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US-12618058-B2 - Blends containing proteases

US12618058B2US 12618058 B2US12618058 B2US 12618058B2US-12618058-B2

Abstract

Described are compositions, in particular lyophilizates, containing proteolytic enzymes, and methods for producing the compositions. Typically these compositions contain one or more proteases with collagenase activity and a neutral protease, for example, thermolysin. The compositions are free of acetate salts. Surprisingly, such compositions can be dissolved in water more rapidly than lyophilized protease mixtures of the state of the art.

Inventors

  • Werner Hoelke
  • Michaela Fischer
  • Johann-Peter Thalhofer
  • Markus Weber

Assignees

  • ROCHE DIAGNOSTICS OPERATIONS, INC.

Dates

Publication Date
20260505
Application Date
20220826
Priority Date
20090319

Claims (16)

  1. 1 . A solid composition obtained or obtainable by the steps of (a) preparing a first homogeneous solution of an acetate-free preparation of a neutral protease (NP) in an aqueous acetate-free low-salt solution, the aggregate concentration of salt(s) in said low-salt solution being in the range of about 1 mM to about 250 mM; (b) adding a neutral salt to the first 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 (C), and making a second homogeneous solution; and (d) freeze-drying the solution of step (c), thereby obtaining the solid composition, wherein: the neutral protease is thermolysin; the neutral salt is sodium chloride; and a total protein content in the second homogeneous solution is in the range of 1 mg/ml to 150 mg/ml, a concentration of the calcium chloride is in the range of 1 mM to about 10 mM, and a concentration of sodium chloride in the second homogeneous solution is in the range of about 50 mM to about 500 mM.
  2. 2 . The solid composition of claim 1 , wherein the neutral protease is thermolysin from Bacillus thermoproteolyticus.
  3. 3 . The solid composition of claim 1 , wherein the acetate-free low-salt solution in step (a) comprises a buffer salt buffering in the range of about pH 6 to about pH 8.5.
  4. 4 . The solid composition of claim 1 , wherein the acetate-free low-salt solution in step (a) further comprises calcium chloride.
  5. 5 . The solid composition of claim 1 , wherein the stabilized solution in step (b) comprises the neutral protease at a concentration in the range of about 0.5 mg/ml to about 5 mg/ml.
  6. 6 . The solid composition of claim 1 , wherein in step (b) the conductivity of the stabilized solution is in the range of about 20 mS/cm to about 23 mS/cm.
  7. 7 . The solid composition of claim 1 , wherein in step (b) the buffer salt is a compound selected from the group consisting of BES (N,NBis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Tris (2-Amino-2-hydroxymethyl)propane-1,3-diol), BisTris (Bis(2-hydroxyethyl)amino-tris(hy-droxymethyl)methane), BisTris propane (1,3-bis(tris (hydroxymethyl)methylamino)propane), HEPES (N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid), MES (2-(Nmorpholino) ethanesulfonic acid), MOPS (3-(Nmorpholino) propanesulfonic acid), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), PIPES (Piperazine-1,4-bis(2-ethanesulfonic acid)), TAPS (N-Tris(hydroxymethyl) methyl-3-aminopropanesulfonic acid), TES (N-Tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid), TEA (Trietha-nolamine), and Tricine (N-(2-Hydroxy-1,1-bis (hydroxymethyl)ethyl)glycine.
  8. 8 . The solid composition of claim 1 , wherein said method further comprises removing small proteolytic fragments from the solution produced by step (b) via diafiltration before performing step (c).
  9. 9 . The solid composition of claim 1 , wherein the composition comprises sodium chloride, calcium chloride, and an organic buffer salt.
  10. 10 . The solid composition of claim 1 , wherein the solid composition is crystalline matter consisting of lamellae which are aligned in parallel.
  11. 11 . The solid composition of claim 1 , wherein the weight-by-weight ratio of all proteases present in the composition and sodium chloride (=(NP+C)/NaCl [w/w]) is in the range of about 0.1 to about 5.
  12. 12 . The solid composition of claim 1 , wherein the weight-by-weight ratio of all proteases present in the composition and calcium chloride hexahydrate (=(NP+C)/CaCl 2 [w/w]) is in the range of about 10 to about 500.
  13. 13 . The solid composition of claim 1 , wherein the weight-by-weight ratio of all proteases present in the composition and the buffer salt (=(NP+C)/buffer [w/w]) is in the range of about 0.05 to about 2.
  14. 14 . The solid composition of claim 1 , wherein step (a) comprises preparing a homogeneous solution of an acetate-free preparation of thermolysin in an aqueous acetate-free low-salt solution, wherein the acetate-free low-salt solution comprises a buffer salt buffering in the range of about pH 6 to about pH 8.5, wherein the acetate-free low-salt solution further comprises calcium chloride, and wherein the aggregate concentration of salt(s) in the acetate-free low-salt solution is in the range of about 1 mM to about 250 mM.
  15. 15 . The solid composition of claim 1 , wherein step (b) comprises adding sodium chloride to the homogeneous solution of step (a) and dissolving the sodium chloride, thereby making a solution, wherein the solution comprises the buffer salt buffering in the range of about pH 6 to about pH 8.5, and wherein the solution comprises the thermolysin at a concentration in the range of about 0.5 mg/ml to about 5 mg/ml, and calcium chloride, and the conductivity of the solution is in the range of about 20 mS/cm to about 23 mS/cm, wherein the buffer salt is a compound selected from the group consisting of BES (N,NBis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Tris (2-Amino-2-hydroxymethyl)propane-1,3-diol), BisTris (Bis(2-hydroxyethyl)amino-tris(hy-droxymethyl)methane), BisTris propane (1,3-bis(tris (hydroxymethyl)methylamino)propane), HEPES (N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid), MES (2-(Nmorpholino) ethanesulfonic acid), MOPS (3-(Nmorpholino)propanesulfonic acid), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), PIPES (Piperazine-1,4-bis(2-ethanesulfonic acid)), TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), TES (N-Tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid), TEA (Trietha-nolamine), and Tricine (N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine.
  16. 16 . The solid composition of claim 1 , wherein said method comprises the steps of (a) preparing a homogeneous solution of an acetate-free preparation of thermolysin in an aqueous acetate-free low-salt solution, wherein the acetate-free low-salt solution comprises a buffer salt buffering in the range of about pH 6 to about pH 8.5, wherein the acetate-free low-salt solution further comprises calcium chloride, and wherein the aggregate concentration of salt(s) in the acetate-free low-salt solution is in the range of about 1 mM to about 250 mM; (b) adding sodium chloride to the homogeneous solution of step (a) and dissolving the sodium chloride, thereby making a solution, wherein the solution comprises the buffer salt buffering in the range of about pH 6 to about pH 8.5, and wherein the solution comprises the thermolysin at a concentration in the range of about 0.5 mg/ml to about 5 mg/ml, and calcium chloride, and the conductivity of the solution is in the range of about 20 mS/cm to about 23 mS/cm, wherein the buffer salt is a compound selected from the group consisting of BES (N,NBis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Tris (2-Amino-2-hydroxymethyl)propane-1,3-diol), BisTris (Bis(2-hydroxyethyl)amino-tris(hy-droxymethyl)methane), BisTris propane (1,3-bis(tris (hydroxymethyl)methylamino)propane), HEPES (N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid), MES (2-(Nmorpholino) ethanesulfonic acid), MOPS (3-(Nmorpholino)propanesulfonic acid), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), PIPES (Piperazine-1,4-bis(2-ethanesulfonic acid)), TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), TES (N-Tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid), TEA (Trietha-nolamine), and Tricine (N-(2-Hydroxy-1,1-bis (hydroxymethyl)ethyl)glycine; (b1) removing small proteolytic fragments from the solution produced by step (b) via diafiltration; (c) mixing the solution of step (b1) with an acetate-free preparation of one or more proteolytic enzymes with collagenase activity, and making a homogeneous solution, wherein the total protein content in the homogeneous solution is in the range of 1 mg/ml to 150 mg/ml, the concentration of calcium chloride is in the range of 1 mM to about 10 mM, and the concentration of sodium chloride is in the range of 50 mM to 500 mM; and (d) freeze-drying the solution of step (c), thereby obtaining the solid composition, wherein the solid composition is crystalline matter consisting of lamellae which are aligned in parallel.

Description

RELATED APPLICATIONS This application is a continuation application of U.S. application Ser. No. 16/865,487 filed May 4, 2020, which is a continuation of U.S. application Ser. No. 15/595,126 filed May 15, 2017 (abandoned), which is a continuation of U.S. application Ser. No. 14/699,299 filed Apr. 29, 2015 (abandoned), which is a continuation of U.S. application Ser. No. 13/230,927 filed Sep. 13, 2011 (abandoned), which is a continuation of PCT/EP2010/001687 filed Mar. 17, 2010, and claims priority to European Application Nos. EP 09005257.2 filed Apr. 9, 2009 and EP 09003967.8 filed Mar. 19, 2009, the disclosures of which are hereby incorporated by reference in their entity. FIELD The present invention provides compositions, preferably lyophilizates, comprising proteolytic enzymes. Preferred compositions comprise one or more proteases with collagenase activity and a neutral protease, preferably thermolysin. According to the invention, the compositions are free of acetate salts. Surprisingly, such compositions can be dissolved in water more rapidly than lyophilized protease mixtures of the state of the art. BACKGROUND 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. Despite the widespread use of enzymes for these applications over the years, many parameters influencing the tissue dissociation process and the harvesting of dissociated target cells are not well understood. As a result, the skilled person's choice of one particular protease or blend of proteases, or one certain technique over another has often been arbitrary and based more on past experience than on an understanding of why the protease-mediated process works and what modifications could lead to even better results. Due to the fact that collagen has a major structural role in the preferred tissues, 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. Several 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. In aqueous solution, 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 Ca2+ ions that are essential in the collagenase structure. 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. Besides, 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. However, the low stability of collagenase isoforms in the presence of water and particularly in the added presence of a further protease such as 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 t