US20120003714A1 - Blends containing proteases - Google Patents

Blends containing proteases Download PDF

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US20120003714A1
US20120003714A1 US13/230,927 US201113230927A US2012003714A1 US 20120003714 A1 US20120003714 A1 US 20120003714A1 US 201113230927 A US201113230927 A US 201113230927A US 2012003714 A1 US2012003714 A1 US 2012003714A1
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Prior art keywords
solution
composition
salt
range
proteases
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Werner Hoelke
Michaela Fischer
Johann-Peter Thalhofer
Markus Weber
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Roche Diagnostics Operations Inc
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Roche Diagnostics Operations Inc
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Assigned to ROCHE DIAGNOSTICS GMBH reassignment ROCHE DIAGNOSTICS GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: THALHOFER, JOHANN-PETER, WEBER, MARKUS, HOELKE, WERNER, FISCHER, MICHAELA
Assigned to ROCHE DIAGNOSTICS OPERATIONS, INC. reassignment ROCHE DIAGNOSTICS OPERATIONS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ROCHE DIAGNOSTICS GMBH
Publication of US20120003714A1 publication Critical patent/US20120003714A1/en
Priority to US14/699,299 priority Critical patent/US20160319268A1/en
Priority to US15/595,126 priority patent/US20170247679A1/en
Priority to US16/865,487 priority patent/US11459556B2/en
Priority to US17/822,587 priority patent/US20230052545A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • 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)
    • 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)
    • C12Y304/24027—Thermolysin (3.4.24.27)

Definitions

  • compositions preferably lyophilizates, comprising proteolytic enzymes.
  • Preferred compositions comprise one or more proteases with collagenase activity and a neutral protease, preferably thermolysis.
  • the compositions are free of acetate salts.
  • such compositions can be dissolved in water more rapidly than lyophilized protease mixtures of the state of the art.
  • tissue dissociation The process of disintegrating a mass of interconnected cells (tissue) wherein the cells are separated from each other is known as “tissue dissociation”. Tissue dissociation is a principal application for certain proteolytic enzymes in tissue culture research and cell biology studies. Blends of proteolytic enzymes, rather than single proteases, are used for the dissociation of biological tissue.
  • the biological tissue is preferably obtained (i.e., explanted) from an animal, preferably from a mammal, and more preferred from a human.
  • the biological tissue is incubated in an aqueous medium containing the proteolytic enzymes in active form. By way of hydrolyzing peptidic bonds in the extracellular matrix, the interconnected cells become separable from each other.
  • proteolytic enzymes with collagenase activity are used with advantage in many processes of tissue dissociation known to the art.
  • Blends containing a plurality of proteases usually comprise collagenases.
  • Collagenases (EC 3.4.24.3) are metalloproteinases, proteolytic enzymes which are able to hydrolyze collagen, both in its native triple-helix and denatured conformation, by dissociating its peptidic bonds under physiological conditions of pH and temperature.
  • Collagenases produced by bacteria are well known in the state of the art.
  • Collagenases produced by bacteria of the Clostridium species, in particular Clostridium hystolyticum are of major interest for applications in tissue dissociation.
  • the collagenases and particularly collagenase I are stable only to a limited extent, even at low temperatures. Particular care is in fact necessary when preparing and handling collagenase solutions, in order to prevent inactivation of enzymatic activity: a temperature above 56° C. is detrimental, as well as the presence of several metal ions and of chelating agents interacting with the
  • the optimal pH value for the storage of collagenases ranges from about 6 to about 8 for crude preparations, while the interval is much narrower when the collagenase isoforms are purified; low pH values can inactivate enzymatic activity.
  • collagenases are sensitive to physical treatment such as freezing, thawing, lyophilization and drying. These treatments, which are often necessary for the purification and preparation of dry products, pose a technical problem in that they may reduce the desired enzymatic activity or may even provoke inactivation of the collagenase enzymes.
  • Collagenase I and/or II isoforms in their lyophilized powder form maintain reasonable stability if kept at temperatures which are lower than 25° C., preferably between about 2° C. and 8° C., in sealed bottles and avoiding exposure to humidity.
  • temperatures which are lower than 25° C., preferably between about 2° C. and 8° C.
  • thermolysin or dispase determines several problems in the preparation of blends, lyophilizates, and compositions for use in tissue dissociation.
  • Thermolysin [EC 3.4.24.27; CAS registry number 9073-78-3] is a thermostable neutral metalloproteinase (also referred to herein as “neutral protease”) produced in the culture broth of Bacillus thermoproteolyticus (Endo, S., J., Ferment. Technol. 40 (1962) 346-353; Matsubara, H., Feder, J., in: 3rd ed., Boyer, P., D., (Ed.), The Enzymes, Vol. 3, Academic Press, New York, 1971, pp. 721-795).
  • LIBERASE enzymes commercially available from Roche Diagnostics GmbH, Mannheim, Germany
  • LIBERASE enzyme technology comprises the methods for purifying Clostridial collagenase isoforms to high specific activity, and for blending them together with high specific activity neutral protease in optimal ratios for effective dissociation of primary tissues.
  • highly purified collagenase I and collagenase II are collected. These two collagenase isoforms are blended in a predetermined ratio with each other, and with a non-Clostridial neutral protease.
  • the type of neutral protease is specifically chosen, and differs according to the final product.
  • LIBERASE Blendzyme 1 contains the neutral protease dispase
  • LIBERASE Blendzyme 2 contains the neutral protease thermolysin.
  • LIBERASE enzymes are available for customers as lyophilizates; the same applies to a number of products from other manufacturers (e.g., Worthington Biochemical Corporation, Lakewood, N.J., USA) for the same intended use.
  • the lyophilizates known to the art comprise one or more collagenase enzyme and/or one or more further protease, such as (but not limited to) thermolysin and dispase.
  • the lyophilizates comprise certain helper substances which are present in the enzyme preparation or enzyme mixture, and which stabilize one or more enzymes in solution and/or during lyophilization.
  • certain proteolytic enzymes can be crystallized in the presence of a helper substance. The solid material obtained upon crystallization can therefore also contain the helper substance.
  • Freeze-drying also referred to as lyophilization or cryodesiccation, is a dehydration process typically used to preserve a perishable material or make the material more convenient for storage and/or transport. Freeze-drying works by freezing the material and then reducing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to gas.
  • any lyophilisate comprising one or more proteases has to be dissolved.
  • the inventors have surprisingly found that one can dramatically reduce the time needed to dissolve a lyophilisate comprising a blend of collagenase enzymes and a neutral protease.
  • the key to an enhanced solubility appears to be certain ionic compounds.
  • lyophilized protease material gains an improved contact with the aqueous solvent which aids dissolving the proteases.
  • the time between contacting the lyophilisate with the aqueous solvent and complete solubilization of the lyophilizate is a crucial parameter limiting the quality of the proteolytic agents.
  • a first embodiment of the invention is a solid composition obtainable by the steps of (a) preparing a homogeneous solution of an acetate-free preparation of a neutral protease in an aqueous acetate-free low-salt solution; (b) adding a neutral salt to the homogeneous solution of step (a) and dissolving the neutral salt, thereby making a stabilized solution wherein said stabilized solution additionally comprises a buffer salt buffering in the range of about pH 6 to about pH 8.5, and wherein the stabilized solution further comprises calcium chloride; (c) mixing the stabilized solution of step (b) with an acetate-free preparation of one or more proteolytic enzymes with collagenase activity, and making a homogeneous solution; (d) freeze-drying the solution of step (c), thereby obtaining the solid composition of the invention.
  • a further embodiment of the invention is a solid composition obtainable by the steps of (a) dissolving an acetate-free preparation of a neutral protease, preferably thermolysin from Bacillus thermoproteolyticus , in an aqueous acetate-free low-salt solution, wherein the solution comprises a buffer salt capable of buffering in the range of about pH 6 to about pH 8.5 and further comprises calcium chloride, and making a homogeneous solution; (b) adding a neutral salt to the homogeneous solution of step (a) and dissolving the neutral salt, thereby making a stabilized solution; (c) mixing the stabilized solution of step (b) with an acetate-free preparation of one or more proteolytic enzymes with collagenase activity, and making a homogeneous solution; (d) freeze-drying the solution of step (c); thereby obtaining the solid composition of the invention.
  • a neutral protease preferably thermolysin from Bacillus thermoproteolyticus
  • a further embodiment of the invention is a method to produce a composition according to the invention, the method comprising the steps of (a) dissolving an acetate-free preparation of a neutral protease, preferably thermolysin from Bacillus thermoproteolyticus , in an aqueous acetate-free low-salt solution, wherein the solution comprises a buffer salt capable of buffering in the range of about pH 6 to about pH 8.5 and further comprises calcium chloride, and making a homogeneous solution; (b) adding a neutral salt to the homogeneous solution of step (a) and dissolving the neutral salt, thereby making a stabilized solution; (c) mixing the stabilized solution of step (b) with an acetate-free preparation of one or more proteolytic enzymes with collagenase activity, and making a homogeneous solution; (d) freeze-drying the solution of step (c); thereby producing the solid composition of the invention.
  • a neutral protease preferably thermolysin from Bacillus thermo
  • Another embodiment of the invention is a solid composition comprising one or more proteolytic enzymes with collagenase activity and a neutral protease characterized in that the composition is free of acetate salt.
  • a further embodiment of the invention is a method to prepare a solution with proteases, comprising the step of contacting a composition according to the invention with water.
  • a further embodiment of the invention is an aqueous solution comprising water and a composition according to the invention.
  • a further embodiment of the invention is the use of an aqueous solution according to the invention for treating a biological tissue.
  • kits comprising one or more containers containing a composition according to the invention.
  • FIG. 1 shows photographs of lyophilizates of protease blends.
  • FIG. 1A is a lyophilizate with unordered clusters of crystals.
  • FIG. 1B is a lyophilizate with crystals having a lamellae- or blade-like structure.
  • the surface area of a lyophilizate was greatly increased when the lyophilizate was prepared free of acetate salts.
  • the lyophilizate according to the invention has a lamellar structure with an increased surface, compared to the lyophilizates known to the state of the art.
  • the lyophilizates according to the invention have enhanced characteristics as to the time needed to dissolve them. This property is of great advantage because the time during which the proteases in solution degrade each other is significantly reduced.
  • the lyophilizates according to the invention are dissolved to form clear, homogeneous solutions. That is to say, no precipitate occurs. Thereby protease solutions of high, reproducible quality are provided.
  • a compound means one compound or more than one compound.
  • a range of numerical values such as a concentration range
  • the range is indicated by the word “between”, followed by a first value n1 and a second value n2.
  • the lower boundary of the designated range is understood as being the value equal to or higher than the first value.
  • the higher boundary of the designated range is understood as being the value which is equal to or lower than the second value.
  • a value x in the designated range is given by n1 ⁇ x ⁇ n2.
  • a “mixture” is a substance made by combining two or more different materials with no chemical reaction occurring. The objects do not bond together in a mixture. A mixture can usually be separated back into its original components. Mixtures are the product of a mechanical blending or mixing of chemical substances like elements and compounds, without chemical bonding or other chemical change, so that each ingredient substance retains its own chemical properties and makeup. While there are no chemical changes in a mixture, physical properties of a mixture, such as its melting point, may differ from those of its components.
  • a lyophilizate is an example for a mixture which is a solid.
  • such a mixture comprises one or more proteinaceous compounds.
  • these are one or more proteolytic enzymes with collagenase activity and a neutral protease.
  • the neutral protease is thermolysis.
  • a lyophilizate additionally comprises a buffer salt, and further helper compounds which stabilize the proteinaceous compounds.
  • the helper compounds can be ionic or non-ionic. Examples for ionic compounds are an organic salt and an inorganic salt. Examples for non-ionic compounds are an organic polymer (such as, but not limited to polyethylene glycol, and starch) and a polyol (such as, but not limited to a sugar and a sugar alcohol). Further helper compounds are possible.
  • Mixtures are either heterogeneous or homogeneous.
  • Homogeneous mixtures are mixtures that have definite, consistent properties. Particles are uniformly spread. For example, any amount of a given mixture has the same composition and properties.
  • a homogeneous mixture is a uniform mixture consisting of only one phase.
  • a solution is a homogeneous mixture of one or more substances (the solutes) dissolved (i.e., dissociated) in another substance (the solvent).
  • the solutes i.e., dissociated
  • a common example would be a solid dissolving into a liquid (i.e., salt or protein dissolving in water).
  • Solubility is a compound property.
  • the amount of a substance that can dissolve in a solution can be variable.
  • non-homogeneous (heterogeneous) mixtures are a colloid and a suspension.
  • a suspension is understood as being a heterogeneous fluid containing solid particles that are sufficiently large for sedimentation. Unlike colloids, the suspended particles settle over time if left undisturbed. This distinguishes a suspension from a colloid in which the suspended particles are smaller and do not settle.
  • the dissolved substance does not exist as a solid, and solute(s) and solvent are homogeneously mixed.
  • the term “stability” of a solution refers to the tendency of the dissolved substance to remain in the dissolved state. That is to say, the term refers to the ability of the solution to remain homogeneous during a given time interval. Stability can therefore be characterized in a quantifying way by determining said time interval.
  • the dissolved substance in a first solution characterized by a lower stability exhibits a higher tendency to precipitate or form a colloid, as opposed to a second solution characterized by a higher stability in which said tendency is lower.
  • said first solution becomes a heterogeneous mixture whereas said second solution remains a homogeneous mixture.
  • Turbidity is a measure of water cloudiness caused by the presence of particles in a suspension or a colloid.
  • There are several practical ways of determining turbidity the most direct being some measure of attenuation (that is, reduction in strength) of light as it passes through a sample column of water.
  • one way to determine turbidity is visual inspection, i.e., inspection by eye.
  • Optical density (also referred to as “OD”) denotes a unit-less measure of the transmittance of an optical element for a given length at a given wavelength ⁇ :
  • composition of a lyophilizate has a consequence for the amount of the solid matter which can be dissolved in a given solvent.
  • composition of a lyophilizate also significantly impacts on the time needed for dissolving the solid matter in the solvent.
  • a central object of the invention was to provide compositions and conditions which accelerate the formation of aqueous solutions of lyophilizates containing a certain amount of proteinaceous constituents. To this end, some further theoretical background is presented aiding the understanding of the invention.
  • the term “sink conditions” describes a dissolution system that is sufficiently dilute so that the dissolution process is not impeded by approach to saturation of the compound of interest.
  • the compound of interest can be each constituent of a particular lyophilizate or the lyophilizate itself.
  • solubility of a compound of interest at a given temperature is that of solubility of a compound of interest at a given temperature.
  • the volume of solvent or the acceptability of a particular solvent can be determined.
  • sink conditions are considered to exist if, at the dissolution of 100% of the highest strength of the lyophilizate to be tested, a concentration of preferably not more than about 2/3, more preferred not more than about 1/2, even more preferred not more than about 1/3 of saturation is achieved.
  • the dissolution of a solid in a bulk liquid is a dynamic process, since molecules migrate from the solid particle into the diffusion layer that surrounds the particle. Then, these molecules diffuse from the diffusion layer into the bulk solution. Provided that during the dissolution of the particles so-called sink conditions are met, the dissolution kinetics are described by Equation (1).
  • the concentration of the molecule in the bulk solution (c(t)) can be calculated from the concentration of the molecule in the diffusion layer or the so-called solubility of the drug (c s ), the time (t) and the rate constant of dissolution (k).
  • the latter can be calculated by Equation (2) from the surface of the particles (S), the diffusion coefficient of the dissolved molecule ( ⁇ ), the volume of the bulk solution (V s ) and the thickness of the diffusion layer (h).
  • the diffusion coefficient ( ⁇ ) can be calculated from the Boltzmann constant (k b ), the temperature (T), the viscosity of the bulk solution ( ⁇ ) and the hydrodynamic radius of the dissolved molecule (r).
  • the surface area (S) of a given volume of a solid lyophilizate is determined by the surface area of the particles.
  • Substitution in Equation (2) shows that for ideal particles the rate constant of dissolution (k) is inversely proportional to the diameter of the particles.
  • the dissolution behavior of a lyophilizate is the result of the cumulative effect of all particles in the solid matter.
  • a first embodiment of the invention is a solid composition obtainable by the steps of (a) preparing a homogeneous solution of an acetate-free preparation of a neutral protease in an aqueous acetate-free low-salt solution; (b) adding a neutral salt to the homogeneous solution of step (a) and dissolving the neutral salt, thereby making a stabilized solution wherein said stabilized solution additionally comprises a buffer salt buffering in the range of about pH 6 to about pH 8.5, and wherein the stabilized solution further comprises calcium chloride; (c) mixing the stabilized solution of step (b) with an acetate-free preparation of one or more proteolytic enzymes with collagenase activity, and making a homogeneous solution; (d) freeze-drying the solution of step (c), thereby obtaining the solid composition of the invention.
  • compositions according to the invention are lyophilizates, that is to say products of a freeze-drying process.
  • Said compositions typically have a residual moisture content which is in the range of about 0.01% [w/w] to about 5% [w/w], preferred in the range of 0.1% to 3% [w/w], even more preferred in the range of 1% to 2% [w/w].
  • the skilled person aims at minimizing the moisture content since this factor advantageously influences product shelf life.
  • the lyophilized composition comprising one or more proteolytic enzymes with collagenase activity and one or more neutral proteases is characterized by an enhanced solubility in an aqueous solvent, preferably water, if the composition is free of acetate salt.
  • an acetate salt are sodium acetate, potassium acetate and calcium acetate.
  • thermolysin crystals according to this state-of-the-art process form in the presence of calcium acetate, and this acetate salt is comprised in the crystals.
  • this acetate salt is comprised in the crystals.
  • the acetate crystals can be dissolved but the solution of the neutral protease is instable in that thermolysin tends to precipitate.
  • thermolysin is based on the first finding that the stability of a homogeneous solution of thermolysin is enhanced by the absence of acetate ions. For this reason, an acetate-free preparation of thermolysin has to be provided and used, in order to practice the present invention. To this end, the THERMOASE preparation of thermolysin can be used.
  • thermolysin a stable solution of thermolysin is obtained when (i) THERMOASE is dissolved in a low-salt buffer to yield a homogeneous solution, and (ii) a neutral salt, preferably sodium chloride, is dissolved subsequently in said homogeneous solution of (i).
  • an embodiment of the invention is a solid composition obtainable by the steps of (a) dissolving an acetate-free preparation of a neutral protease, preferably thermolysin from Bacillus thermoproteolyticus , in an aqueous acetate-free low-salt solution, wherein the solution comprises a buffer salt capable of buffering in the range of about pH 6 to about pH 8.5 and further comprises calcium chloride, and making a homogeneous solution; (b) adding a neutral salt to the homogeneous solution of step (a) and dissolving the neutral salt, thereby making a stabilized solution; (c) mixing the stabilized solution of step (b) with an acetate-free preparation of one or more proteolytic enzymes with collagenase activity, and making a homogeneous solution; (d) freeze-drying the solution of step (c); thereby obtaining the solid composition of the invention.
  • a neutral protease preferably thermolysin from Bacillus thermoproteolyticus
  • the low-salt solution of step (a) preferably comprises (and more preferred consists of) water, CaCl 2 and an organic buffer salt.
  • the preferred buffer salt is HEPES but other buffer salts are possible.
  • the aggregate concentration of dissolved salts in the low-salt solution of step (a) is preferably in the range of about 1 mM to about 250 mM, more preferred in the range of about 5 mM to about 100 mM, even more preferred in the range of about 10 mM to about 50 mM, and most preferred about 25 mM.
  • the neutral salt in step (b) is preferably sodium chloride.
  • the solution of the neutral protease prior to step (c) is subjected to an adjustment of dissolved ions and/or a removal of low molecular weight protein fragments. This can be done, for example, by way of diafiltration.
  • a very much preferred stabilized solution obtained in step (b) preferably comprises thermolysin at a concentration in the range of about 0.5 mg/ml to about 5 mg/ml, more preferred in the range of about 1 mg/ml to about 3 mg/ml.
  • the non-proteinaceous compounds in the solution preferably comprise CaCl 2 , a neutral salt, and an organic buffer salt capable of buffering in the range of about pH 6 to about pH 8.5.
  • the conductivity of the stabilized solution obtained in step (b) is preferably in the range of about 20 mS/cm to about 23 mS/cm.
  • a main advantage of the stabilized solution is that the neutral protease, particularly thermolysin, remains stable in homogeneous solution for a longer time, compared to the situation before when state-of-the-art methods involving acetate-containing preparations were used.
  • the neutral protease particularly thermolysin
  • thermolysin In blending processes aiming at the formulation of mixtures of several proteases including thermolysin, enhanced stability of the latter protease in solution allows the handling of larger quantities. Thus, more efficient and economic blending processes are possible on the basis if the present invention.
  • the total protein content in the homogeneous solution made in step (c) is preferably in the range of about 1 mg/ml to about 150 mg/ml, more preferred in the range of about 5 mg/ml to about 100 mg/ml.
  • the concentration of CaCl 2 in said homogeneous solution made in step (c) is preferably in the range of about 1 mM to about 10 mM, more preferred in the range of about 3 mM to about 5 mM.
  • the concentration of the neutral salt in said homogeneous solution made in step (c) is preferably in the range of about 50 mM to about 500 mM, more preferred in the range of about 50 mM to about 250 mM, even more preferred in the range of about 50 mM to about 200 mM or less than 200 mM.
  • a solid composition (lyophilizate) is obtained, wherein the composition is free of acetate salt and wherein the composition comprises one or more proteolytic enzymes with collagenase activity and a neutral protease.
  • the lyophilizate consists of crystalline matter consisting of lamellae which are aligned in parallel (see also FIG. 1 ).
  • the structure of the lyophilizate provides a very large surface. As a result, upon contacting the lyophilizate with water, the solid matter dissolves very rapidly, usually within 3 min or less.
  • protease solution Rapid formation of a protease solution is an important factor when the protease mixture is to be used in the dissociation of tissue into single cells.
  • the invention provides an important basis for improved methods of tissue dissociation.
  • the present invention comprises the following items which are preferred embodiments:
  • Clostridium histolyticum collagenases I and II were provided separately, in purified form. Before any of the blending procedures described herein, collagenase I and collagenase II were each purified to homogeneity. Each collagenase was provided in dissolved form, at a concentration of 55 mg/ml in a buffer containing 1 mM CaCl 2 , 5 mM HEPES, pH 7.5.
  • the major parts of a freeze drying (lyophilization) procedure include freezing, primary drying (sublimation), and secondary drying (desorption).
  • freezing is to produce a frozen matrix with sufficient crystal structure to allow the sublimating material to escape.
  • Some products form a glassy material and annealing may be required during the freezing process. Annealing, first lowering the temperature then raising the temperature and then lowering it again, locks the constituents in place and then allows the crystals to grow. Freezing can range from 1 hour to 24 hours, depending on the application.
  • Primary drying (sublimation) drives the unbound moisture out of the product. Sublimation occurs under vacuum with the product temperature below its critical temperature. At the end of the primary drying cycle, the product will usually have 3% to 5% moisture content.
  • Secondary drying (desorption) drives the water which is bound by electrostatic and/or ionic forces from the material. This is done by heating the product. Secondary drying can result in moisture levels of 0.5% or less.
  • Lyophilizates of protease blends according to the invention were prepared using a lyophilizator containing a chamber with deep-frozen plates and a separate chamber consisting of a condenser all manufactured by HSK (Germany), and according to the instructions by the manufacturer.
  • An exemplary lyophilization process comprised the following steps and conditions:
  • Step time (hh:mm) temp. pressure comment 1. loading of 00:03 2° C. ambient enzyme solution pressure 2. Freezing 00:30 ⁇ 50° C. ambient temperature may pressure fluctuate by ⁇ 4° C. 3. Freezing 04:00 ⁇ 50° C. ambient pressure 4. Evacuating 00:20 ⁇ 50° C. 0.01 mbar 5. primary drying 00:40 ⁇ 50° C. 0.01 mbar
  • Thermolysin (EC 3.4.24.27) was obtained as a freeze-dried, amorphous powder from Daiwa Kasei K. K. (Japan) containing at least about 60% [w/w] of enzyme protein, about 20% [w/w] of anhydrous Calcium acetate (Ca-acetate or CaAc), and about 10% [w/w] of anhydrous Sodium acetate (Na-acetate or NaAc). In case thermolysin in the presence of acetate was used, twice crystallized thermolysin was used.
  • thermolysin lyophilizate was suspended at a concentration in the range of between about 1% [w/v] to about 3% [w/v] in an ice-cold aqueous solution of Calcium acetate at a concentration of 0.01 M.
  • thermolysin was dissolved by adding 0.2 N NaOH drop-wise and under agitation, until the pH of the solution was between pH 11.0 and pH 11.4. After removal of any undissolved residue (if found to be present), the solution was neutralized to pH 6.0 with 0.2 N acetic acid. Crystallization usually was complete after about 2 days. During the whole process ice-cold temperatures were maintained. The crystals were recovered and freeze-dried under standard conditions.
  • An exemplary lyophilizate of crystallized thermolysin had a protein content of about 66.1% [w/w] and was used in blending experiments.
  • the crystallized thermolysin lyophilizate was suspended in aqueous HEPES buffer (1 mM CaCl 2 , 5 mM HEPES, pH 7.5).
  • HEPES buffer 1 mM CaCl 2 , 5 mM HEPES, pH 7.5.
  • about 7.2 mM NaOH were added (as 0.1 N NaOH) to raise the pH to a value of about pH 11.
  • the pH was lowered to pH 7.5 by admixing 0.5 M HEPES solution which was not titrated with hydroxide and therefore acidic.
  • the final volume of the solution was adjusted with HEPES buffer (1 mM CaCl 2 , 5 mM HEPES, pH 7.5).
  • the final protein concentration in the solution was between 13.1 mg/ml and 13.2 mg/ml.
  • thermolysin solution was homogeneous during the whole process.
  • thermolysin in the present context is a protein mixture consisting of
  • a volume of 6.5 l of an aqueous buffer containing 5 mM CaCl 2 , 20 mM HEPES, pH 7.5 was prepared. An amount of 200 g dry THERMOASE lyophilisate was dissolved in the aqueous buffer and a clear solution was obtained. Subsequently, 935 g solid NaCl was added and dissolved in the solution. The volume of the solution was adjusted to 8 l by adding a further volume of the aqueous buffer containing 5 mM CaCl 2 , 20 mM HEPES, pH 7.5, and mixing by stirring. A homogeneous solution was obtained.
  • the final NaCl concentration in the solution was about 2.3 M.
  • the final concentration of crude thermolysin (about 33% [w/w] of the lyophilisate) in the solution was about 8.25 mg/ml corresponding to a concentration of substantially undegraded thermolysin of about 5.8 mg/ml in the solution.
  • thermolysin produced as described above was stable for at least 20 hours and up to 48 hours. That is to say, no precipitate was formed in the solution during this time. Under other conditions, particularly when the amount of NaCl was dissolved in the buffer prior to adding THERMOASE lyophilizate, beginning precipitation of thermolysin could be observed after about an hour. Therefore the stabilized solution containing thermolysin allowed extensive further processing, including removal of small proteolytic fragments by way of diafiltration, and subsequent blending using larger volumes of thermolysin solution.
  • Diafiltration was performed using a filter with an exclusion limit of about 10 kDa and against an aqueous buffer containing 5 mM CaCl 2 , 170 mM NaCl, 20 mM HEPES, pH 7.5.
  • the final protein concentration after diafiltration was between about 2.25 mg/ml and about 2.75 mg/ml in the diafiltration buffer.
  • the conductivity of the diafiltrated thermolysin solution was 21.2 ⁇ 1 mS/cm.
  • thermolysin was either used directly in blending processes, or the solution was aliquoted and aliquots were frozen and stored at ⁇ 20° C. Frozen aliquots were thawed before use and remained stable afterwards for 6 hours or more.
  • the blend contained collagenase I, collagenase II and thermolysin.
  • Collagenase solutions according to Example 1 and thermolysin solution according to Example 2 were mixed according to Table 1.
  • the resulting mixture had the final volume as indicated in the table and was lyophilized immediately after addition of the last component.
  • the lyophilizate which was obtained consisted of white crystals which in the freeze-drying process formed unordered clusters.
  • the morphology of the lyophilizate corresponded to the lyophilizate depicted in FIG. 1A .
  • the blend contained collagenase I, collagenase II and thermolysin.
  • Collagenase solutions according to Example 1 and thermolysin solution according to Example 2 were mixed according to Table 2.
  • the resulting mixture had the final volume as indicated in the table and was lyophilized immediately after addition of the last component.
  • the lyophilizate which was obtained consisted of white crystals which in the freeze-drying process formed unordered clusters.
  • the morphology of the lyophilizate corresponded to the lyophilizate depicted in FIG. 1A .
  • the blend contained collagenase I, collagenase II and thermolysin.
  • Collagenase solutions according to Example 1 and thermolysin solution according to Example 2 were mixed according to Table 3.
  • the resulting mixture had the final volume as indicated in the table and was lyophilized immediately after addition of the last component.
  • the lyophilizate which was obtained consisted of white crystals which in the freeze-drying process formed unordered clusters.
  • the morphology of the lyophilizate corresponded to the lyophilizate depicted in FIG. 1A .
  • the blend contained collagenase I, collagenase II and thermolysin.
  • Collagenase solutions according to Example 1 and diafiltrated stabilized thermolysin solution according to Example 3 were mixed according to Table 4.
  • the resulting mixture had the final volume as indicated in the table and was lyophilized immediately after addition of the last component.
  • the lyophilizate which was obtained consisted of white crystals which in the freeze-drying process formed lamellae or blade-like structures of which most were aligned in parallel.
  • the morphology of the lyophilizate corresponded to the lyophilizate depicted in FIG. 1B .
  • the blend contained collagenase I, collagenase II and thermolysin.
  • Collagenase solutions according to
  • Example 1 and diafiltrated stabilized thermolysin solution according to Example 3 were mixed according to Table 5.
  • the resulting mixture had the final volume as indicated in the table and was lyophilized immediately after addition of the last component.
  • the lyophilizate which was obtained consisted of white crystals which in the freeze-drying process formed lamellae or blade-like structures of which most were aligned in parallel.
  • the morphology of the lyophilizate corresponded to the lyophilizate depicted in FIG. 1B .
  • the blend contained collagenase I, collagenase II and thermolysin.
  • Collagenase solutions according to Example 1 and diafiltrated stabilized thermolysin solution according to Example 3 were mixed according to Table 6.
  • the resulting mixture had the final volume as indicated in the table and was lyophilized immediately after addition of the last component.
  • the lyophilizate which was obtained consisted of white crystals which in the freeze-drying process formed lamellae or blade-like structures of which most were aligned in parallel.
  • the morphology of the lyophilizate corresponded to the lyophilizate depicted in FIG. 1B .
  • Lyophilized blends in sealed bottles were dissolved in different amounts of purified water, in order to yield solutions with different protein concentrations.
  • the bottles were put on a roller device and agitated at 32 revolutions per minute at 20° C.
  • the time needed to dissolve the lyophilizates was recorded. Recordings were stopped after 75 min, even if solubilization was not complete at this point.
  • Table 7 indicates the time intervals needed for dissolving the lyophilizates according to each of Example 4 to 9. Completeness of solubilization (i.e., the fact whether or not a homogeneous solution was obtained) was assessed by visual inspection or by turbidity measurements.

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US16/865,487 US11459556B2 (en) 2009-03-19 2020-05-04 Methods of preparing a solid composition containing proteases
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US20110021611A1 (en) * 2007-05-18 2011-01-27 Hvidovre Hospital Efficient cell culture system for hepatitis c virus genotype 5a
US8618275B2 (en) 2007-05-18 2013-12-31 Hvidovre Hospital Efficient cell culture system for hepatitis C virus genotype 5A
US20110059512A1 (en) * 2007-12-20 2011-03-10 Gottwein Judith M Efficient cell culture system for hepatitis c virus genotype 6a
US8569472B2 (en) 2007-12-20 2013-10-29 Hvidovre Hospital Efficient cell culture system for hepatitis C virus genotype 6A
US8506969B2 (en) 2008-08-15 2013-08-13 Hvidovre Hospital Efficient cell culture system for hepatitis C virus genotype 7a
US8663653B2 (en) 2008-08-15 2014-03-04 Hvidovre Hospital Efficient cell culture system for hepatitis C virus genotype 2B
US8772022B2 (en) 2008-10-03 2014-07-08 Hvidovre Hospital Hepatitis C virus expressing reporter tagged NS5A protein

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