US20220305094A1 - Collagenase formulations and methods of producing the same - Google Patents

Collagenase formulations and methods of producing the same Download PDF

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US20220305094A1
US20220305094A1 US17/419,899 US202017419899A US2022305094A1 US 20220305094 A1 US20220305094 A1 US 20220305094A1 US 202017419899 A US202017419899 A US 202017419899A US 2022305094 A1 US2022305094 A1 US 2022305094A1
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formulation
collagenase
lyophilized
mannitol
formulations
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Karunakar Sukuru
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Endo Usa Inc
Endo Operations Ltd
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Endo Global Aesthetics Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24003—Microbial collagenase (3.4.24.3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24007—Interstitial collagenase (3.4.24.7), i.e. matrix metalloprotease 1 or MMP1

Definitions

  • collagenase-comprising formulations with improved stability and storage properties Disclosed herein are collagenase-comprising formulations with improved stability and storage properties.
  • XIAFLEX® (collagenase from Clostridium histolyticum (CCH)) is currently approved for the treatment of Dupuytren's Contracture (DC) and Peyronie's Disease (PD).
  • DC Dupuytren's Contracture
  • PD Peyronie's Disease
  • the currently approved XIAFLEX® formulation is supplied as a lyophilized cake containing 0.9 mg CCH in 3 CC vials along with a diluent vial.
  • the current XIAFLEX® formulation pre-lyophilization
  • has a lyophilization cycle time of about 72 hours in vials. Efficient lyophilization is required for shelf life and enzyme stability.
  • formulations comprising: 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 formulations comprising: a collagenase; a disaccharide; mannitol; and Tris-HCl.
  • Reconstituted formulations comprising: a collagenase; a disaccharide; mannitol; Tris-HCl; calcium chloride; and sodium chloride are also disclosed.
  • Kits are also provided, wherein the kits comprise: a container comprising any of the disclosed lyophilized formulations; and a container comprising a sterile diluent comprising calcium chloride and sodium chloride.
  • FIG. 1 illustrates the effect of pH on protein interactions in exemplary formulations comprising trehalose, mannitol, and various collagenases.
  • FIG. 2A and FIG. 2B illustrate an exemplary hydrogen peroxide challenges analyzing the effect of pH and excipients on turbidity.
  • NTU Nephelometric Turbidity Units
  • PS Polysorbate
  • T Tehalose
  • S Sucrose
  • M Mesmarate
  • H 2 O 2 Hydrogen peroxide
  • 7.5, 8.0, and 8.5 refers to formulation pH.
  • 3R illustrate scanning electron microscopy (SEM) images of cakes from various lyophilized formulations (Va: 0.93 mg/ml CCH; 60 mM Sucrose; 112.5 mM Mannitol; 10 mM Tris/HCl buffer pH 8.5; Vb: 0.93 mg/ml CCH; 60 mM Sucrose; 225 mM Mannitol; 10 mM Tris/HCl buffer pH 8.5; and Vc: 0.93 mg/ml CCH; 60 mM Sucrose; 337.5 mM Mannitol; 10 mM Tris/HCl buffer pH 8.5).
  • SEM scanning electron microscopy
  • FIG. 4A , FIG. 4B , and FIG. 4C illustrate images of cakes from various lyophilized formulations under a pressure of 128 ⁇ bar ( FIG. 4A ), 380 ⁇ bar ( FIG. 4B ), and 1030 ⁇ bar ( FIG. 4C ).
  • FIG. 5A and FIG. 5B illustrate images of cakes from various lyophilized formulations under a pressure of 128 ⁇ bar ( FIG. 5A ) and 4000 ⁇ bar ( FIG. 5B ).
  • FIG. 6 illustrates the moisture content over time in various lyophilized formulations.
  • any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed formulations are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.
  • range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited.
  • range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the invention as described herein.
  • CH collagenase Clostridium histolyticum
  • USP United States Pharmacopeia
  • NTU Nephelometric Turbidity Units
  • PS Polysorbate
  • H 2 O 2 Hydrogen peroxide
  • formulations comprising, or consisting of:
  • the formulations can contain between about 0.2 mg/ml to about 50 mg/ml of collagenase.
  • 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.
  • collagenase produced by fermentation of Clostridium histolyticum also known as Hathewaya histolytica
  • CCH CCH
  • collagenase having at least 50% sequence alignment with collagenase I also referred as class I collagenase
  • collagenase II also referred as class II collagenase
  • collagenase produced by fermentation of other source organisms i.e., non- Clostridium histolyticum ), e.g., mammalian, crustacean, fungal, bacterial or microbial collagenase
  • collagenase obtained by recombinant techniques e.g., mammalian, crustacean, fungal, bacterial or microbial collagenase
  • collagenase obtained by recombinant techniques e.g., collagenase with a molecular mass from about 65 kDa to about 130 kDa; (i) collagenase designated as collagenase I or collagenase II;
  • collagenase from Vibrio alginolyticus (o) collagenase from Vibrio alginolyticus ; (p) collagenase from Streptomyces ; (q) collagenase from Pseudomonas ; (r) collagenase from Achromobacter iophagus ; (s) collagenase described by Worthington Biochemical Corp. (www.Worthington-biochem.com; “Product Highlights”); (t) collagenase described by Sigma-Aldrich (www.sigma-aldrich.com); (u) collagenase having one or more of the following characteristics:
  • the collagenase can comprise a collagenase I.
  • a suitable collagenase I includes, for example, a 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.
  • the collagenase I comprises the amino acid sequence of SEQ ID NO: 1.
  • the collagenase can comprise a collagenase II.
  • a suitable collagenase II includes, for example, a 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.
  • the collagenase II comprises the amino acid sequence of SEQ ID NO: 2.
  • the collagenase can comprise a mixture of collagenase I and collagenase II.
  • the collagenase can comprise, for example, a mixture of a 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 a 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.
  • the collagenase comprises a mixture of the collagenase I comprising the amino acid sequence of SEQ ID NO: 1 and the collagenase II comprising the amino acid sequence of SEQ ID NO: 2.
  • Suitable mixtures of the collagenase I and collagenase II include, for example, a collagenase I:collagenase II mass ratio 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 the 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 by, for example, reverse phase HPLC.
  • the collagenase can comprise collagenase Clostridium histolyticum (CCH).
  • CCH refers to collagenase Clostridium histolyticum containing a mixture of collagenase I (SEQ ID NO: 1) and collagenase II (SEQ ID NO: 2) in an approximate 1:1 mass ratio.
  • SEQ ID NO: 1 collagenase I
  • SEQ ID NO: 2 collagenase II
  • CCH is obtained by the fermentation of Clostridium histolyticum (also known as Hathewaya histolytica ).
  • Suitable disaccharides include those that:
  • the disaccharide comprises sucrose or trehalose.
  • the formulation comprises: a collagenase; about 30 mM to about 240 mM sucrose; about 50 mM to about 800 mM of mannitol; and about 6 mM to about 10 mM of a Tris-HCl.
  • the formulation comprises: a collagenase; about 30 mM to about 240 mM of trehalose; about 50 mM to about 800 mM of mannitol; and about 6 mM to about 10 mM of a Tris-HCl.
  • the disaccharide can 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 can be present at a concentration of about 30 mM, 60 mM, 90 mM, 120 mM, 150 mM, 180 mM, 210 mM, or 240 mM.
  • the mannitol can be present at a concentration of 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
  • the mannitol can be present at a concentration of about 50 mM, 100 mM, 150 mM, 200 mM, 225 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, or 800 mM.
  • 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.
  • the formulation can comprise: CCH; about 60 mM sucrose; about 225 mM mannitol; and about 10 mM Tris-HCl, wherein the formulation has a pH of about 8.5.
  • the formulation can comprise: about 0.9 mg CCH/ml; about 60 mM sucrose; about 225 mM mannitol; and about 10 mM Tris-HCl, wherein the formulation has a pH of about 8.5.
  • the formulation can consist of: CCH; about 60 mM sucrose; about 225 mM mannitol; and about 10 mM Tris-HCl, wherein the formulation has a pH of about 8.5.
  • the formulation can consist of: about 0.9 mg CCH/ml; about 60 mM sucrose; about 225 mM mannitol; and about 10 mM Tris-HCl, wherein the formulation has a pH of about 8.5.
  • the disclosed formulation can further comprise a surfactant.
  • Suitable surfactants include, for example, polysorbate 20, polysorbate 80, or poloxamer 188.
  • the surfactant can be present at a concentration of 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 can be present at a concentration of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, or 2%.
  • the formulation further comprises polysorbate 20 at a concentration of about 0.02%.
  • the formulation further comprises polysorbate 80 at a concentration of about 0.02%.
  • the formulation further comprises poloxamer 80 at a concentration of about 0.02%.
  • the above formulations can be liquid.
  • the disclosed formulations can be lyophilized under more aggressive conditions compared to previous collagenase-containing formulations, such as XIAFLEX®.
  • the disclosed formulations can be lyophilized in shorter times, using higher pressures, and/or with fewer drying steps (e.g., single temperature drying), resulting in a lyophilized formulation that exhibits increased stability and that maintains acceptable collagenase activity upon reconstitution.
  • the pH and mannitol lead to the formation of a more robust formulation that can subsequently undergo more aggressive lyophilization.
  • lyophilized formulations can be formed by the lyophilization of any of the above formulations.
  • the lyophilized formulations comprise, or consist of:
  • the lyophilized formulation can contain between about 0.2 mg to about 50 mg of collagenase.
  • 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 collagenase can comprises a collagenase I.
  • a suitable collagenase I includes, for example, a 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.
  • the collagenase I comprises the amino acid sequence of SEQ ID NO: 1.
  • the collagenase can comprise a collagenase II.
  • a suitable collagenase II includes, for example, a 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.
  • the collagenase II comprises the amino acid sequence of SEQ ID NO: 2.
  • the collagenase can comprise a mixture of collagenase I and collagenase II.
  • the collagenase can comprise, for example, a mixture of a 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 a 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.
  • the collagenase comprises a mixture of the collagenase I comprising the amino acid sequence of SEQ ID NO: 1 and the collagenase II comprising the amino acid sequence of SEQ ID NO: 2.
  • Suitable mixtures of the collagenase I and collagenase II include, for example, a collagenase I:collagenase II mass ratio 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.
  • the collagenase is collagenase Clostridium histolyticum (CCH).
  • Each of the 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 by, for example, reverse phase HPLC.
  • Suitable disaccharides include, for example, sucrose or trehalose.
  • the lyophilized formulation comprises, or consists of, a collagenase, sucrose, mannitol, and Tris-HCl.
  • the lyophilized formulation comprises, or consists of, a collagenase, trehalose, mannitol, and Tris-HCl.
  • the lyophilized formulations can be in a unit-dose vial, multi-dose vial, cartridge, or syringe.
  • the lyophilized formulation can contain between about 0.2 mg to about 50 mg of collagenase.
  • 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 can contain between about 0.2 mg to about 50 mg of collagenase.
  • the vial, cartridge, or syringe can 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 can contain between about 0.2 mg to about 50 mg of the lyophilized formulation.
  • the vial, cartridge, or syringe can 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.
  • the formulation Prior to lyophilization, can comprise, or consist of: 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. Prior to lyophilization, the formulation can comprise, or consist of: about 0.9 mg collagenase/ml; 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.
  • the formulation Prior to lyophilization, can comprise, or consist of: CCH; 60 mM sucrose; 225 mM mannitol; 10 mM Tris-HCl, and have 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 have a pH of about 8.5.
  • the disclosed lyophilized formulations have increased stability compared to previous collagenase-containing formulations, such as XIAFLEX®.
  • the disclosed lyophilized formulations are stable at pressures above 380 ⁇ bar, above 400 ⁇ bar, above 450 ⁇ bar, above 500 ⁇ bar, above 550 ⁇ bar, above 600 ⁇ bar, above 650 ⁇ bar, above 700 ⁇ bar, above 750 ⁇ bar, above 800 ⁇ bar, above 850 ⁇ bar, above 900 ⁇ bar, above 950 ⁇ bar, above 1000 ⁇ bar, above 1500 ⁇ bar, above 2000 ⁇ bar, above 2500 ⁇ bar, above 3000 ⁇ bar, above 3500 ⁇ bar, or above 4000 ⁇ bar.
  • the lyophilized formulation is stable at a pressure of about 4000 ⁇ bar.
  • the disclosed lyophilized formulations also exhibit improved shelf life and storage conditions compared to previous collagenase-containing formulations.
  • the disclosed lyophilized formulations exhibit an extended shelf life at low temperatures, such as 2-8° C., and at elevated temperatures, such as room temperature (40° C./75% relative humidity).
  • the disclosed lyophilized formulation can be stable at, for example:
  • the disclosed lyophilized formulation can be formed by a 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 the lyophilized formulation.
  • Suitable temperatures for the freezing step include about ⁇ 25° C., ⁇ 30° C., ⁇ 35° C., ⁇ 40° C., ⁇ 45° C., ⁇ 50° C., or ⁇ 55° C.
  • Suitable temperatures for the drying step include about 25° C., 30° C., 35° C., 40° C., 45° C., or 50° C.
  • the lyophilized formulations can be formed using a single temperature freezing step and a single temperature drying step.
  • the lyophilized formulation can be formed by a method comprising: 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 the lyophilized formulation.
  • the single temperature freezing step can be performed at a temperature between about ⁇ 25° C. and about ⁇ 55° C.
  • 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.
  • the single temperature drying step can be performed at about 25° C., 30° C., 35° C., 40° C., 45° C., or 50° C.
  • the method may further comprise a “ramp up” step between the freezing and drying to allow the lyophilizer to reach the suitable drying temperature.
  • the disclosed lyophilized formulations can be formed by a lyophilization method that is much faster than the lyophilization method used to form other collagenase-containing lyophilized formulations.
  • the lyophilized formulation can be formed by a lyophilization method that is performed for less than 72 hours. In some embodiments, the methods can be performed for less than 30 hours. In some embodiments, the methods can be performed for less than 18 hours. In some embodiments, the method can be performed for about 15 hours to about 25 hours.
  • the disclosed lyophilized formulations can be formed by a lyophilization method that uses a much higher pressure than the lyophilization method used to form other collagenase-containing lyophilized formulations.
  • the lyophilized formulation can be formed by a lyophilization method that is performed at a pressure of between about 380 ⁇ bar to about 4000 ⁇ bar.
  • the disclosed methods can be performed at a pressure of between about 500 ⁇ bar to about 4000 ⁇ bar, between about 750 ⁇ bar to about 4000 ⁇ bar, between about 1000 ⁇ bar to about 4000 ⁇ bar.
  • the disclosed methods can be performed at 380 ⁇ bar, 500 ⁇ bar, 750 ⁇ bar, 1000 ⁇ bar, 1500 ⁇ bar, 2000 ⁇ bar, 2500 ⁇ bar, 3000 ⁇ bar, 3500 ⁇ bar, or 4000 ⁇ bar.
  • the disclosed lyophilized formulations once reconstituted, can be used to treat or reduce collagen-mediated conditions, including the severity of cellulite (also known as edematous fibrosclerotic panniculopathy (EFP)), Dupuytren's contracture (DC) with a palpable cord, or Peyronie's disease (PD) with a palpable plaque and curvature deformity of at least 30 degrees.
  • EFP edematous fibrosclerotic panniculopathy
  • DC Dupuytren's contracture
  • PD Peyronie's disease
  • reconstituted formulations comprising, or consisting of: a collagenase; a disaccharide; mannitol; Tris-HCl; calcium chloride; and sodium chloride.
  • the collagenase can comprise a collagenase I.
  • a suitable collagenase I includes, for example, a 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.
  • the collagenase I comprises the amino acid sequence of SEQ ID NO: 1.
  • the collagenase can comprise a collagenase II.
  • a suitable collagenase II includes, for example, a 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.
  • the collagenase II comprises the amino acid sequence of SEQ ID NO: 2.
  • the collagenase can comprise a mixture of collagenase I and collagenase II.
  • the collagenase can comprise, for example, a mixture of a 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 a 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.
  • the collagenase comprises a mixture of the collagenase I comprising the amino acid sequence of SEQ ID NO: 1 and the collagenase II comprising the amino acid sequence of SEQ ID NO: 2.
  • Suitable mixtures of the collagenase I and collagenase II include, for example, a collagenase I:collagenase II mass ratio 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.
  • the collagenase is collagenase Clostridium histolyticum (CCH).
  • Each of the 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 by, for example, reverse phase HPLC.
  • Suitable disaccharides include, for example, sucrose or trehalose.
  • the reconstituted formulation comprises, or consists of, a collagenase, sucrose, mannitol, Tris-HCl, calcium chloride, and sodium chloride.
  • the lyophilized formulation comprises, or consists of, a collagenase, trehalose, mannitol, Tris-HCl, calcium chloride, and sodium chloride.
  • Suitable amounts of calcium chloride and sodium chloride include those that enable the reconstituted formulation to be isotonic to human blood.
  • 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% of calcium chloride.
  • 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% of sodium chloride.
  • the reconstituted formulation can also comprise water for injection (WFI).
  • WFI water for injection
  • the disclosed reconstituted formulations can be used to treat or reduce collagen-mediated conditions, including the severity of cellulite (also known as edematous fibrosclerotic panniculopathy (EFP)), Dupuytren's contracture (DC) with a palpable cord, or Peyronie's disease (PD) with a palpable plaque and curvature deformity of at least 30 degrees.
  • EFP edematous fibrosclerotic panniculopathy
  • DC Dupuytren's contracture
  • PD Peyronie's disease
  • the reconstituted formulations can comprise about 0.01 mg to about 50 mg of the collagenase in a single or divided dose.
  • the reconstituted formulation can comprise, 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 the collagenase in a single or divided dose.
  • the reconstituted formulation can comprise, 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 1.00 mg, 1.05 mg, about 1.10 mg, about 1.15 mg, about 1.20 mg, about 1.25 mg, about 1.30 mg, about 1.35 mg, about 1.40 mg, about 1.45 mg, about 1.50 mg, about 1.55 mg, about 1.60 mg, about 1.65 mg, about 1.70 mg, about 1.75 mg, about 1.80 mg, about 1.85 mg, about 1.90 mg, about 1.95 mg, about 2.00 mg, 2.05 mg, about 2.10 mg, about 2.15 mg, about 2.20 mg, about 2.25 mg, about 0.30 mg, about 2.35 mg, about 0.40 mg, about 0.45 mg,
  • the reconstituted formulation can have a total volume of about 0.1 mL to about 50 mL.
  • 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.
  • Kits comprising the disclosed lyophilized formulations and a sterile diluent are also provided.
  • the kits can comprise: a container comprising any of the disclosed lyophilized formulations; and a container comprising a sterile diluent comprising calcium chloride and sodium chloride.
  • Suitable containers for the lyophilized formulation and/or the sterile diluent include, for example, a vial, cartridge, or syringe.
  • the vial can be a unit-dose vial or a multi-dose vial.
  • Suitable container sizes include, for example, 2 mL to 50 mL containers, such as 5 mL, 7.5 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, or 50 mL.
  • the container comprising the disclosed lyophilized formulation can comprise between about 0.2 mg to about 50 mg collagenase.
  • the container can comprise 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 collagenase.
  • the container comprising the disclosed lyophilized formulation can comprise between about 0.2 mg to about 50 mg of the lyophilized formulation.
  • the container can comprise 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.
  • the container comprising the sterile diluent can comprise an amount of sterile diluent that, upon reconstitution of the lyophilized formulation, results in a solution that is isotonic to human blood.
  • 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% of calcium chloride.
  • 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% of sodium chloride.
  • the volume of the sterile diluent can be about 0.1 mL to about 50 mL.
  • the volume of the sterile diluent can be 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.
  • methods of lyophilizing any of the disclosed formulations 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 the lyophilized formulation.
  • the freezing is performed at a single temperature and the drying is performed at a single temperature.
  • the methods may further comprise a “ramp up” step between the freezing and drying to allow the lyophilizer to reach the suitable drying temperature.
  • the single temperature freezing step can be performed at a temperature between about ⁇ 25° C. and about ⁇ 55° C.
  • 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.
  • the single temperature drying step can be performed at about 25° C., 30° C., 35° C., 40° C., 45° C., or 50° C.
  • the disclosed methods can be performed for less than 72 hours. In some embodiments, the methods can be performed for less than 30 hours. In some embodiments, the methods can be performed for less than 18 hours. In some embodiments, the method can be performed for about 15 hours to about 25 hours.
  • the disclosed methods can be performed at a pressure of between about 128 ⁇ bar to about 4000 ⁇ bar, between about 380 ⁇ bar to about 4000 ⁇ bar, between about 500 ⁇ bar to about 4000 ⁇ bar, between about 750 ⁇ bar to about 4000 ⁇ bar, between about 1000 ⁇ bar to about 4000 ⁇ bar.
  • the disclosed methods can be performed at 380 ⁇ bar, 500 ⁇ bar, 750 ⁇ bar, 1000 ⁇ bar, 1500 ⁇ bar, 2000 ⁇ bar, 2500 ⁇ bar, 3000 ⁇ bar, 3500 ⁇ bar, or 4000 ⁇ bar.
  • the XIAFLEX® formulation (CCH, 10 mM Tris/HCl pH 8.0, 60 mM sucrose) has a relatively long lyophilization cycle time of 72 hours.
  • An objective of the below studies was to achieve a more efficient lyophilization process with reduced cycle time and improved yields.
  • pre-lyophilization As a part of the work to identify an optimal formulation composition for lyophilization, several different solution formulations (“pre-lyophilization”) containing various combinations of excipients such as sucrose, trehalose, and mannitol with and without surfactant at different levels were evaluated. In addition, untreated and siliconized (baked on) vials were also evaluated.
  • Standard glass vials and siliconized glass vials were used for the test to investigate the influence of hydrophobic surfaces on the protein stability.
  • the formulations were supplemented with two types of surfactants (polysorbate 20 and poloxamer 188) to examine the influence of surfactants on the stability of the formulations during the challenge testing.
  • a strong oxidizing agent hydrogen peroxide
  • a matrix of 21 variants were used for the challenge testing.
  • CCH was formulated in three different formulation variants.
  • the formulation variants were exposed to thermal stress with agitation and freeze/thaw stress to challenge the formulation candidates. Hydrogen peroxide was added to the samples to force oxidative stress to the proteins. Each sample was analyzed after stress exposure by turbidity measurements to monitor the formation of aggregates.
  • Vials were washed in a laboratory dishwasher with purified water. Afterwards, the vials were dried and heat-treated at 300° C. for 2 hours for depyrogenization/sterilization. Stoppers were autoclaved at 2 bar and 121° C. for 20 min in sterilization bags and dried for 8 hours at 80° C.
  • First XIAFLEX® drug substance was dialyzed against the formulation variants A or B. The dialysis was accomplished in three independent dialysis steps to achieve a quantitative buffer exchange. 60 ml of the XIAFLEX® drug substance was dialyzed against formulation variant A and 40 ml of the XIAFLEX® drug substance was dialyzed against formulation variant B. XIAFLEX® drug substance was transferred into two preconditioned (in dialysis buffer) Slide-A-LyzerTM cassettes (Thermo Scientific, Rockford, USA).
  • Filled Slide-A-LyzerTMcassettes were incubated in 2000 ml or 1000 ml of the target buffer for 2 hours before a first buffer change (2000 ml/1000 ml) was performed. After dialysis for two additional hours, the buffer was changed a second time (2000 ml for both formulation variants) to finalize the dialysis overnight. The protein sample was removed from the Slide-A-LyzerTM cassettes and the concentration was adjusted to 1 mg/ml by dilution.
  • Each formulation was sterile filtrated under laminar flow and exposed to thermal/agitation and freeze/thaw stress.
  • the freeze/thaw stability of the formulations was tested by running 3 freeze/thaw cycles in total. Siliconized 6R glass vials or 2R standard glass vials were filled with 1.0 ml of each formulation. 3 vials were prepared for each variant. Liquid samples were frozen from 25° C. to ⁇ 30° C. within 55 min at a controlled freezing rate and warmed up again to room temperature within 55 min at a controlled heating rate. For an adequate temperature control, the samples were loaded into a pilot freeze dryer. After each freeze/thaw cycle the turbidity of the samples was determined. For the turbidity measurement, 1 ml of each sample was filled in single-use turbidity cuvettes and analyzed. After analyzing, the liquid was returned to the glass vial and the experiment was continued.
  • Test samples of all formulations were stressed at 40° C. for 4 days under agitation (200 rpm) in 2R vials (1 ml fill volume). Turbidity of the stressed samples was analyzed afterwards.
  • the turbidity of the samples was determined using a 2100AN turbidity meter (Hach Lange, Dusseldorf, Germany) according to the European Pharmacopeia. The system was calibrated as follows:
  • Table 4 displays the result of the freeze/thaw testing. None of the variants showed an increase in turbidity with increasing number of freeze/thaw cycles. The enzymes seemed to be stable against freeze/thaw stress. The addition of surfactants or hydrogen peroxide showed no influence on the turbidity of the samples.
  • Table 5 shows the results of the thermal stress at 40° C. and agitation at 200 rpm. Variants containing hydrogen peroxide showed a clear increase of turbidity (as shown by the increased nephelometric turbidity units (NTU)) after thermal stress. In variants V A and variants V B (mannitol is present in both formulations) the resulting turbidity was remarkably lower (decreased NTU) than in the original XIAFLEX® formulation variant (“Or”—without mannitol). The beneficial effect of mannitol is believed to be caused by its radical scavenger properties.
  • the samples were analyzed by turbidity measurements to monitor the formation of aggregates.
  • the turbidity of the samples did not increase by freeze/thaw cycling.
  • the formulations seemed to be stable against the formation of aggregates under freeze/thaw stress (up to 3 cycles).
  • Hydrophobic surface contact as well as addition of hydrogen peroxide had no influence on the turbidity of the samples either with or without surfactants during freeze/thaw stress.
  • thermal stress a clear increase in turbidity (as measured by NTU) under forced oxidative stress was observed (addition of hydrogen peroxide).
  • Variants containing mannitol and having higher repulsive interactions between the molecules exhibited a less pronounced increase in turbidity under forced oxidative stress.
  • Collagenase I and collagenase II was dialyzed against the corresponding formulation buffer. After dialysis, the sample solutions were further diluted. Colloidal stability and thermodynamic stability of collagenase I, collagenase II, and the mixture was determined by CG-MALS and nanoDSC, respectively.
  • the stability of the proteins in each formulation variant was additionally investigated in a stress test (freeze/thaw and thermal/shear stress) study. To force oxidative stress during the stability test, each formulation variant was examined in the presence and absence of 0.1% H 2 O 2 (sub-variant without hydrogen peroxide are marked with a; sub-variant with hydrogen peroxide are marked with b).
  • the dialysis of collagenase I and collagenase II was accomplished in three independent dialysis steps to achieve a quantitative buffer exchange. 14 ml of the collagenase I intermediate sample and 14 ml of the collagenase II intermediate sample was transferred into two preconditioned (in dialysis buffer) Slide-A-LyzerTM cassettes (Thermo Scientific, Rockford, USA). Filled Slide-A-LyzerTM cassettes were incubated in 1000 ml of the target buffer for 2 hours before a first buffer change (1000 ml) was performed. After dialysis for two further hours the buffer was changed a second time (1000 ml) to finalize the dialysis overnight. The protein sample was removed from the Slide-A-LyzerTM cassettes and processed further. Each dialysis step represents a 1/35 fold buffer exchange leading to a calculated buffer exchange factor of approx. 2 ⁇ 10 5 in total.
  • DSC Differential Scanning calorimetry
  • a buffer scan was conducted prior to each sample run to generate a baseline.
  • Dialyzed samples were diluted with the corresponding formulation buffer to 1 mg/ml.
  • the corresponding dialysis buffer was used as buffer scan and buffer reference.
  • A2 is a measure of protein-protein interactions in solution. Negative A2 values indicate attractive protein-protein interactions while positive values indicate repulsive protein interactions. A protein solution is “colloidally unstable” when the A2 values are negative. A higher A2 value indicates greater repulsion, which is indicative of less protein interaction and less potential for protein aggregation and better stability. A2 values for various formulations were determined by CG-MALS and used as measures of non-specific protein-protein interactions
  • the apparent weight average molecular weight (Mw app ) is determined for each step in the concentration gradient by analyzing the light scattering and concentration data. Significant interactions between macromolecules manifest as changes in Mw app vs. concentration. A 2 calculation was conducted via Zimm plot analysis by an extrapolation to 0 mg/ml concentration according to formula I below:
  • Dialyzed samples were used undiluted for CG-MALS measurements.
  • the corresponding dialysis buffer was used as diluent for the CG-MALS experiment.
  • the samples and the buffer were passed over a 0.1 ⁇ m filter.
  • concentration of the samples was determined by UV-absorption measurement. The concentration was used to calculate the concentrations of each gradient step.
  • a Calypso II CG-MALS system was used to supply the MALS detector with the concentration gradient of the analyte.
  • the samples were loaded on syringe pump 1 and syringe pump 2 of the system and the dialysis buffer on syringe pump 3.
  • the CG-MALS measurement consisted of three steps. In the first step, a concentration gradient of sample 1 ranging from 10% to 100% was applied to determine the self-virial coefficient of sample 1 in the formulation.
  • the second step consisted of a cross-over gradient, in which the concentration of sample 1 was reduced from 90% to 10% while the concentration of sample 2 was increased from 10% to 90%. This step was conducted to determine the cross-virial coefficient.
  • a concentration gradient of sample 2 ranging from 100% to 10% was applied to determine the self-virial coefficient of sample 2 in the formulation.
  • 0.7 ml of sample was injected into the MALS detector.
  • the resulted light scattering signal was recorded over a time period of 180 sec.
  • Multi component Zimm plot analysis with fixed molecular weight was performed with Calypso software version 2.1.5.
  • the concentration of collagenase I and collagenase II in solution was determined using an 8452A UV spectrometer (Agilent Technologies, Santa Clara, USA). Samples were measured at a concentration of about 3 mg/ml using plastic cuvettes with an optical path thickness of 0.2 cm. The concentration was calculated according to Lambert-Beer's law using an extinction coefficient of 1.52 ml/(mg*cm) for collagenase I and 1.48 ml/(mg*cm) for collagenase II, respectively.
  • the freeze/thaw stability of the formulations was tested by running 3 freeze/thaw cycles in total. Dialyzed collagenase I and dialyzed collagenase II was mixed to exhibit a solution containing 0.5 mg/ml collagenase I and 0.5 mg/ml collagenase II.
  • 2R glass vials were filled with 1.0 ml of each formulation.
  • 3 vials were prepared for each variant. Liquid samples were frozen from 25° C. to ⁇ 30° C. within 55 min at a controlled freezing rate and warmed up again to room temperature within 55 min at a controlled heating rate. For an adequate temperature control the samples were loaded into a pilot freeze dryer. After each freeze/thaw cycle, the turbidity of the samples was determined. For the turbidity measurement, 1 ml of each sample was filled in single-use turbidity cuvettes and analyzed. After analyzing, the liquid was returned to the glass vial and the experiment was continued.
  • Dialyzed collagenase I and dialyzed collagenase II was mixed to exhibit a solution containing 0.5 mg/ml collagenase I and 0.5 mg/ml collagenase II.
  • Test samples of all formulations were stressed at 40° C. for 4 days under agitation (200 rpm) in 2R vials (1 ml fill volume). Turbidity of the stressed samples was analyzed afterwards.
  • the turbidity of the samples was determined as described above.
  • Table 7 shows the results of the CG-MALS and nanoDSC measurements of each formulation variant.
  • Colloidal stability The pH value of the formulation showed a strong impact on the colloidal stability of collagenase I, collagenase II, and its mixture. Strongest repulsive interactions were observed at pH 8.5. At pH 7.5, the repulsive interactions were lower. At pH 7.5, collagenase I showed attractive interactions when mannitol was not present in the formulation and the repulsive interactions become stronger with higher concentrations of mannitol. At more basic pH values, the effect of mannitol became subordinated.
  • Table 8 shows the turbidity values of the samples during the subsequent freeze thaw cycles.
  • Table 9 shows the turbidity values of each formulation before and after thermal stress.
  • Turbidity [NTU] 4 days/ Tris Mannitol Sucrose H 2 O 2 200 rpm/ # pH mM mM mM % T0 40° C. 1a 7.5 10 0 60 0 2.1 2.3 2a 8.0 10 0 60 0 2.2 2.3 3a 8.5 10 0 60 0 2.8 2.4 4a 7.5 10 112.5 60 0 2.4 2.9 5a 8.0 10 112.5 60 0 3.0 2.4 6a 8.5 10 112.5 60 0 2.6 2.7 7a 7.5 10 225 60 0 1.8 2.8 8a 8.0 10 225 60 0 2.5 2.7 9a 8.5 10 225 60 0 2.7 2.6 10a 7.5 10 337.5 60 0 2.8 2.8 11a 8.0 10 337.5 60 0 2.6 2.7 12a 8.5 10 337.5 60 0 2.5 2.7 1b 7.5 10 0 60 0.1 1.9 388.0 2b 8.0 10 0 60 0.1 2.6 34.0 3
  • sucrose and trehalose were evaluated and the concentration used for a given sample is noted in the data tables below.
  • concentrations of surfactant were evaluated and the concentration used for a given sample is noted in the data tables below.
  • the effect of pH on various formulations pre-lyophilization was evaluated as a part of the formulation optimization.
  • CG-MALS Composition-Gradient Multi-Angle Light Scattering
  • DSC Nano Differential Scanning calorimetry
  • Trehalose, sucrose, and mannitol also do not adversely affect the repulsive forces and are therefore were appropriate potential components of the formulation.
  • Nano-DSC The variables studied using Nano-DSC to assess protein unfolding (pH, type and concentration of excipient) are presented in Table 16.
  • T onset Effect of Composition and pH on Onset Temperature (T onset ) for Protein Unfolding and Protein Unfolding Temperature (T m ) Variable Parameters Sodium Nano DSC Chloride Trehalose Mannitol Sucrose T onset [° C.] T m [° C.] pH (mM) (mM) (mM) (mM) Coll. I Coll. II Coll. I Coll.
  • T onset onset temperature
  • Tm protein melting temperature
  • compositions of the formulations Fill Variant # Packaging system Formulation volume Va 5 ml glass vial 0.93 mg/ml CCH; 60 mM 1 ml Sucrose; 112.5 mM Mannitol; 10 mM Tris/HCl buffer pH 8.5 Vb 5 ml glass vial 0.93 mg/ml CCH; 60 mM 1 ml Sucrose; 225 mM Mannitol; 10 mM Tris/HCl buffer pH 8.5 Vc 5 ml glass vial 0.93 mg/ml CCH; 60 mM 1 ml Sucrose; 337.5 mM Mannitol; 10 mM Tris/HCl buffer pH 8.5 Placebo 5 ml glass vial 60 mM Sucrose; 337.5 mM 1 ml Mannitol; 10 mM Tris/HCI buffer pH 8.5
  • Lyophilization stoppers were autoclaved at 121° C. for 15 min and dried for 8 hours at 105° C. Vials were rinsed with purified water and depyrogenized at 300° C. for 2 hours.
  • the formulation variants were prepared by dialysis.
  • the dialysis of XIAFLEX® drug substance was accomplished in three independent dialysis steps to achieve a quantitative buffer exchange. 125 ml of the XIAFLEX® drug substance was dialyzed against variant a, b and c.
  • XIAFLEX® drug substance was transferred into two preconditioned (in dialysis buffer) dialysis tubes. Filled dialysis tubes were incubated in 1 L of the target buffer for 2 hours before a first buffer change (1 L) was performed. After dialysis for two additional hours the buffer was changed a second time (2 L) to finalize the dialysis overnight.
  • the protein sample was removed from the dialysis tube and the concentration was adjusted to 0.93 mg/ml ( ⁇ 10%) by dilution (concentration was checked by UV 280 nm).
  • concentration was checked by UV 280 nm.
  • XIAFLEX® drug substance was used without dialysis.
  • the lyophilization solutions were passed over a 0.22 ⁇ m filter before filling. 1 ml of the corresponding lyophilization solution was filled into the vial.
  • the filled vials with lyophilization-stoppers attached to the vials in “lyo-position” were loaded into the pilot freeze dryer. About 125 vials per formulation and 100 placebo vials of Vb were loaded.
  • the lyophilization cycle was run with the following parameters:
  • thermo couples were inserted into product vials. Pressure was controlled during lyophilization by using a Pirani-pressure sensor. Pressure regulation was managed via vacuum and dosing valve (nitrogen injection). Vials were closed at a pressure of 750 mbar under nitrogen atmosphere.
  • the content of one vial of the corresponding lyophilizate was weighed into a glass vial which was sealed with a crimp cap.
  • the sample was transferred into the oven of the Karl-Fischer coulometer (756/774; Metrohm) which was heated to 100° C.
  • the septum of the cap was penetrated by an injection needle, and the generated water vapor was directly transferred into the titration chamber of the Karl-Fischer coulometer via dry nitrogen. Measurement was repeated one time. Empty glass vials were used for blank correction.
  • the oven sample processor 774 enables a unique temperature ramping method in Karl Fischer titration.
  • the sample was heated up by a defined heating rate and the released water was directly transferred into the titration chamber of the Karl-Fischer coulometer. By recording the generated water and the water drift ( ⁇ g water/min) depending on the oven temperature, specified events where water was released (e.g. the release of hydrate water) can be detected.
  • 50 to 100 mg of the lyophilizates was weighted into an empty 6R type 1 glass vial and closed with an alu-crimp-cab.
  • the sample was transferred into the oven of the sample processor. There the sample is heated up by a defined temperature ramp from 50° C. to 140° C. in 45 min (2° C./min). The ramp was concluded at 140° C. to exclude undesired Maillard-reactions.
  • Lyophilizates were removed from the vial by carefully breaking the glass vial and the lyophilization cake was cut vertically to screen its inner layer for collapse zones.
  • Lyophilizates were analyzed by SEM to evaluate their microstructure.
  • Lyophilizates were cut, and the vertical cross sections and top/bottom surfaces were analyzed via SEM at 50 ⁇ and 150 ⁇ magnification.
  • Vials were reconstituted with 4 ml of a solution containing 0.03% calcium chloride and 0.66% sodium chloride. Time for complete dissolution of the lyophilizates was recorded.
  • Nano DSC was performed as described above.
  • Lyophilization solutions and reconstituted lyophilizates were analyzed at a concentration of 0.93 mg CCH/ml.
  • the corresponding buffer was used as buffer scan and buffer reference.
  • the primary drying step was terminated after about 17 hours of total lyophilization time. After 18 hours of secondary drying one vial of each variant was removed from the freeze dryer and the residual moisture level was determined by Karl-Fischer titration while the secondary drying step was prolonged for the rest of the batch. The Karl-Fischer analysis revealed that the residual moisture level was already below the desired value. Thus, the rest of the batch was unloaded immediately after receiving these results. Total duration of secondary drying was 19 hours.
  • the objective of this work was to update the lyophilization cycle to result in a shorter process time and more efficient and robust process.
  • formulation #1 was buffer exchanged using dialysis to introduce the new excipients (trehalose, mannitol, and polysorbate 20) into the formulation in Tris buffer, pH 8.5.
  • the lyophilized cakes for all formulations rapidly dissolved in the diluent with a reconstitution time of less than 10 seconds and had moisture contents (KF) of less than 0.4%.
  • the XIAFLEX® cake appeared shrunken compared to the experimental formulation variants, which all showed a well formed and robust cake. All of the experimental formulations were tested for various characteristics including protein concentration, collagenase I and collagenase II mass composition, and biologic activity. The test results for all of the formulations aligned with the XIAFLEX® formulation.
  • Experimental formulations #3 and #5 were selected for further testing. These formulations were used to conduct informal short term stability studies at 5° C. and at accelerated storage conditions of 25° C./60% RH. Samples were analyzed per approved test methods. All results generated for both formulations were in line with historical data for the XIAFLEX® formulation, indicating that the formulation changes being studied do not adversely affect product quality (data not shown). As noted previously, the currently approved limits may be revised based on a statistical analysis of data generated for the experimental formulations.
  • formulation optimization development work and data generated to date indicated that the two studied protein stabilizers (sucrose, trehalose), the addition of a bulking agent (mannitol), and a surfactant (polysorbate 20), do not adversely affect product quality and that these formulations would be suitable to pursue further.
  • Pressure tests were performed to facilitate identification of the optimal lyophilization process conditions. This test set out to identify the maximum tolerable pressure and other reliable process parameters for freezing, primary drying, and secondary drying during lyophilization under process relevant conditions to realize a fast and robust lyophilization cycle.
  • Sample vials filled with the experimental formulations were loaded onto one freeze dryer shelf (middle position). After freezing, the chamber pressure was set to initial value of 128 ⁇ bar and shelf temperature was increased to an initial value ( ⁇ 10° C. in test 1/+10° C. in test 2). Lyophilization was allowed to run for a period of time to generate a small volume of lyophilizate above the ice interface. Chamber pressure was then increased step wise (e.g., 380 ⁇ bar, 1030 ⁇ bar etc.) and the sample vials were video monitored for cake collapse or other visual adverse effects. The shelf temperature was set to ⁇ 10° C. to achieve a moderate energy input.
  • FIG. 4A - FIG. 4C Pictures of the vials at the end of each pressure step are presented in FIG. 4A - FIG. 4C .
  • the Control #1, XIAFLEX® in the vial shown on the far right
  • the maximum tolerable pressure for the XIAFLEX® formulation was therefore between 128 ⁇ bar and 380 ⁇ bar, equivalent to an ice interface temperature of between ⁇ 40° C. and ⁇ 30° C.
  • Experimental Formulations #3 and #5 remained intact over the entire pressure range investigated.
  • a second pressure test was conducted, in which experimental formulations #3 and #5 were further investigated.
  • the pressure range was expanded to 4 mbar.
  • the temperature of the shelf during the test was set to +10° C. to provide sufficient energy input for enabling efficient sublimation at this pressure range.
  • the mannitol-containing formulations can be freeze dried at chamber pressures up to 4 mbar with no risk of structural collapse during sublimation
  • the lyocycles for the optimization trials were run using a chamber pressure of 1 mbar to take advantage of high sublimation rates.
  • Run #1 Utilized experimental formulation #3 only. Results from this run indicated that a higher temperature during secondary drying is required to reach a residual moisture level below 0.5%. Results for other physical attribute tests were acceptable (data not shown). A summary of the lyophilization parameters used are presented below:
  • Drying temperature (sublimation and secondary drying) (shelf): 35° C.
  • Run #2 Utilized experimental formulation #3 only. This run identified adequate conditions for drying using 40° C. All results were acceptable (data not shown). A summary of the lyophilization parameters used are presented below:
  • the lyocycle optimization studies identified basic parameters for an efficient freeze drying cycle. All tests for the physical attributes of the lyophilized cakes were acceptable.
  • the 6 process validation lots were monitored on long-term stability.
  • Storage conditions included long-term stability at various storage condition (2-8° C.; 25° C./60% relative humidity (RH); and 40° C./75% RH) to support product shelf life as well as short-term stability studies at various storage conditions to support evaluation of potential temperature excursions during shipping or storage.
  • Stability batches were tested for appearance (pre- and post-reconstitution), reconstitution time, osmolality, pH, concentration by UV A280 , quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reverse-phase high performance liquid chromatography (RP-HPLC) [purity], mass and ratio by composition by RP-HPLC, size-exclusion high-performance liquid chromatography (SEC-HPLC), soluble rat tail collagen (SRC) assay for collagenase I potency, glycyl-L-prolyl-L-alanine (GPA) assay for collagenase II potency, moisture, particulates, endotoxin, and container closure integrity helium leak.
  • SRC reverse-phase high performance liquid chromatography
  • GPA soluble rat tail collagen
  • Osmolality The results for osmolality at the storage conditions tested showed no significant changes or unexpected trends (data not shown).
  • Container Closure Integrity/Helium Leak The results for container closure integrity/helium leak showed no significant changes or unexpected trends (data not shown).
  • Available stability data for the 6 process validation lots of CCH formulation demonstrated stability of the material through at least the 18-month pull point at both 5° C. and 25° C./60% RH storage conditions, as well as through the 6-month pull point at the 40° C./75% RH storage condition. Additionally, available data for a formulation development lot of the CCH formulation in a smaller vial demonstrated stability through the 24-month pull point at both the 5° C. and 25° C./60% RH storage conditions as well as through the 6-month pull point at the 40° C./75% RH storage condition.
  • the CCH formulation demonstrated acceptable photostability as all results generated following exposure were comparable to a control (unexposed) sample.
  • CCH formulation Following reconstitution with sterile diluent, CCH formulation showed acceptable stability for up to 24 hours when stored at 25° C./60% RH and for up 120 hours when stored at 5° C. Reconstituted CCH formulation also demonstrated acceptable stability when stored at 25° C./60% RH for 24 hours, then at 2° C. to 8° C. for 96 hours and then at 25° C./60% RH for an additional 24 hours.

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