the larger group of values ​​within the described range to the same extent that each of the smaller ranges is explicitly described. When a range of numerical values ​​is described herein as being larger than the stated value, the range is nevertheless finite and bounded at its upper end by a value operable within the context of the invention described herein. When a range of numerical values ​​is described herein as being smaller than the stated value, the range is nevertheless bounded at its lower end by a non-zero value. It is not intended that the scope of the present disclosure be limited to the specific values ​​described in defining the range. All ranges are inclusive and combinable.

[0015] When values ​​are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. Reference to a particular value includes at least that particular value unless the context clearly dictates otherwise.

[0016] It will be understood that certain features of the disclosed formulations that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed formulations that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0017] As used herein, the singular forms "a," "an," and "the" include the plurals.

[0018] In this specification and claims, various terms are used in connection with aspects of this specification. Such terms are intended to have their ordinary meaning in the art unless otherwise specified. Other specifically defined terms are to be interpreted in a manner consistent with the definitions set forth herein.

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

[0020] Collagenase: Clostridium histolyticum (CCH), United States Pharmacopeia (USP), Nephelometric Turbidity Units (NTU), Polysorbate (PS), Hydrogen peroxide (H2O2), etc. are used as abbreviations.

[0021] Provided herein are formulations comprising or consisting of: Collagenase, About 30 mM to about 240 mM disaccharides; About 50 mM to about 800 mM mannitol, and About 6 mM to about 10 mM Tris-HCl.

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

[0023] As used herein, "collagenase" refers to any of the following: (a) a collagenase (including variants) having activity as defined in EC 3.4.24.3 (www.brenda-enzymes.org / enzyme.php?ecno=3.4.24.3 (accessed July 3, 2019); (b) a collagenase produced by fermentation of Clostridium histolyticum (also known as Hathewaya histolytica); (c) a CCH (as described herein); (d) a collagenase having at least 50% sequence identity with collagenase I (also known as class I collagenase) as determined by BLAST; (e) a collagenase having at least 50% sequence alignment with collagenase II (also known as class II collagenase) as determined by BLAST; (f) a collagenase produced by fermentation of other source organisms (i.e., non-Clostridium histolyticum), e.g. mammalian, crustacean, fungal, bacterial or microbial collagenases, (g) collagenases obtained by recombinant techniques, (h) collagenases having a molecular weight of about 65 kDa to about 130 kDa, (i) collagenases designated as collagenase I or collagenase II, (j) mixtures of collagenases I and II, (k) collagenases from JCM 1403 strain (ATCC 19401) or derivatives thereof, (l) collagenases from ATCC 21000 strain or derivatives thereof, (m) collagenases from ATCC 69334 or derivatives thereof, (n) collagenases from C. perfringens, (o) collagenases from Vibrio alginolyticus, (p) collagenases from Streptomyces, (q) collagenases from Pseudomonas, (r) collagenases from Achromobacter iophagus collagenase, (s) collagenases described by Worthington Biochemical Corp. (www.Worthington-biochem.com; "Product Highlights"); (t) collagenases described by Sigma-Aldrich (www.sigma-aldrich.com); (u) collagenases having one or more of the following characteristics: ·Vmax (min -1) is approximately 0.08 to 7.70 (SRC assay) or approximately 0.3 to 30.5 (GPA assay). ·K M , about 4.1 to 410 nM (SRC assay), or about 0.03 to 3.1 mM (GPA assay) ·K cat (sec -1 ) is about 1.1 to 107 (SRC assay) or about 93 to 9,179 (GPA assay) 1 / K cat , about 376–37,222 (SRC assay), or about 4–428 (GPA assay) microseconds, or ·K cat / K M , mM -1 sec -1 is approximately 5,140 to 508,814 (SRC assay) or approximately 60 to 5,934 (GPA assay), (v) collagenase as described by Nordmark Arzneimittel GmbH & Co.KG, (w) collagenase from strain 004, or (x) equivalents of any of the foregoing or some combination thereof. Non-limiting examples of collagenases that can be used in the present disclosure 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, including suitable collagenase I, for example, collagenase I that comprises 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, collagenase I comprises the amino acid sequence of SEQ ID NO: 1.

[0025] In some embodiments, the collagenase can include collagenase II. Suitable collagenase II includes, for example, collagenase II that comprises 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:2. In some embodiments, collagenase II consists of the amino acid sequence of SEQ ID NO:2. [Table 1] TIFF2025016453000003.tif65141

[0026] In some embodiments, the collagenase can comprise a mixture of collagenase I and collagenase II. The collagenase can comprise, for example, a mixture of collagenase I comprising 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 and collagenase II comprising 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:2. In some aspects, the collagenase comprises a mixture of collagenase I comprising the amino acid sequence of SEQ ID NO:1 and collagenase II comprising the amino acid sequence of SEQ ID NO: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 can include collagenase Clostridium histolyticum (CCH). "CCH" as used herein refers to collagenase Clostridium histolyticum, which includes a mixture of collagenase I (SEQ ID NO: 1) and collagenase II (SEQ ID NO: 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: -Stabilize proteins, Protects proteins during both freezing and dehydration - Suppresses unfolding caused by freeze-drying are non-reducing, and / or -Tends to become amorphous when freeze-dried.

[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] The disaccharides may be present at a concentration of about 30 mM to about 240 mM, about 60 mM to about 240 mM, about 90 mM to about 240 mM, about 120 mM to about 240 mM, about 150 mM to about 240 mM, about 180 mM to about 240 mM, about 210 mM to about 240 mM, about 30 mM to about 210 mM, about 30 mM to about 180 mM, about 30 mM to about 150 mM, about 30 mM to about 120 mM, about 30 mM to about 90 mM, or about 30 mM to about 60 mM. The disaccharide may be present at a concentration of about 30 mM, about 60 mM, about 90 mM, about 120 mM, about 150 mM, about 180 mM, about 210 mM, or about 240 mM.

[0031] Mannitol is about 50 mM to about 800 mM, about 100 mM to about 800 mM, about 150 mM to about 800 mM, about 200 mM to about 800 mM, about 250 mM to about 800 mM, about 300 mM to about 800 mM, about 350 mM to about 800 mM, about 400 mM to about 800 mM, about 450 mM to about 800 mM, about 500 mM to about 800 mM, about 550 mM to about 800 mM, about 600 mM to about 800 mM, about 650 mM to about 800 mM, about 700 mM to about 800 mM , about 750 mM to about 800 mM, about 50 mM to about 750 mM, about 50 mM to about 700 mM, about 50 mM to about 650 mM, about 50 mM to about 600 mM, about 50 mM to about 550 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 150 mM, or about 50 mM to about 100 mM. Mannitol can be present at a concentration of about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 225 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, or about 800 mM.

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

[0033] The formulation comprises: CCH, about 60 mM sucrose, about 225 mM mannitol, and about 10 mM Tris-HCl, said formulation having a pH of about 8.5.The formulation comprises: about 0.9 mg CCH / ml, about 60 mM sucrose, about 225 mM mannitol, and about 10 mM Tris-HCl, said formulation having a pH of about 8.5.

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

[0035] The disclosed formulations can further include a surfactant. Suitable surfactants include, for example, polysorbate 20, polysorbate 80, or poloxamer 188. The surfactant is about 0.01% to about 2%, about 0.05% to about 2%, about 0.1% to about 2%, about 0.15% to about 2%, about 0.2% to about 2%, about 0.25% to about 2%, about 0.3% to about 2%, about 0.4% to about 2%, about 0.5% to about 2%, about 1% to about 2%, about 1.5% to about 2%, about 0.01% to about 1.5%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.01% to about 0.1%, or about 0.01% to about 0.05%. The surfactant may be present at a concentration 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 formulations may be liquid.

[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 less time, at higher pressures, and / or with fewer drying steps (e.g., drying at a single temperature), resulting in lyophilized formulations with improved stability and that maintain acceptable collagenase activity upon reconstitution. As indicated herein, the pH and mannitol lead to the formation of more robust formulations that can then be subjected to more aggressive lyophilization.

[0038] Also provided herein are lyophilized formulations. The lyophilized formulations can be formed by lyophilizing any of the above formulations. In some embodiments, the lyophilized formulations comprise the following: Collagenase, Disaccharides, Mannitol, and Tris-HCl, or consisting of:

[0039] The lyophilized formulation may contain about 0.2 mg to about 50 mg of collagenase. For example, the lyophilized formulation may contain about 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] The collagenase may include collagenase I. Suitable collagenase I includes, for example, collagenase I that comprises 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 embodiments, collagenase I comprises the amino acid sequence of SEQ ID NO: 1.

[0041] The collagenase may include collagenase II. Suitable collagenase II includes, for example, collagenase II that comprises 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:2. In some embodiments, collagenase II comprises the amino acid sequence of SEQ ID NO:2.

[0042] The collagenase may comprise a mixture of collagenase I and collagenase II. The collagenase may comprise, for example, a mixture of collagenase I comprising 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 and collagenase II comprising 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:2. In some embodiments, the collagenase comprises a mixture of collagenase I comprising the amino acid sequence of SEQ ID NO:1 and collagenase II comprising the amino acid sequence of SEQ ID NO: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] 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.

[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. In some embodiments, the lyophilized formulation comprises or consists of collagenase, trehalose, mannitol, and Tris-HCl.

[0045] The lyophilized formulation can be placed in a unit dose vial, a multi-dose vial, a cartridge, or a syringe. The lyophilized formulation can contain about 0.2 mg to about 50 mg of collagenase. For example, the lyophilized formulation can contain about 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. The vial, cartridge, or syringe can have a volume of 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. The vial, cartridge, or syringe may contain about 0.2 mg to about 50 mg of collagenase. For example, the vial, cartridge, or syringe may contain about 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. The vial, cartridge, or syringe may contain about 0.2 mg to about 50 mg of the lyophilized formulation. For example, a vial, cartridge, or syringe may contain about 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 the lyophilized formulation.

[0046] The formulation before lyophilization comprises or consists of collagenase, about 30 mM to about 240 mM disaccharides, about 50 mM to about 800 mM mannitol, and about 6 mM to about 10 mM Tris-HCl. The formulation before lyophilization comprises or consists of about 0.9 mg collagenase / ml, about 30 mM to about 240 mM disaccharides, about 50 mM to about 800 mM mannitol, and about 6 mM to about 10 mM Tris-HCl. Prior to lyophilization, the formulation comprises or consists of CCH, 60 mM sucrose, 225 mM mannitol, 10 mM Tris-HCl, and a pH of about 8.5. In some embodiments, prior to lyophilization, the formulation can comprise or consist of about 0.9 mg CCH / ml, 60 mM sucrose, 225 mM mannitol, 10 mM Tris-HCl, and has a pH of about 8.5.

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

[0048] The disclosed lyophilized formulations have improved shelf life and storage conditions compared to conventional collagenase-containing formulations. For example, the disclosed lyophilized formulations exhibit 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 formulations can be stable, for example, in the following locations: (a) at least 36 months at 2 to 8°C; (b) at least 36 months at 25°C / 60% relative humidity; (c) at least 6 months at 40°C / 75% relative humidity, or (d) Any combination of (a) through (c).

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

[0050] It is shown that the lyophilized formulation can be formed using a single-temperature freezing step and a single-temperature drying step. For example, the lyophilized formulation can be formed by a method including freezing the formulation at a single temperature between about -25°C and -55°C to form a frozen formulation, and drying the frozen formulation at a single temperature between about 25°C and about 50°C to form a lyophilized formulation. The single-temperature freezing step can be performed at a temperature between about -25°C and about -55°C. For example, the single-temperature freezing step can be performed at about -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, or -55°C. The single-temperature drying step can be performed at a temperature between about 25°C and about 50°C. For example, the single-temperature drying step can be performed at about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, or about 50°C. When the method is carried out with a single temperature freezing step and a single temperature drying step, the method may further include a "ramp-up" step between freezing and drying for the freeze-dryer to reach a suitable 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 that is 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 hours to about 25 hours.

[0052] The disclosed lyophilized formulations can be formed by lyophilization methods that use much higher pressures than lyophilization methods used to form other collagenase-containing lyophilized formulations. The lyophilized formulations can be formed by lyophilization methods that are performed at pressures between about 380 μbar and about 4000 μbar. The disclosed methods can be performed at pressures between about 500 μbar and about 4000 μbar, between about 750 μbar and about 4000 μbar, between about 1000 μbar and about 4000 μbar. The disclosed methods 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.

[0053] The disclosed lyophilized formulations, once reconstituted, can be used to treat or reduce the severity of collagen-mediated conditions such as cellulite (also known as edematous fibrosclerotic plaque (EFP)), Dupuytren's contracture (DC) with palpable cords, or Peyronie's disease (PD) with palpable plaques and curve deformity of 30 degrees or more.

[0054] Also provided herein is a reconstituted formulation comprising or consisting of collagenase, a disaccharide, mannitol, Tris-HCl, calcium chloride, and sodium chloride.

[0055] The collagenase can include collagenase I, with suitable collagenase I including, for example, collagenase I including 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 embodiments, collagenase I includes the amino acid sequence of SEQ ID NO:1. The collagenase can include collagenase II. Suitable collagenase II includes, for example, collagenase II including 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:2. In some embodiments, collagenase II includes the amino acid sequence of SEQ ID NO:2. The collagenase can include a mixture of collagenase I and collagenase II. The collagenase can include, for example, a mixture of collagenase I that includes 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 and collagenase II that includes 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: 2. In some embodiments, the collagenase includes a mixture of collagenase I that includes the amino acid sequence of SEQ ID NO: 1 and collagenase II that includes the amino acid sequence of SEQ ID NO: 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] Each of collagenase I and collagenase II may be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure 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] Suitable amounts of calcium chloride and sodium chloride include those that allow the reconstituted formulation to be isotonic with human blood. In some embodiments, the reconstituted formulation comprises 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 formulation comprises 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] The reconstituted formulation may also include water for injection (WFI).

[0060] The disclosed reconstituted formulations can be used to treat or reduce the severity of collagen-mediated conditions, including cellulite (also known as edematous fibrosclerotic plaque (EFP)), Dupuytren's contracture (DC) with palpable cords, or Peyronie's disease (PD) with palpable plaques and a curve deformity of 30 degrees or more.

[0061] The reconstituted formulation may contain, in a single or divided dose, about 0.01 mg to about 50 mg of collagenase. The reconstituted formulation may contain, for example, about 0.05 mg to about 15 mg, about 0.10 mg to about 10 mg, about 0.15 mg to about 5 mg, about 0.20 mg to about 3 mg, or about 0.25 mg to about 2 mg of collagenase in a single or divided dose. Reconstituted formulations may include, for example, about 0.05 mg, about 0.10 mg, about 0.15 mg, about 0.20 mg, about 0.25 mg, about 0.30 mg, about 0.35 mg, about 0.40 mg, about 0.45 mg, about 0.50 mg, about 0.55 mg, about 0.60 mg, about 0.65 mg, about 0.70 mg, about 0.75 mg, about 0.80 mg, about 0.85 mg, about 0.90 mg, about 0.95 mg, about 0.05 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 55 mg, about 56 mg, about 57 mg, about 58 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, About 5.25 mg, about 5.30 mg, about 5.35 mg, about 5.40 mg, about 5.45 mg, about 5.50 mg, about 5.55 mg, about 5.60 mg, about 5.65 mg, about 5.70 mg, about 5.75 mg, about 5.80 mg, about 5.85 mg, about 5.90 mg, about 5.95 mg, about 6.00 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50 mg of collagenase.

[0062] The reconstituted formulation can have a total volume of about 0.1 mL to about 50 mL. For example, the reconstituted formulation can have a total volume of about 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] Kits containing the disclosed lyophilized formulations and a sterile diluent are also provided. The kits can include a container containing any of the disclosed lyophilized formulations and a container containing a sterile diluent that includes calcium chloride and sodium chloride.

[0064] Suitable containers for the lyophilized formulation and / or the sterile diluent include, for example, vials, cartridges, or syringes. The vials can be unit dose vials or multi-dose vials. Suitable container sizes include, for example, containers of 2mL to 50mL, such as 5mL, 7.5mL, 10mL, 15mL, 20mL, 30mL, 40mL, or 50mL.

[0065] The container containing the disclosed lyophilized formulation may contain about 0.2 mg to about 50 mg of collagenase. For example, the container may contain about 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. The container containing the disclosed lyophilized formulation may contain about 0.2 mg to about 50 mg of the lyophilized formulation. For example, the container may contain about 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 the lyophilized formulation.

[0066] The container containing the sterile diluent may contain an amount of the sterile diluent that, upon reconstitution of the lyophilized formulation, results in a solution that is isotonic with human blood. In some embodiments, the sterile diluent comprises 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 comprises 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 volume of the sterilized diluent can be about 0.1 mL to about 50 mL, for example, 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] Provided herein is a method of lyophilizing any of the disclosed formulations, the method comprising freezing the formulation at a temperature between about -25°C and -55°C to form a frozen formulation, and drying the frozen formulation at a temperature between about 25°C and about 50°C to form a lyophilized formulation.

[0069] In some embodiments, the freezing is performed at a single temperature and the 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 the appropriate drying temperature. The single temperature freezing step may be performed at a temperature between about -25°C and about -55°C. For example, the single temperature freezing step may be performed at about -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, or -55°C. The single temperature drying step may be performed at a temperature between about 25°C and about 50°C. For example, the single temperature drying step may be performed at about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, or about 50°C.

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

[0071] The disclosed methods can be performed at pressures between about 128 μbar and about 4000 μbar, between about 380 μbar and about 4000 μbar, between about 500 μbar and about 4000 μbar, between about 750 μbar and about 4000 μbar, between about 1000 μbar and about 4000 μbar. The disclosed methods 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, but not to limit, the disclosed embodiments.

[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 reduce cycle time and improve yield.

[0074] In optimizing the formulation and the lyophilization process, the following criteria were considered: formulation: - pH that optimizes the thermodynamic stability of the protein, Trehalose or sucrose as a cryoprotectant / cryoprotectant to stabilize proteins, Addition of bulking / isotonic agents (e.g. mannitol), and · If necessary, use a non-ionic detergent to reduce protein aggregation. Freeze-drying process: -Prevents protein unfolding during freezing and drying. The glass transition temperature of the product exceeds the intended storage temperature; Ability to maintain a relatively low moisture content, and Elegant cake structure.

[0075] As part of identifying the optimal formulation for lyophilization, several solution formulations ("pre-lyophilization") were evaluated that contained various combinations of excipients, including sucrose, trehalose, and mannitol, and different levels of surfactants, as well as untreated and siliconized (baked) vials.

[0076] Formulation Robustness Study – Study 1 The objective of these studies was to challenge two formulation variants with freeze / thaw testing, shear stress, heat stress, peroxide stress, and hydrophobic surface contact in comparison to the original XIAFLEX® formulation. The following formulations were subjected to heat / 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® bulk drug formulation (Or): CCH, 10 mM Tris / HCl pH 8.0, 60 mM sucrose

[0077] Standard and siliconized glass vials were used in the study to investigate the effect of hydrophobic surfaces on protein stability. Two surfactants (polysorbate 20 and poloxamer 188) were added to the formulations to investigate their effect on the formulation stability during challenge testing. Also, tests were performed in the presence of a strong oxidizing agent (hydrogen peroxide) to enforce oxidative stress. In total, 21 different matrices were used in the challenge testing.

[0078] Experimental design CCH was formulated in three different formulation variants. To challenge the candidate formulations, the formulation variants were subjected to heat stress with agitation and freeze / thaw stress. Hydrogen peroxide was added to the samples to subject the protein to oxidative stress. To monitor aggregate formation, each sample after stress exposure was analyzed by turbidity measurements.

[0079] Processing of glass vials and stoppers The vials were washed with pure water in a laboratory dishwasher. Afterwards, the vials were dried and depyrogenated / sterilized by heat treatment at 300 °C for 2 h. The stoppers were autoclaved in sterilization bags at 2 bar and 121 °C for 20 min and dried at 80 °C for 8 h.

[0080] Sample preparation To achieve quantitative buffer exchange, dialysis was performed in three independent dialysis steps. 60 ml of XIAFLEX® drug substance was dialyzed against formulation variant A and 40 ml of XIAFLEX® drug substance was dialyzed against formulation variant B. XIAFLEX® drug substance was transferred to two preconditioned Slide-A-Lyzer® cassettes (Thermo Scientific, Rockford, USA). The loaded Slide-A-Lyzer® cassettes were incubated with 2000 ml or 1000 ml of target buffer for 2 hours before the first buffer exchange (2000 ml / 1000 ml). After another 2 hours of dialysis, a 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 adjust the concentration to 1 mg / ml.

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

[0082] Each formulation was sterile 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. The liquid samples were frozen from 25°C to -30°C within 55 minutes at a controlled freezing rate and warmed again to room temperature within 55 minutes at a 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 measurements were performed by placing 1 ml of each sample into a single-use turbidity cuvette. After analysis, the liquid was transferred back into the glass vial and the experiment continued.

[0084] Thermal Stress Test Test samples of all formulations were placed in 2R vials (filling volume 1 ml) and stressed for 4 days at 40° C. under stirring (200 rpm), after which the turbidity of the stressed samples was analyzed.

[0085] Turbidity measurement The turbidity of the samples was measured according to the European Pharmacopoeia using a 2100AN turbidimeter (Hach Lange, Dusseldorf, Germany). 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. The solution was left to stand for 4 to 6 hours. · Hexamethylenetetramine solution: 2.5 g of hexamethylenetetramine was dissolved in 25.0 ml of pure water and placed in a 100 ml glass volumetric flask. Primary Opalescent Suspension: 25.0 ml of the hydrazine sulfate solution was added to the hexamethylenetetramine solution in a volumetric flask. Mix and allow to stand for 24 hours. Milky standard: 15.0 ml of the primary milky suspension was diluted to 1000.0 ml with purified water. This suspension was freshly prepared (stored for a maximum of 24 hours). Reference Suspension According to Table 3, a reference suspension was prepared. [Table 3]

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

[0087] Table 5 shows the results of heat stress at 40°C and agitation at 200 rpm. In the variant containing hydrogen peroxide, a clear increase in turbidity was observed after heat stress (indicated by an increase in Nephelometric Turbidity Units (NTU)). A and variant V B (both formulations contained mannitol) resulted in significantly lower turbidity (reduced NTU) than the original XIAFLEX® formulation variant ("Or" - no mannitol). The beneficial effect of mannitol is believed to be due to its radical scavenging properties. [Table 4] [Table 5]

[0088] conclusion The increase in turbidity is V A and V B This is probably because the intermolecular repulsion is stronger at pH 8.5 than at pH 8.0, which reduces the tendency for aggregate formation due to oxidative stress.

[0089] The addition of surfactants did not affect sample turbidity, either positively or negatively. The hydrophobic glass vial surface did not affect sample turbidity after heat stress.

[0090] To monitor the formation of aggregates, samples were analyzed by turbidity measurements. Sample turbidity did not increase with freeze / thaw cycles. The formulation appeared to be stable against aggregate formation even under freeze / thaw stress (up to 3 cycles). Hydrophobic surface contact or addition of hydrogen peroxide, with or without detergent, did not affect the turbidity of samples under freeze / thaw stress. Under thermal stress, a clear increase in turbidity (measured in NTU) was observed under forced oxidative stress (addition of hydrogen peroxide). In variants containing mannitol and with higher intermolecular repulsion, the increase in turbidity was less pronounced even under forced oxidative stress. The stability of the formulations against oxidative stress was highest in the following order: variant A > variant B > original XIAFLEX® formulation. The presence of detergents under forced oxidative stress did not affect the turbidity of the samples. In the absence of oxidative stress, all samples remained transparent even under heat / agitation stress. Hydrophobic surface contact did not affect the turbidity of samples, with or without detergent, under heat / agitation stress.

[0091] Formulation Robustness Study – Study 2 Twelve formulations with different pH values ​​and mannitol concentrations were investigated by CG-MALS and nanoDSC. In addition, each variant was subjected to thermal stress by stirring and freeze-thaw stress tests. Also, 0.1% H2O2 was added to reduce oxidative stress. The objectives of this study are as follows: · To clarify the molecular interactions between collagenase I and collagenase II in various formulations using CG-MALS and nano-DSC. Test the stability of XIAFLEX® and additional formulation variants against freeze / thaw and heat stress.

[0092] The following CCH formulations were prepared and tested: [Table 6]

[0093] Collagenase I and collagenase II were dialyzed against the corresponding formulation buffer. After dialysis, 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 investigated by stress tests (freeze / thaw and heat / shear stress). To enforce oxidative stress during the stability tests, each formulation was examined in the presence and absence of 0.1% H2O2 (the variant without hydrogen peroxide is shown in a, and the variant with hydrogen peroxide is shown in b).

[0094] dialysis Dialysis of collagenase I and collagenase II was performed in three independent dialysis steps to achieve quantitative buffer exchange. 14 ml of intermediate collagenase I sample and 14 ml of intermediate collagenase II sample were transferred to two preconditioned (in dialysis buffer) Slide-A-Lyzer® cassettes (Thermo Scientific, Rockford, USA). The loaded Slide-A-Lyzer® cassettes were incubated for 2 h in 1000 ml of target buffer before the first buffer exchange (1000 ml) was performed. After another 2 h of dialysis, a second buffer exchange (1000 ml) was performed, finally terminating the dialysis overnight. The protein samples were removed from the Slide-A-Lyzer® cassettes and further processed. Each dialysis step represented a 1 / 35-fold buffer exchange, resulting in a calculated buffer exchange factor of approximately 2 × 10 in total. 5 It becomes.

[0095] Nano Differential Scanning Calorimetry Differential scanning calorimetry (DSC) is a technique used to assess the stability of proteins in their native form by measuring the thermal changes associated with the thermal denaturation of molecules when heated at a constant rate. Proteins in solution are in equilibrium between their native (folded) and denatured (unfolded) conformations. When heated, native proteins undergo unfolding (thermal denaturation) at a characteristic temperature (T onset ). The more intrinsically stable a biopolymer is, the higher the onset temperature of the unfolding transition. DSC measures the enthalpy of unfolding resulting from thermal denaturation and can elucidate factors that contribute to the folding and stability of native biomolecules. These include hydrophobic interactions, hydrogen bonding, conformational entropy, and physical environment. The following experimental methods were performed: Mode: Scan Temperature parameters: Lower limit 20℃ Upper limit: 100℃ Rate: 1℃ / min Heating / Cooling Equilibration: 600s Pressure parameters: Manual, 3.0 atmospheres Data interval: 1s

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

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

[0098] CG-MALS The interactions between protein molecules in solution were characterized by analyzing the change in light scattering behavior at different concentrations by calculating the second virial coefficient (A2). A2 is an index of protein-protein interactions in solution. Negative values ​​of A2 indicate attractive protein-protein interactions, while positive values ​​indicate repulsive protein interactions. When A2 values ​​are negative, the protein solution is "colloidally unstable". Higher A2 values ​​indicate greater repulsion, which indicates less protein interaction, less chance of protein aggregation, and greater stability. The A2 values ​​of various formulations were measured using CG-MALS as an index 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 macromolecules are observed at Mw app The calculation of A2 was performed via Zimm plot analysis by extrapolating to a concentration of 0 mg / ml according to the following formula I:

number

[0100] Sample preparation The dialyzed samples were used as is for CG-MALS measurements. The corresponding dialysis buffer was used as a diluent in the CG-MALS experiments. Samples and buffers were passed through a 0.1 μm filter. Before loading the samples into the CG-MALS system, the exact concentration of the samples was determined by UV absorption measurements. This concentration was used to calculate the concentration of each gradient step.

[0101] Sample measurement A Calypso II CG-MALS system was used to deliver a concentration gradient of the analytes to the MALS detector. The samples were loaded into syringe pump 1 and syringe pump 2 of the system, and the dialysis buffer was loaded into 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% to measure the self-virial coefficient of sample 1 in the formulation. In the second step, a crossover gradient was performed in which the concentration of sample 1 was decreased from 90% to 10% and the concentration of sample 2 was increased from 10% to 90%. This step was performed to determine the cross-virial coefficient. In the third step, a concentration gradient was performed in which the concentration of sample 2 was increased from 100% to 10% to measure the self-virial coefficient of sample 2 in the formulation. For each gradient step, 0.7 ml of sample was injected into the MALS detector. The resulting light scattering signal was recorded for a period of 180 seconds. Multicomponent Zimm plot analysis with fixed molecular weights was performed using Calypso software version 2.1.5.

[0102] UV measurement The concentrations of collagenase I and collagenase II in the solutions were measured using an 8452A UV spectrometer (Agilent Technologies, Santa Clara, USA). Samples were measured in plastic cuvettes with a light path thickness of 0.2 cm at a concentration of approximately 3 mg / ml. The concentrations were 1.52 ml / (mg * cm), and 1.48 ml / (mg) for collagenase II. *The extinction coefficient was 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 represent a solution containing 0.5 mg / ml collagenase I and 0.5 mg / ml collagenase II.

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

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

[0106] Turbidity measurement The turbidity of the samples was measured as described above.

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

[0108] Colloidal stability-The pH value of the formulation showed a strong effect on the colloidal stability of collagenase I, collagenase II, and their mixtures. 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 attractive interactions 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 of the onset temperature of unfolding. It showed 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 was independent of the pH values ​​investigated. The presence of mannitol seemed to be beneficial for the thermodynamic stability of collagenase II. [Table 7]

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

[0111] None of the formulations showed an increase in turbidity after the freeze / thaw test. The presence of hydrogen peroxide was well tolerated 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, the turbidity was constant for all formulation variants investigated. In the presence of hydrogen peroxide, the turbidity of the samples increased strongly, indicating that oxidative stress induces the formation of aggregates. A basic solution at pH 8.5 stabilized the protein against aggregation due to oxidative stress. Mannitol showed an additional beneficial effect on the turbidity of the samples after thermal stress in the presence of hydrogen peroxide. It is believed that mannitol acts as a radical scavenger and improves the stability of the protein against oxidative stress.

[0114] Formulation Robustness Study – Study 3 In the studies below, 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 tables below. As part of the formulation optimization, the effect of pH prior to lyophilization of the various formulations was evaluated.

[0115] The solution robustness of the proposed formulation 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] The stressed samples were then examined for turbidity and the main quality attributes of the product were evaluated by the following methods: UV A280 Protein concentration by Mass and ratio of collagenase I and collagenase II by reversed-phase high-performance liquid chromatography (as described herein) - Soluble rat tail collagen (SRC) enzyme activity assay (collagenase I; described herein) Glycyl-L-prolyl-alanine (GPA) enzyme activity assay (collagenase II: as described herein)

[0117] result TurbidityData obtained from freeze / thaw cycles are 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, indicating that the excipients tested are suitable for further evaluation.

[0119] Data on exposure to shaking and high temperatures are shown in Table 11. [Table 11]

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

[0121] In a second study, the effect of different concentrations of polysorbate 20 and high pH (8.8) on the turbidity of the product under stress conditions was evaluated. The results of this study 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) investigated do not adversely affect protein aggregation, resulting in consistent turbidity values ​​upon freeze / thaw cycles and heat / shake. These results suggest that thermodynamically stable formulations are possible and that polysorbate 20 can be used as a surfactant in the concentration range investigated. Furthermore, it was also shown that the use of trehalose and mannitol alone or in combination does not adversely affect protein aggregation as measured by sample turbidity.

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

[0124] These data confirm that formulation modification at various pH levels with the three excipients tested did not adversely affect protein stability / concentration and enzyme activity, making all of these components suitable for further evaluation in lyophilized formulations, and did not adversely affect product quality over the range of pH values ​​tested.

[0125] Formulation Robustness Study – Study 4 The various formulations were also characterized using composition gradient multi-angle light scattering (CG-MALS) to assess self- and hetero-protein interactions, and nano-differential scanning calorimetry (DSC) to assess protein unfolding. CG-MALS and nanoDSC were performed as described above.

[0126] A study was also performed to determine whether hydrogen peroxide, as an oxidizing agent, had any effect on the investigated formulation (pre-lyophilized solution) based on the turbidity values.

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

[0128] For the formulations containing trehalose, the most repulsive interactions within and between proteins (collagenase I and collagenase II) were observed at pH 8.5, as shown in FIG.

[0129] Trehalose, sucrose, and mannitol were also candidates for suitable formulation ingredients since they did not adversely affect the repulsive force.

[0130] Nano-DSC - The variables (pH, excipient type and concentration) investigated using Nano-DSC to assess protein unfolding are shown in Table 16. [Table 16]

[0131] As a result, the various formulation components examined showed a high protein unfolding temperature (T onset ) and protein thawing temperature (T m ) was shown to have no effect on T onsetの The data showed that a temperature of 40°C could be successfully utilized in the secondary drying step of the lyophilization process without affecting protein stability.

[0132] Hydrogen Peroxide Challenge-The formulations were challenged with hydrogen peroxide in a short-term study (Figure 2A and Figure 2B). Trehalose-containing formulations (with or without mannitol) and sucrose-containing formulations (with or without mannitol) showed significantly lower turbidity at pH 8.5 after exposure to hydrogen peroxide compared to the same formulations at pH 8.0 and pH 7.5. Lower turbidity values ​​indicate stable formulations with less protein aggregation. These results are consistent with the higher A2 values ​​(higher repulsive interactions) seen in the pH 8.5 formulations, indicating potential for improved stability. It was also shown that upon exposure to hydrogen peroxide, formulations containing trehalose and mannitol with or without polysorbates (pH 8.0 or 8.5) showed significantly lower turbidity compared to the formulation containing sucrose without mannitol (pH 8.0). The presence of surfactants did not affect turbidity positively or negatively.

[0133] conclusion Based on the results of the formulation optimization work, which identified the optimal excipients in terms of quality and quantity, the following formulations (pre-lyophilization) were advanced for lyophilization cycle evaluation: CCH and sucrose mixed with mannitol (with or without polysorbate 20) at pH 8.5 CCH and trehalose mixed with mannitol (with or without polysorbate 20) at pH 8.5

[0134] In future analyses, the lowest concentration of polysorbate 20 (0.02%) was used because higher concentrations of polysorbate 20 were ineffective.

[0135] Lyophilization cycle development for both formulations was performed in 5 mL vials. Table 17 provides details of the formulations evaluated. [Table 17]

[0136] Optimization of the freeze-drying cycle - Study 1 Varying mannitol concentration The aim of this work was to investigate the effect of varying the ratio of mannitol to a fixed concentration of sucrose, in the absence of surfactant, on the freeze-drying process and cycle time. The following experimental variants were prepared along with a placebo Lyo sample: [Table 18]

[0137] A medium freeze-drying cycle ("Lyo cycle") was used to freeze-dry the product (targeting a total cycle time of approximately 36 hours). The information for the pilot freeze-dryer used in this study is as follows: ·Manufacturer: Hof Sonderanlagenbau(Lohra,Germany) Shelf area 0.5m 2 Ice storage capacity: 10kg Adjustable ice condenser temperature In-vial temperature record Differential pressure measurement -Connectable radiation cage

[0138] Preparing packaging materials The freeze-dried stoppers were autoclaved at 121 °C for 15 min and dried at 105 °C for 8 h. The vials were washed with pure water and dehydrated at 300 °C for 2 h.

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

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

[0141] Freeze drying The vials were filled with lyophilization stoppers in the "Lyo-position" vials and loaded into a pilot lyophilizer. Approximately 125 vials of each formulation and 100 placebo vials of Vb were loaded. The lyophilization cycle was run with the following parameters: ·Freezing temperature (shelf): -50℃ ·Primary drying temperature (shelf): -10℃ ·Secondary drying temperature (shelf): 40℃ Pressure: 0.25mbar Accumulation time: Approx. 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 to control the pressure during freeze-drying. Pressure regulation was controlled by vacuum and a dosing valve (nitrogen injection). The vial was closed at 750 mbar pressure under nitrogen atmosphere.

[0143] Karl Fischer titration The contents of one vial of the corresponding lyophilisate were weighed into a glass vial sealed with a crimp cap. The sample was transferred to the oven of a Karl Fischer coulometer (756 / 774; Metrohm) heated to 100 °C. The septum of the cap was pierced with a syringe needle and the evolved water vapour was delivered directly into the titration chamber of the Karl Fischer coulometer via dry nitrogen. The measurements were repeated once. An empty glass vial was used for blank correction.

[0144] Analysis of water of crystallization The Oven Sample Processor 774 allows a unique temperature ramp method for Karl Fischer titration. The sample is heated at a defined heating rate and the released water is transferred directly to the titration chamber of the Karl Fischer titrator. By recording the water produced and the water drift with oven temperature (μg water / min), specific events of water release (e.g. release of water of hydration) can be detected. 50-100 mg of lyophilisate were weighed into empty 6R type 1 glass vials and closed with an Al Crimp Carb. The sample was transferred to the oven of the sample processor. There, the sample was heated by a defined temperature ramp from 50 °C to 140 °C in 45 min (2 °C / min). The temperature ramp was terminated at 140 °C to avoid undesired Maillard reactions.

[0145] exterior The lyophilisates were removed from the glass vials by carefully breaking them, and the lyophilised cakes were cut vertically and screened for any collapsed zones in their inner layers.

[0146] Scanning electron microscope The freeze-dried products were analyzed by SEM to evaluate their microstructure. The freeze-dried products were cut and the longitudinal and top and bottom surfaces were analyzed by SEM at magnifications of 50x and 150x.

[0147] Reconstruction The vials were reconstituted with 4 ml of a solution containing 0.03% calcium chloride and 0.66% sodium chloride, and the time required for the lyophilisate to completely dissolve was recorded.

[0148] Nano Differential Scanning Calorimetry Nano-DSC was performed as described above.

[0149] Sample preparation Lyophilisates and reconstituted lyophilisates were analysed at a concentration of 0.93 mg CCH / ml, and the corresponding buffers were used as buffer scans and buffer references.

[0150] result Digital data acquisition proved that the freeze-drying process occurred as intended. Sublimation of the batch was complete after approximately 15 hours of total freeze-drying time, as indicated by the Pirani and conductive pressure sensor differentials. The completion of sublimation of each sublot was indicated by a sudden increase in product temperature during primary drying.

[0151] The primary drying step was finished after approximately 17 hours of total lyophilization time. After 18 hours of secondary drying, one vial of each variant was removed from the lyophilizer and the residual moisture level was measured by Karl Fischer titration while the secondary drying step of the remaining batches was extended. Karl Fischer analysis showed that the residual moisture content was already below the desired value. Following this result, the remaining batches were immediately unloaded. The total time of secondary drying was 19 hours.

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

[0153] Reconstruction behavior -All lyophilized sublots dissolved rapidly and spontaneously within seconds (<30 sec).

[0154] Residual moistureTable 19 shows the residual water content of the samples, determined 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 comparable. The top surface of variant Va (which had the lowest amount of sucrose) 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 - Table 20 summarizes the results obtained. [Table 20]

[0157] To demonstrate the precision of the unfolding temperature and denaturation enthalpy, variants Vc (before lyophilization) were analyzed six times. Samples were obtained from a single preparation, eliminating any uncertainty in concentration. T onset and Tm results differed less than 1%, while enthalpy variations were more than 7%, which is in good agreement with the instrument specifications (TA Instruments specifies an uncertainty of 5% for the enthalpy of unfolding of lysozyme) (data not shown). It can be concluded that before and after lyophilization, all formulations were comparable within the error of the method in terms of unfolding temperature and transition enthalpy, indicating that the freeze-drying process conditions used do not adversely affect CCH. Increasing the amount of mannitol seems to increase the thermodynamic stability, as represented by slightly higher values ​​of Tm and T onset.

[0158] conclusion From these observations and all the data, it was found that lyophilized formulations of CCH could be produced with various concentrations of mannitol in the presence of sucrose in Tris buffer. This study also identified gentler lyophilization conditions to achieve 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 studies were performed in 5 cc vials using four experimentally developed formulations and the XIAFLEX® formulation as a control. These variants are shown in Table 21 below. [Table 21]

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

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

[0163] Results and Conclusions The lyophilized cakes of all formulations dissolved rapidly in diluent with reconstitution times of 10 seconds or less and moisture contents (KF) of less than 0.4%.

[0164] At shorter experimental cycle times, the XIAFLEX® cakes appeared to shrink compared to the experimental formulation variants, however, all of these formulations demonstrated well-formed and robust cakes. All experimental formulations were tested for a variety of properties, including protein concentration, collagenase I and collagenase II mass composition, and biological activity. Test results for all formulations were consistent with the XIAFLEX® formulation.

[0165] Experimental formulations #3 and #5 were selected for further testing. Informal short-term stability studies were performed with these formulations at accelerated storage conditions of 5°C and 25°C / 60% RH. Samples were analyzed according to the approved test method. All results for both formulations were consistent with historical data for the XIAFLEX® formulation, indicating that this formulation change does not adversely affect product quality (data not shown). As previously mentioned, the currently approved limits are subject to revision based on statistical analysis of experimentally derived data.

[0166] In conclusion, the development work and data conducted to date for formulation optimization indicates that the addition of the two investigated protein stabilizers (sucrose, trehalose), bulking agent (mannitol), and surfactant (polysorbate 20) did not adversely affect product quality and are suitable for further pursuit of these formulations.

[0167] Freeze-drying cycle optimization – Study 3 Pressure analysis Research purpose To facilitate the identification of optimal freeze-drying process conditions, pressure tests were performed to determine the maximum allowable pressures and other reliable process parameters during freezing, primary drying, and secondary drying at process-appropriate conditions to achieve a fast and robust freeze-drying cycle.

[0168] Experimental design Three formulations in 5 cc vials were used, as shown in Table 23. [Table 23]

[0169] To identify the pressure of the sublimation stage, two pressure tests were performed with different shelf temperatures and different pressure steps. Sample vials filled with the experimental formulation were loaded onto one freeze-dryer shelf (middle position). After freezing, the chamber pressure was set to an initial value of 128 μbar and the shelf temperature was increased to the initial value (-10 °C for test 1, +10 °C for test 2). Freeze-drying was performed for a certain time to generate a small amount of lyophilisate above the ice interface. The chamber pressure was then increased in steps (e.g. 380 μbar, 1030 μbar, etc.) and the sample vial was monitored by video for cake collapse or other visual adverse effects. Moderate energy input was performed with the shelf temperature set at -10 °C.

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

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

[0172] The results are shown in Figures 5A and 5B. No cake collapse or other defects were observed in all 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] This initial lyophilization cycle development work supported the use of mannitol in pursuit of more efficient and robust lyophilization cycles.

[0174] Optimizing the Freeze-Drying Cycle – Study 4 Further Optimization Based on the results of the compression tests described above, optimization of the lyophilization parameters was performed for experimental formulations #3 and #5 (data not shown).

[0175] Pressure tests showed that formulations containing mannitol could be lyophilized at chamber pressures up to 4 mbar without risk of structural collapse during sublimation, so in optimization studies Ryo cycles were performed at a chamber pressure of 1 mbar to take advantage of the high sublimation rate.

[0176] Two freeze cycle experiments were performed to identify the fundamental parameters for a robust and efficient freeze-drying cycle. After freeze-drying, samples were analyzed for appearance, residual moisture, physical stability, and by scanning electron microscopy (SEM).

[0177] Experiment 1 : Only experimental formulation #3 was used. experiment The results showed that higher temperatures during secondary drying were required to achieve residual moisture levels below 0.5%. Other physical attribute test results were within acceptable limits (data not shown). The freeze-drying parameters used are outlined below: ·Freezing temperature (shelf): -50℃ ·Drying temperature (sublimation / secondary drying) (shelf): 35℃ Pressure: 1mbar Accumulation time: Approx. 21.5 hours

[0178] Experiment 2 : Only experimental formulation #3 was used. experiment were used to determine suitable conditions for drying at 40°C. All results were within acceptable limits (data not shown). The freeze-drying parameters used are outlined below. ·Freezing temperature (shelf): -50℃ ·Drying temperature (sublimation / secondary drying) (shelf): 40℃ Pressure: 1mbar Accumulation time: Approx. 21.5 hours

[0179] conclusion In the freeze-drying cycle optimization study, the basic parameters for an efficient freeze-drying cycle were identified. All tests on the physical attributes of the freeze-dried cake were acceptable.

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

[0181] Long-term stability monitoring was performed on six process validation lots. Storage conditions included long-term stability at a variety of storage conditions (2-8°C; 25°C / 60% relative humidity (RH); and 40°C / 75% RH) to support the product's shelf life, as well as short-term stability studies at a variety of storage conditions to support evaluation of potential temperature changes during shipping or storage. Also included was data from an earlier formulation development lot with the same formulation, a lower fill weight (0.46 mg), and a comparable container closure system.

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

[0183] Stability batches were analyzed for appearance (before and after reconstitution), reconstitution time, osmolality, pH, UV A280 The following tests were performed: concentration by quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high performance liquid chromatography (RPHPLC) [purity], mass and ratio of components by RP-HPLC, size-exclusion high performance liquid chromatography (SEC-HPLC), collagenase I potency by soluble rat tail collagen (SRC) assay, collagenase II potency by glycyl-L-prolyl-L-alanine (GPA) assay, moisture, particulates, endotoxins, and helium leak by container sealability.

[0184] Results and Analysis An 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) are shown below.

[0185] Appearance (before and after replacement) ) - The appearance (before and after replacement) under the storage conditions tested showed no significant changes or unexpected trends (data not shown).

[0186] Reconfiguration time - Reconstitution times for the storage conditions tested showed no significant changes or unexpected trends (data not shown).

[0187] Osmolality -Osmolality results showed no significant changes or unexpected trends under the storage conditions tested (data not shown).

[0188] pH - pH results showed no significant changes or unexpected trends at the storage conditions tested (data not shown).

[0189] UV A280 Concentration by-UV under tested storage conditions A280 No significant changes or unexpected trends were observed in the enrichment results by (data not shown).

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

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

[0192] Mass and percentage composition by RP-HPLC - Composition RP-HPLC mass and ratio results showed no significant changes or unexpected trends at the storage conditions tested (data not shown).

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

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

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

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

[0197] fine particles - No significant changes or unexpected trends were observed in particulate results across the storage conditions tested (data not shown).

[0198] Container seal / helium leakage -Container tightness / helium leakage results showed no significant changes or unexpected trends (data not shown).

[0199] Photostability Test After exposure to 1.2 million lux of cool white light, the LEDs emit 200 watt-hours per square meter (W / m 2 The photostability of the CCH formulation was examined by exposure to UV light at 10.00 W / m 2. The samples were exposed to cool white light at 8.00 klux for 150 hours and then to UV light at 10.00 W / m 2. 2 UV light for 20 h. All results after this exposure were comparable to unexposed controls (data not shown).

[0200] Reconstitution stability test Several reconstitution stability studies were performed to demonstrate compatibility of the lyophilized CCH formulation with the sterile diluent used for reconstitution and to generate stability data for the reconstituted product at potential conditions of use. The lyophilized CCH formulation was reconstituted with a sterile diluent and analyzed at specific time points after storage at specific temperatures. Table 25 provides a summary of the reconstitution stability studies performed. [Table 25]

[0201] In these studies, potency was measured using the SRC and GPA test methods (data not shown). In these studies, microplate versions of the SRC and GPA test methods were used to measure potency. These test methods provide higher throughput and faster results than cuvette-based test methods. These results demonstrate the compatibility of the lyophilized CCH formulation with the sterile diluent and the stability of the reconstituted CCH formulation under potential use conditions.

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

[0203] The CCH formulation exhibited acceptable photostability as all results obtained after light exposure were comparable to the control (unexposed) samples.

[0204] After reconstitution with sterile diluent, CCH formulations exhibited acceptable stability when stored at 25°C / 60% RH for up to 24 hours and at 5°C for up to 120 hours. Reconstituted CCH formulations also exhibited acceptable stability when stored at 25°C / 60% RH for 24 hours, followed by storage at 2°C-8°C for 96 hours and then at 25°C / 60% RH for an additional 24 hours.

[0205] Future plans call for annual stability studies to be conducted that include storage at 5°C ± 3°C and 25°C ± 2°C / 60% ± 5% RH. Material at both storage conditions will be evaluated over the proposed 36-month shelf life.

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

[0207] The disclosures of each patent, patent application, and publication cited or described herein are incorporated by reference in their entirety into this Agreement.

[0208] Embodiment The following list of embodiments is intended to complement, not replace, the previous description. EMBODIMENT 1 1. A formulation comprising: Collagenase, About 30 mM to about 240 mM disaccharides; About 50 mM to about 800 mM mannitol, and About 6 mM to about 10 mM Tris-HCl; 13. A formulation comprising: EMBODIMENT 2 The formulation of embodiment 1, wherein the collagenase comprises collagenase I. EMBODIMENT 3 The formulation of embodiment 2, wherein the collagenase I comprises the amino acid sequence of SEQ ID NO:1. EMBODIMENT 4 The formulation of embodiment 1, wherein the collagenase comprises collagenase II. EMBODIMENT 5 The formulation of embodiment 4, wherein the collagenase II comprises the amino acid sequence of SEQ ID NO:2. EMBODIMENT 6 The formulation of embodiment 1, wherein the collagenase comprises a mixture of collagenase I and collagenase II. EMBODIMENT 7 The formulation of embodiment 6, wherein the collagenase I comprises the amino acid sequence of SEQ ID NO:1 and the collagenase II comprises the amino acid sequence of SEQ ID NO:2. EMBODIMENT 8 The formulation of embodiment 6 or 7, wherein the collagenase is collagenase Clostridium histolyticum (CCH). EMBODIMENT 9 9. The formulation according to any one of embodiments 1 to 8, wherein the disaccharide comprises sucrose or trehalose. EMBODIMENT 10 10. The formulation according to any one of embodiments 1-9, wherein the pH of the formulation is from about 7.8 to about 8.8. EMBODIMENT 11 A formulation according to any one of embodiments 1 to 10, wherein the formulation comprises: CCH and About 60 mM sucrose, About 225 mM mannitol, and About 10 mM Tris-HCl; The formulation has a pH of about 8.5. EMBODIMENT 12 12. The formulation of any one of embodiments 1-11, further comprising a surfactant comprising polysorbate 20, polysorbate 80, or poloxamer 188. EMBODIMENT 13 The formulation of embodiment 12, comprising from about 0.01% to about 2% of the surfactant. EMBODIMENT 14 The formulation of embodiment 13, comprising about 0.02% of said surfactant. EMBODIMENT 15 The formulation according to any one of embodiments 1 to 14, wherein the formulation is a liquid formulation. EMBODIMENT 16 A lyophilized formulation comprising: Collagenase, Disaccharides, Mannitol, and Tris-HCl, 13. A lyophilized formulation comprising: EMBODIMENT 17 The lyophilized formulation of embodiment 16, wherein the collagenase comprises collagenase I. EMBODIMENT 18 The lyophilized formulation of embodiment 17, wherein the collagenase I comprises the amino acid sequence of SEQ ID NO:1. EMBODIMENT 19 The lyophilized formulation of embodiment 16, wherein the collagenase comprises collagenase II. EMBODIMENT 20 The lyophilized formulation of embodiment 19, wherein the collagenase II comprises the amino acid sequence of SEQ ID NO:2. EMBODIMENT 21 The lyophilized formulation of embodiment 16, wherein the collagenase comprises a mixture of collagenase I and collagenase II. EMBODIMENT 22 The lyophilized formulation of embodiment 21, wherein the collagenase I comprises the amino acid sequence of SEQ ID NO:1 and the collagenase II comprises the amino acid sequence of SEQ ID NO:2. EMBODIMENT 23 The lyophilized formulation of embodiment 21 or 22, wherein the collagenase is collagenase Clostridium histolyticum (CCH). EMBODIMENT 24 The lyophilized formulation according to any one of embodiments 16 to 23, wherein the disaccharide comprises sucrose or trehalose. EMBODIMENT 25 In any one of embodiments 16 to 24, the lyophilized formulation is, prior to lyophilization, CCH and 60 mM sucrose, 225 mM mannitol, and 10 mM Tris-HCl, and A lyophilized formulation having a pH of about 8.5. EMBODIMENT 26 The lyophilized formulation according to any one of embodiments 16 to 25, wherein the lyophilized formulation is stable at a pressure of 380 μbar or more. EMBODIMENT 27 The lyophilized formulation of embodiment 26, wherein the lyophilized formulation is stable at a pressure of about 4000 μbar. EMBODIMENT 28 In any one of embodiments 16 to 27, the freeze-dried preparation comprises: (i) at least 36 months at 2–8°C; (j) At least 36 months at 25°C / 60% relative humidity; (k) At least 6 months at 40°C / 75% relative humidity, or (l) A freeze-dried formulation stable in any combination of (a) to (c). EMBODIMENT 29 In any one of embodiments 16 to 28, the freeze-dried preparation comprises: freezing the formulation at a temperature between about -25°C and -55°C to form a frozen formulation; and and drying the frozen formulation at a temperature between about 25°C and about 50°C to form the lyophilized formulation. EMBODIMENT 30 29. The lyophilized formulation of embodiment 29, comprising: freezing the formulation at a single temperature between about -25°C and -55°C to form a frozen formulation; and and drying the frozen formulation at a single temperature between about 25°C and about 50°C to form the freeze-dried formulation. EMBODIMENT 31 The lyophilized formulation according to any one of embodiments 16 to 30, wherein the lyophilized formulation is formed by a lyophilization process carried out in less than 72 hours. EMBODIMENT 32 32. The lyophilized formulation according to any one of embodiments 16 to 31, wherein the lyophilized formulation is formed by a lyophilization process carried out at a pressure between about 380 μbar and about 4000 μbar. EMBODIMENT 33 The lyophilized formulation according to any one of embodiments 16 to 32, which is contained in a unit dose vial, a multi-dose vial, a cartridge, or a syringe. EMBODIMENT 34 1. A reconstituted formulation comprising: Collagenase, Disaccharides, Mannitol, Tris-HCl, Calcium chloride, and Sodium chloride, 23. A reconstituted formulation comprising: EMBODIMENT 35 The reconstituted formulation of embodiment 34, wherein the collagenase is collagenase Clostridium histolyticum (CCH). EMBODIMENT 36 The reconstituted formulation of embodiment 34 or 35, wherein the disaccharide comprises sucrose or trehalose. EMBODIMENT 37 37. The reconstituted formulation according to any one of embodiments 34 to 36, wherein said reconstituted formulation is isotonic with respect to human blood. EMBODIMENT 38 A kit comprising: A container containing the lyophilized formulation according to any one of embodiments 16 to 32, and a container containing a sterile diluent comprising calcium chloride and sodium chloride; Including the kit.

Claims

1. A method for producing a lyophilized collagenase formulation, the method comprising: freezing a composition comprising collagenase, a disaccharide, mannitol, and Tris-HCl to form a frozen collagenase formulation; and drying the frozen collagenase formulation at a pressure of about 380 μbar to about 4000 μbar to form the lyophilized collagenase formulation; A method comprising:

2. The method of claim 1, comprising: freezing the composition at a temperature range of about -25°C to about -55°C to form the frozen collagenase formulation; and / or drying the frozen collagenase formulation at a temperature range of about 25° C. to about 50° C.; A method comprising:

3. The method of claim 2, comprising: freezing the composition at a single temperature between about -25°C and about -55°C to form the frozen collagenase formulation; and drying the frozen collagenase formulation at a single temperature between about 25° C. and about 50° C.; A method comprising:

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

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

6. The method according to any one of claims 1 to 5, wherein the drying step is carried out at a pressure of about 500 μbar to about 4000 μbar, about 750 μbar to about 4000 μbar, or about 1000 μbar to about 4000 μbar.

7. A method according to any one of claims 1 to 6, wherein the drying step is carried out at 380 μbar, 500 μbar, 750 μbar, 1000 μbar, 1500 μbar, 2000 μbar, 2500 μbar, 3000 μbar, 3500 μbar, or 4000 μbar.

8. The method according to claim 1, wherein the disaccharide is sucrose or trehalose.

9. The method of claim 1, wherein the collagenase comprises collagenase I.

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

1.

11. The method of claim 1, wherein the collagenase comprises collagenase II.

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

2.

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

14. The method of claim 13, wherein the collagenase I comprises the amino acid sequence of SEQ ID NO:1, and the collagenase II comprises the amino acid sequence of SEQ ID NO:

2.

15. The method of claim 13 or 14, wherein the collagenase is collagenase Clostridium histolyticum (CCH).

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

17. The method according to any one of claims 1 to 16, wherein the composition comprises: about 30 mM to about 240 mM of said disaccharide; about 50 mM to about 800 mM of said mannitol; and About 6 mM to about 10 mM of said Tris-HCl; A method comprising:

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

8.

19. The method of claim 17 or 18, wherein the composition comprises: About 60 mM sucrose; About 225 mM mannitol, and About 10 mM Tris-HCl; The composition has a pH of about 8.

5. method.

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

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

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

24. The method of any one of claims 1 to 23, wherein the lyophilized collagenase formulation does not contain mannitol and has greater stability at pressures above 380 μbar compared to a control lyophilized formulation produced by the same method.

25. The method of any one of claims 1 to 24, wherein the lyophilized collagenase formulation does not contain mannitol and has greater stability at pressures above 1030 μbar compared to a control lyophilized formulation produced by the same method.

26. The method of any one of claims 1 to 25, wherein the lyophilized collagenase formulation does not contain mannitol and has greater stability at pressures above 4000 μbar compared to a control lyophilized formulation produced by the same method.

27. The method according to any one of claims 1 to 26, wherein the lyophilized collagenase preparation comprises: (a) at least 36 months at 2-8°C; (b) at least 36 months at 25° C. and 60% relative humidity; (c) at 40° C. and 75% relative humidity for at least 6 months; or (d) any combination of (a) to (c); How to stabilize it.

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