WO1994024273A1 - Compositions stables a base de collagenase et procedes de preparation - Google Patents

Compositions stables a base de collagenase et procedes de preparation Download PDF

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Publication number
WO1994024273A1
WO1994024273A1 PCT/US1994/004082 US9404082W WO9424273A1 WO 1994024273 A1 WO1994024273 A1 WO 1994024273A1 US 9404082 W US9404082 W US 9404082W WO 9424273 A1 WO9424273 A1 WO 9424273A1
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WIPO (PCT)
Prior art keywords
collagenase
stabilizer
stabilized
composition
solution
Prior art date
Application number
PCT/US1994/004082
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English (en)
Inventor
Catherine Lee
Tan Thanh Dinh
Cynthia Hornacek
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Baxter International Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baxter International Inc. filed Critical Baxter International Inc.
Publication of WO1994024273A1 publication Critical patent/WO1994024273A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to stabilized enzyme compositions and methods for preparing and storing stabilized dry enzyme compositions. More particularly, the present invention is directed toward the proteolytic enzyme, collagenase, and methods for stabilizing collagenase during lyophilization procedures and for producing dry collagenase compositions which are suitable for long-term storage.
  • Proteolytic enzymes are widely utilized in a variety of laboratory and clinical applications. Typically these applications involve cell dissociation and related therapeutic procedures which are benefitted by the ability of proteolytic enzymes to hydrolytically break up or loosen connective tissue networks.
  • bacterial collagenase derived from Clostridium histolyticum has been used to disperse cells in laboratory tissue culture applications. Additionally, collagenase has demonstrated utility in cell isolation procedures such as those associated with isolating pancreatic islets and dispersing a variety of tumor cells.
  • Collagenase has also found use in clinical applications, for example, topically for the treatment of burns, ulcers and other wounds. Collagenase is also utilized in the treatment of Peyronie's disease and as an adjunct to cryoprostatectomy for the removal of retained cryoslough, intervertebral discolysis, and in ophthalmic surgery.
  • collagenase Most commercial collagenase is derived from the bacteria Clostridium histolyticum and, in its crude form, differs widely in hydrolytic activity and purity. Uncontrolled amounts of impurities found in crude collagenase include contaminating bacterial materials, pigment, toxins, and proteolytic enzymes, including clostripain, trypsin, and caseinase.
  • a number of problems are associated with using crude collagenase to digest tissue and isolate embedded cells. For example, proteolytic enzyme impurities will cause the catalytic degradation of the collagenase composition.
  • toxins associated with crude collagenase can be a serious problem for procedures involving both in vivo and in vitro applications. Toxins can disrupt cell membranes, destroy cell viability and generally lower cell yield.
  • Bacterial materials may include variable amounts of bacterial DNA, which potentially can cause immunologic and tumorigenic problems when isolated cells or tissue digestion procedures, involve in vivo applications.
  • noncollagen-specific protein impurities found in crude collagenase may act as sensitizing antigens, which can cause anaphylactic shock if administered to patients.
  • collagenase purification procedures involve chromatographic separation of collagenase from contaminating proteolytic enzymes, toxins, and various unreactive components of crude collagenase.
  • the collagenase is lyophilized to provide a dry composition for storage.
  • the lyophilization procedure and subseguent dry storage is known to result in degradation of the collagenase. This degradation is exemplified by a substantial decrease in its solubility in aqueous systems and a decrease in its specific activity.
  • the present invention accomplishes the above objectives by providing stabilized collagenase compositions and methods for their preparation.
  • Stable collagenase compositions prepared according to the methods of the present invention, retain a high degree of hydrolytic activity and solubility following lyophilization and long term storage. Because these compositions maintain their hydrolytic activity following lyophilization and storage, they are useful in tissue digestion and cell isolation applications which require predictable enzyme performance.
  • the present invention provides for incorporating selected proteins or oligosaccharides with collagenase to stabilize the collagenase. Through the addition of selected concentrations of these components under favorable conditions, the collagenase retains a substantially higher specific activity than it otherwise would have after lyophilization.
  • this composition will continue to retard the degradation of collagenase, ensuring that a higher level of enzyme activity is maintained over extended periods of time.
  • the use of these compositions prevents denaturing during lyophilization and storage, thus increasing the overall solubility of the desiccated enzyme.
  • the consistent specific activity and retention of physical parameters is critical to the development of laboratory reagents and therapeutic clinical applications involving collagenase.
  • As the present invention will enhance the stability of collagenase irrespective of any contaminants, there is no minimum required level of purification. However, a reduction of contaminants will result in a corresponding decrease in the rate of degradation and an increase in the level of stability achieved.
  • any collagenase preparations suitable for use in the compositions of the present invention will be substantially free of pigment, bacterial materials, toxins, and noncollagen-specific enzyme activity. Therefore, a preferred embodiment of the present invention involves the use of collagenase that has been purified to some extent. This may be accomplished in any one of a number of ways well known in the art such as fractionation, centrifugation, and chromatography, or a combination thereof. Following any desired purification of the collagenase, the enzyme is mixed with the stabilizer or stabilizers in a suitable aqueous buffer solution, such as a buffered electrolyte solution, which may contain a variety of different salts to facilitate enzyme solubility, and if desired, a surfactant.
  • a suitable aqueous buffer solution such as a buffered electrolyte solution, which may contain a variety of different salts to facilitate enzyme solubility, and if desired, a surfactant.
  • the mixture is adjusted to the desired pH using a buffer, such as a phosphate or Tris buffer , tris(hydroxymethyl)aminomethane, for example.
  • a buffer such as a phosphate or Tris buffer , tris(hydroxymethyl)aminomethane, for example.
  • a nonionic surfactant preferably is employed, usually at concentrations of about 0.01% to about 0.05% by weight.
  • collagenase may be stabilized by the addition of certain proteins such as globulins or certain oligosaccharides, such as maltose, trehalose, and sucrose.
  • proteins such as globulins or certain oligosaccharides, such as maltose, trehalose, and sucrose.
  • globulins such as human serum albumin and bovine serum albumin are preferred, other animal proteins can be used as well.
  • the sugars in general, any non-reducing sugar can be used in addition to the preferred oligosaccharides.
  • the protein or sugar stabilizers can be present singly or in combination.
  • the present invention combines one or more stabilizers with collagenase in an aqueous buffer for the purpose of stabilization.
  • the concentration of stabilizer can vary, typical values are about 1 mg/mL to about 100 mg/mL of the stabilized collagenase solution.
  • the mixture of stabilizer and buffer is filtered to remove possible contaminants before combining with the collagenase.
  • the volume of liquid used to mix the stabilizer with the collagenase in the final step of the process before lyophilization is referred to as the final mixing volume.
  • the aqueous buffer, containing the mixture of stabilizer and collagenase is gently mixed to insure homogeneity of the solubilized components.
  • the liquid is aliquoted and lyophilized.
  • Lyophilization employs a vacuum to remove any water present in the frozen aqueous solution and results in the deposition of a dry, concentrated film or powder.
  • the concentrated powder consists primarily of the collagenase and stabilizer.
  • the salts used to buffer the solution and small amounts of impurities may also be present.
  • the lyophilization process may be carried out over a range of temperatures extending from 4°C to 37"C and under a high degree of. vacuum, but for the present invention is preferably performed at room temperature (25°C) .
  • the resulting powder, with stabilized enzymatic activity may then be packaged in a variety of forms depending on the intended use. Further objects, features, and advantages of the present invention will become apparent to those skilled in the art from a consideration of the following detailed description of exemplary embodiments.
  • the present invention provides improved stabilization of collagenase and a corresponding increase in specific activity during lyophilization and long-term storage when compared with non-stabilized collagenase.
  • Collagenase treated according to the process herein before lyophilization will reproducibly solubilize and maintain a fairly consistent level of activity over an extended period of time.
  • untreated collagenase will be severely degraded by the lyophilization process and storage, with a corresponding loss of solubility in aqueous solutions.
  • the invention allows these beneficial properties to be realized using small amounts of inexpensive, and easily obtained materials without labor intensive procedures.
  • this process also has the advantage of yielding a product which acts predictably over extended periods of time.
  • concentration and activity of collagenase present in the initial solution will vary with the starting material and enzyme purification procedure, if any.
  • the well known principles of enzyme activity are applicable.
  • Basic experimentation involving techniques designed to optimize and quantify enzyme concentration and total activity can provide the necessary information to determine the initial parameters.
  • Common protein quantification methodology such as enzyme activity assays, electrophoresis, chromatography or spectroscopy may be used for the determination of overall enzyme concentration.
  • the stability of the collagenase may be determined from the specific activity of the enzyme obtained through the use of collagenase assays well known in the art.
  • Collagenase may be stored as an aqueous buffered solution.
  • the collagenase buffer is altered through extensive dialysis of the enzyme.
  • the concentration of the enzyme may be adjusted, for example, through ultrafiltration, to preferred levels which are optimized to increase the efficiency of the process.
  • typical collagenase concentrations in the purified starting material are on the order of about 0.5 mg/ml to about 25 mg/ml.
  • the collagenase is generally concentrated and introduced into a buffer solution in preparation for mixing with the selected stabilizer.
  • the stabilizer is dissolved in the same aqueous buffer solution as the collagenase.
  • the buffers may vary in both their physical properties and volumes added to the mixing vessel. Those familiar with the art will recognize that there are a number of physiologically compatible buffers which can provide a suitable medium for the desired components. Where phosphate buffered saline (PBS) is used it can be used in a range of about pH 5 to pH 8.5.
  • PBS phosphate buffered saline
  • both the buffer containing the stabilizer and the buffer present in the initial collagenase solution are a 0.1 M PBS, pH 7.6, having small amounts of other salts present.
  • the buffer may contain a nonionic surfactant such as Tween-20, available from SIGMA Chemical Co. , present in low concentrations.
  • Tween-20 available from SIGMA Chemical Co.
  • a predetermined amount of collagenase in the desired buffer is combined with a corresponding amount of stabilizing compound or compounds, also in a selected buffer. Additional buffer may then be added to bring the combination up to its final mixing volume. As the final mixing volume will be greater than either the volume of the stabilizing solution or the collagenase solution, allowances must generally be made for the dilution of the enzyme or stabilizer.
  • the concentrated collagenase solution may simply be diluted with buffer to obtain the final mixing volume before a solid form of the stabilizer is added.
  • the final mixing volume which will eventually be lyophilized, may be adjusted according to material limitations and production needs.
  • the preferred stabilizing agents used for the invention consist of selected oligosaccharides or globulins.
  • the globulin is human serum albumin (HSA) , which may be obtained from a number of sources. HSA from Baxter-IV Systems is especially suitable because it has a proven long shelf-life in solution and is approved for in vivo use.
  • HSA human serum albumin
  • the oligosaccharide is either trehalose, maltose, or sucrose.
  • any oligosaccharide with similar properties can be used and will give similar results.
  • Properties indicative of compatibility may include, but are not limited to, hydrophilic and hydrogen bonding properties, solubilization parameter, molecular weight, molecular charge, and isoelectric point.
  • the final concentration of stabilizer sufficient to maintain enzymatic activity is dependent on the physical parameters used in practicing the invention as well as the particular stabilizer selected. Generally favorable stabilization effects have been obtained with stabilizer concentrations of about 0.05% to about 20% of the final mixing volume under compatible conditions. Especially preferred are concentrations of about 0.5%.
  • the lyophilization volumes are determined by the type of apparatus used and optimized according to production demands.
  • a typical lyophilizer is one manufactured by VirTis, which is used with starting aliquot volumes of 5 mL per vial. The lyophilization is continued until the sample is completely desiccated to a dry powder.
  • the vials may be capped, sealed, and stored, or the material may be collected and packaged according to its intended use.
  • enzymatic activity assays are performed.
  • the assay performed can be essentially the same whether the parameters are designed to test the lyophilization recovery or to document the effects of stabilizers on the retention of hydrolytic activity over a long period of time.
  • the collagenase activity is assayed by using a FALGPA, furylacryloyl Leu-Gly-Pro-Ala, synthetic substrate.
  • the activity may be reported in nkat/ml and, when taken as a percentage of a control baseline, may be used as a convenient gauge to determine the retention of hydrolytic activity following lyophilization and/or storage.
  • the nkat/ml unit is defined as nanomoles of substrate hydrolyzed per second by 1.0 ml of enzyme solution under the assay conditions used.
  • As a control aliquots of the final reaction volume containing stabilizer and collagenase are frozen at -80°C and thawed at the appropriate time.
  • the lyophilized samples are rehydrated with the appropriate volume of deionized H 2 0.
  • the volumes are adjusted to provide equivalent concentrations of collagenase in the control and lyophilized samples.
  • the reconstituted collagenase is then assayed to determine the amount of activity remaining.
  • purified collagenase was used because the compositions of the solutions can be better defined than in the case of crude collagenase.
  • the following example illustrates the effect of stabilization of collagenase during lyophilization.
  • Collagenase was purified according to the method described in Bond et al., "Purification and Separation of Individual Collagenases of Clostridium histolyticum Using Red Dye Ligand Chromatography", Biochemistry Vol. 23, No. 13, 3077-3091, 1984. Concentrated solutions of 10% trehalose, 10% sucrose, or 10% HSA stabilizer were mixed in PBS buffer and filtered to remove impurities. A quantity of 0.5 mL of collagenase solution was combined with 5 mL of stabilizer solution or
  • the following example illustrates the retention of hydrolytic activity over a long period of time under different conditions.
  • only one stabilizing agent was used so as to reduce the variation in the study and demonstrate the effects of different temperatures and buffers on stabilized collagenase over time.
  • Example 2 The procedure of Example 1 was followed except that human serum albumin was used as the stabilizer and TWEEN- 20 was not used. Vials of lyophilized samples were stored at 4°C, room temperature, or 37°C during the course of the experiment. At periodic intervals, lyophilized samples were reconstituted with water and assayed along with a frozen control aliquot to determine remaining hydrolytic activity. The results are shown in Table II.
  • the following example illustrates the effect of using a surfactant along with the stabilizer.
  • Example 2 The procedure of Example 2 was followed except that 0.01% TWEEN-20 was used with the buffer. The results are shown in Table 3.

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Abstract

Compositions à base de collagénase stabilisées et procédés destinés à leur préparation. Une solution de collagénase est mélangée avec un tampon et un stabilisateur, puis lyophilisée. Le stabilisateur peut être une protéine ou un olygosaccharide. Ces compositions de collagénase stabilisées retiennent un niveau élevé d'activité hydrolytique et de solubilité suite à la lyophilisation et au stockage de longue durée.
PCT/US1994/004082 1993-04-16 1994-04-13 Compositions stables a base de collagenase et procedes de preparation WO1994024273A1 (fr)

Applications Claiming Priority (2)

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US4901693A 1993-04-16 1993-04-16
US08/049,016 1993-04-16

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041870A1 (fr) * 1995-06-08 1996-12-27 Societa' Cooperativa Centro Ricerche Poly-Tech A Responsabilita' Limitata Compositions hydrosolubles de collagenase et procede de preparation
EP0971232A2 (fr) * 1998-07-05 2000-01-12 Fraunhofer-Gesellschaft Zur Förderung Der Angewandten Forschung E.V. Détermination luminométrique d'ATP
US6818251B2 (en) 2000-09-08 2004-11-16 3M Innovative Properties Company Masking materials and method of use
WO2007006484A1 (fr) * 2005-07-07 2007-01-18 Fidia Farmaceutici S.P.A. Nouvelles compositions pharmaceutiques contenant de l'acide hyaluronique et de la collagénase pour le traitement topique de plaies, de brûlures et d'ulcères

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0115974A1 (fr) * 1983-01-05 1984-08-15 Institut Pasteur Préparation à activité collagénolytique ayant une activité élevée et compositions pharmaceutiques la contenant
EP0260645A1 (fr) * 1986-09-16 1988-03-23 Knoll Ag Système thérapeutique pour l'application locale de composés pharmaceutiques
EP0468180A2 (fr) * 1990-06-13 1992-01-29 SCLAVO S.p.A. Solutions de tampons contenant de la collagenase, leurs préparation et usage pour diluer des serums humains
JPH0494685A (ja) * 1990-08-08 1992-03-26 Morishita Jintan Kk ショウガ酵素製剤及びその製法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0115974A1 (fr) * 1983-01-05 1984-08-15 Institut Pasteur Préparation à activité collagénolytique ayant une activité élevée et compositions pharmaceutiques la contenant
EP0260645A1 (fr) * 1986-09-16 1988-03-23 Knoll Ag Système thérapeutique pour l'application locale de composés pharmaceutiques
EP0468180A2 (fr) * 1990-06-13 1992-01-29 SCLAVO S.p.A. Solutions de tampons contenant de la collagenase, leurs préparation et usage pour diluer des serums humains
JPH0494685A (ja) * 1990-08-08 1992-03-26 Morishita Jintan Kk ショウガ酵素製剤及びその製法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch Week 9219, Derwent World Patents Index; Class B04, AN 92-156055 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041870A1 (fr) * 1995-06-08 1996-12-27 Societa' Cooperativa Centro Ricerche Poly-Tech A Responsabilita' Limitata Compositions hydrosolubles de collagenase et procede de preparation
EP0971232A2 (fr) * 1998-07-05 2000-01-12 Fraunhofer-Gesellschaft Zur Förderung Der Angewandten Forschung E.V. Détermination luminométrique d'ATP
EP0971232A3 (fr) * 1998-07-05 2001-04-18 Fraunhofer-Gesellschaft Zur Förderung Der Angewandten Forschung E.V. Détermination luminométrique d'ATP
US6818251B2 (en) 2000-09-08 2004-11-16 3M Innovative Properties Company Masking materials and method of use
WO2007006484A1 (fr) * 2005-07-07 2007-01-18 Fidia Farmaceutici S.P.A. Nouvelles compositions pharmaceutiques contenant de l'acide hyaluronique et de la collagénase pour le traitement topique de plaies, de brûlures et d'ulcères
US8900576B2 (en) 2005-07-07 2014-12-02 Fidia Farmaceutici S.P.A Pharmaceutical compositions containing hyaluronic acid and collagenase for the topical treatment of wounds, burns and ulcers
US9603910B2 (en) 2005-07-07 2017-03-28 Fidia Farmaceutici S.P.A. Pharmaceutical compositions containing hyaluronic acid and collagenase for the topical treatment of wounds, burns and ulcers
US11040089B2 (en) 2005-07-07 2021-06-22 Fidia Farmaceutici S.P.A Pharmaceutical compositions containing hyaluronic acid and collagenase for the topical treatment of wounds, burns and ulcers

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