U.S. patent application number 15/595126 was filed with the patent office on 2017-08-31 for blends containing proteases.
The applicant listed for this patent is Roche Diagnostics Operations, Inc.. Invention is credited to Michaela Fischer, Werner Hoelke, Johann-Peter Thalhofer, Markus Weber.
Application Number | 20170247679 15/595126 |
Document ID | / |
Family ID | 42211779 |
Filed Date | 2017-08-31 |
United States Patent
Application |
20170247679 |
Kind Code |
A1 |
Hoelke; Werner ; et
al. |
August 31, 2017 |
BLENDS CONTAINING PROTEASES
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: |
Hoelke; Werner; (Penzberg,
DE) ; Fischer; Michaela; (Geretsried, DE) ;
Thalhofer; Johann-Peter; (Weilheim, DE) ; Weber;
Markus; (Habach, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Diagnostics Operations, Inc. |
Indianapolis |
IN |
US |
|
|
Family ID: |
42211779 |
Appl. No.: |
15/595126 |
Filed: |
May 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14699299 |
Apr 29, 2015 |
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15595126 |
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13230927 |
Sep 13, 2011 |
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14699299 |
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PCT/EP2010/001687 |
Mar 17, 2010 |
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13230927 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/54 20130101; C12N
9/6491 20130101; C12Y 304/24003 20130101; C12N 9/52 20130101; C12N
9/96 20130101; C12Y 304/24027 20130101 |
International
Class: |
C12N 9/96 20060101
C12N009/96; C12N 9/54 20060101 C12N009/54; C12N 9/52 20060101
C12N009/52 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 19, 2009 |
EP |
09003967.8 |
Apr 9, 2009 |
EP |
09005257.2 |
Claims
1. A method for producing a solid composition comprising the steps
of: preparing a homogeneous solution of an acetate-free preparation
of a neutral protease (NP) in an aqueous acetate-free, low-salt
solution, wherein the low-salt solution has an aggregate
concentration of salt(s) in the range of about 1 mM to about 250
mM; adding a neutral salt to the homogeneous solution and
dissolving the neutral salt, thereby making a stabilized solution,
wherein the stabilized solution additionally comprises a buffer
salt buffering in a range of about pH 6 to about pH 8.5, and
wherein the stabilized solution further comprises calcium chloride;
mixing the stabilized solution with an acetate-free preparation of
one or more proteolytic enzymes with collagenase activity (C) and
making a homogeneous solution; and freeze-drying the homogeneous
solution, thereby obtaining the solid composition.
2. The method according to claim 1, wherein the neutral protease is
thermolysin from Bacillus thermoproteolyticus.
3. The method according to claim 1, wherein a weight/weight ratio
of the neutral protease versus all proteases present in the
composition (NP/(NP+C)) is in a range of about 1 to about 25.
4. The method according to claim 1, wherein the neutral salt is
sodium chloride and a weight/weight ratio of all proteases present
in the composition versus sodium chloride ((NP+C)/NaCl) is in a
range of about 0.1 to about 5.
5. The method according to claim 1, wherein a weight/weight ratio
of all proteases present in the composition versus calcium chloride
((NP+C)/CaCl.sub.2) is in a range of about 10 to about 500.
6. The method according to claim 1, wherein a weight/weight ratio
of all proteases present in the composition versus the buffer salt
((NP+C)/buffer) is in a range of about 0.05 to about 2.
7. The method of claim 1, wherein the solid composition further
comprises sodium chloride, calcium chloride, and an organic buffer
salt.
8. The method of claim 1 further comprising the step of contacting
the solid composition obtained in said step of freeze-drying with
water.
9. The method of claim 8, wherein upon said further step of
contacting, the proteases in solution have a concentration in a
range of about 5 mg/ml to about 50 mg/ml.
10. The method of claim 9, wherein upon said further step of
contacting, a homogeneous solution is obtained within a period of
about 3 minutes or less than 3 minutes.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/699,299 filed Apr. 29, 2015, now 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 applications 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 entirety.
FIELD
[0002] 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
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.degree.
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.degree.
C., preferably between about 2.degree. C. and 8.degree. 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.
[0009] 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). It requires one zinc ion for enzyme activity and four
calcium ions for structural stability (Latt, S., A., et al.,
Biochem. Biophys. Res. Commun. 37 (1969) 333-339; Feder, J., et
al., Biochemistry 10 (1971) 4552-4556; Tajima, M., et al., Eur. J.
Biochem. 64 (1976) 243-247) and catalyzes specifically the
hydrolysis of peptide bonds containing hydrophobic amino acid
residues (Morihara, K., and Tsuzuki, H., Eur. J. Biochem. 15 (1970)
374-380; Inouye, K., et al., Biochem. J. 315 (1996) 133-138).
[0010] Roche Applied Science has developed LIBERASE enzymes
(commercially available from Roche Diagnostics GmbH, Mannheim,
Germany) which are blends of highly purified enzymes, designed to
improve the quality and reproducibility of tissue dissociation, and
improve the health of isolated cells. 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. In the manufacturing
process 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. For example,
LIBERASE Blendzyme 1 contains the neutral protease dispase, and
LIBERASE Blendzyme 2 contains the neutral protease thermolysin.
[0011] 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. In addition, 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. In addition, 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.
[0012] 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.
[0013] Before use, 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. In the presence of these 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. The shorter this
period, the less the proteolytic enzymes degrade each other, the
more proteolytic activity is applied to the target tissue to be
dissociated. Apart from shortening the absolute time span needed
for solubilizing the lyophilizate, the variation of said time span
could be minimized, too. This is particularly advantageous in terms
of reproducibility of the enzymatic activity applicable in the
subsequent tissue dissociation workflow. The smaller the variation,
the higher the reproducibility.
SUMMARY
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] Yet, 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.
[0019] Yet, a further embodiment of the invention is an aqueous
solution comprising water and a composition according to the
invention.
[0020] Yet, a further embodiment of the invention is the use of an
aqueous solution according to the invention for treating a
biological tissue.
[0021] Yet, a further embodiment of the invention is a kit
comprising one or more containers containing a composition
according to the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1A shows a photograph of a lyophilizate with unordered
clusters of crystals.
[0023] FIG. 1B is a lyophilizate with crystals having a lamellae-
or blade-like structure.
DETAILED DESCRIPTION
[0024] It was a surprising finding by the inventors that the
surface area of a lyophilizate was greatly increased when the
lyophilizate was prepared free of acetate salts. Typically, 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. Thus, 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.
[0025] In addition, 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.
[0026] Certain technical terms are used with particular meaning, or
are defined for the first time, in this description of the present
invention. For the purposes of the present invention, the terms
used are defined by their art-accepted definitions, when such
exist, except that when those definitions conflict or partially
conflict with the definitions set forth below. In the event of a
conflict in definition, the meaning of a term is first defined by
any of the definitions set forth below.
[0027] The term "comprising" is used in the description of the
invention and in the claims to mean "including, but not necessarily
limited to".
[0028] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "a compound" means one compound
or more than one compound.
[0029] When designating 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. Thus, a value x in the designated
range is given by n1.ltoreq.x.ltoreq.n2.
[0030] If not stated otherwise, it is understood that the term
"about" and the character ".about." in combination with a numerical
value n ("about n", ".about.n") indicates a value x in the interval
given by the numerical value.+-.5% of the value, i.e.,
n-0.05*n.ltoreq.x.ltoreq.n+0.05*n. In case the term "about" or the
character ".about." in combination with a numerical value n
describes a preferred embodiment of the invention, the value of n
is most preferred, if not indicated otherwise.
[0031] 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.
[0032] A lyophilizate is an example for a mixture which is a solid.
In the context of the present invention such a mixture comprises
one or more proteinaceous compounds. Preferably, these are one or
more proteolytic enzymes with collagenase activity and a neutral
protease. Preferably, the neutral protease is thermolysin. Also
preferred, 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.
[0033] 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.
[0034] A solution is a homogeneous mixture of one or more
substances (the solutes) dissolved (i.e., dissociated) in another
substance (the solvent). A common example would be a solid
dissolving into a liquid (i.e., salt or protein dissolving in
water). Solubility is a compound property. Depending on the
composition of the mixture to be dissolved and conditions (e.g.,
temperature, type of solvent, solutes present in the solvent), the
amount of a substance that can dissolve in a solution can be
variable.
[0035] Examples for non-homogeneous (heterogeneous) mixtures are a
colloid and a suspension. In the context of the invention, 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.
[0036] In a solution, 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. Thus, 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. As a consequence, after a certain amount of time said first
solution becomes a heterogeneous mixture whereas said second
solution remains a homogeneous mixture.
[0037] 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. Thus, one
way to determine turbidity is visual inspection, i.e., inspection
by eye.
[0038] Another way of determination is measurement of light
attenuation with a photometer. In this regard, the term "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 .lamda.:
OD.sub..lamda.=log.sub.10O=-log.sub.10T=-log.sub.10(I/I.sub.0)
wherein [0039] O=the per-unit opacity [0040] T=the per-unit
transmittance [0041] I.sub.0=the intensity of the incident light
beam [0042] I=the intensity of the transmitted light beam.
[0043] The higher the optical density, the lower the transmittance.
Owing to the scattering of a light beam focused on the particles
the optical density of a suspension or a colloid is increased
compared to a clear solution.
[0044] The composition of a lyophilizate has a consequence for the
amount of the solid matter which can be dissolved in a given
solvent. However, the 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.
[0045] 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. In
the present context, the compound of interest can be each
constituent of a particular lyophilizate or the lyophilizate
itself.
[0046] An important physical measurement required is that of
solubility of a compound of interest at a given temperature. Once
the solubility is known, the volume of solvent or the acceptability
of a particular solvent can be determined. For example, 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.
[0047] 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).
C(t)=c.sub.s.times.(1-e.sup.-k.times.t) (1)
[0048] With the so-called Noyes-Whitney equation (Noyes, A. A.
& Whitney, W. R., J., Am. Chem. Soc. 19 (1897) 930-934), 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.sub.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 (.xi.) the
volume of the bulk solution (V.sub.s) and the thickness of the
diffusion layer (h).
k = S .times. .xi. Vs .times. h ( 2 ) ##EQU00001##
[0049] According to the Stokes Equation (3), the diffusion
coefficient (.xi.) can be calculated from the Boltzmann constant
(k.sub.b), the temperature (T), the viscosity of the bulk solution
(.eta.) and the hydrodynamic radius of the dissolved molecule
(r).
.xi. = k b .times. T 6 .pi. .times. .eta. .times. r ( 3 )
##EQU00002##
[0050] 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.
[0051] For several particle sizes one finds that small particles
will dissolve much quicker than bigger particles as with smaller
particles a larger surface area gets into contact with the solvent.
In case the size distribution of the particles gets broader, the
average rate constant of dissolution will become less accurate,
thus leading to a less accurate prediction of the dissolution
profile. That is to say, the dissolution behavior of a lyophilizate
is the result of the cumulative effect of all particles in the
solid matter.
[0052] 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.
[0053] The 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]. Generally, the skilled person aims at minimizing
the moisture content since this factor advantageously influences
product shelf life.
[0054] According to the invention, 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. Examples of an acetate salt
are sodium acetate, potassium acetate and calcium acetate.
[0055] In Example 2 below a first crystallization process for
thermolysin is disclosed. The 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. In a
blending process the acetate crystals can be dissolved but the
solution of the neutral protease is instable in that thermolysin
tends to precipitate.
[0056] The present invention 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.
[0057] A further important finding of the inventors was that 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).
[0058] In view of the inventor's basic findings, 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.
[0059] The low-salt solution of step (a) preferably comprises (and
more preferred consists of) water, CaCl.sub.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.
[0060] The neutral salt in step (b) is preferably sodium chloride.
In a very much preferred embodiment of the invention, prior to step
(c) the solution of the neutral protease 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.sub.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.
[0061] 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. 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.
[0062] Prior to step (d) 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.sub.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.
[0063] As a result of the freeze-drying step (d) of the inventive
process 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. Typically, the lyophilizate consists of
crystalline matter consisting of lamellae which are aligned in
parallel (see also FIG. 1). Apparently, 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.
[0064] 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 shorter the time to dissolve the
proteases, the less proteolytic activity is lost due to
auto-proteolysis occurring prior to the application of the
proteases to the tissue. Thus, the invention provides an important
basis for improved methods of tissue dissociation.
[0065] Yet, in more detail, the present invention comprises the
following items which are preferred embodiments: [0066] 1. A solid
composition comprising one or more proteolytic enzymes with
collagenase activity (C), and a neutral protease (NP),
characterized in that the composition is free of acetate salt.
[0067] 2. The composition according to item 1, characterized in
that the composition comprises sodium chloride, calcium chloride,
and a buffer salt, preferably an organic buffer salt. [0068] 3. The
composition according to any of the items 1 and 2, characterized in
that the buffer salt is capable of buffering in the range of about
pH 6 to about pH 8.5. [0069] 4. The composition according to any of
the items 1 to 3, characterized in that the buffer salt is a
compound selected from the group consisting of BES
(N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Tris
(2-Amino-2-(hydroxymethyl)propane-1,3-diol), BisTris
(Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane), BisTris
propane (1,3-bis(tris(hydroxymethyl)methylamino)propane), HEPES
(N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid), MES
(2-(N-morpholino)ethanesulfonic acid), MOPS
(3-(N-morpholino)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
(Triethanolamine), and Tricine
(N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine. [0070] 5. The
composition according to item 4, characterized in that the buffer
salt is HEPES. [0071] 6. The composition according to any of the
items 1 to 5, characterized in that the composition is a
lyophilizate, i.e., obtained by a freeze-drying process. [0072] 7.
The composition according to any of the items 1 to 6, characterized
in that the proteolytic enzymes with collagenase activity in the
composition are collagenase I (C1) and/or collagenase II (CII) from
Clostridium histolyticum. [0073] 8. The composition according to
any of the items 1 to 7, characterized in that the NP in the
composition is thermolysin from Bacillus thermoproteolyticus.
[0074] 9. The composition according to any of the items 1 to 8,
characterized in that the weight-by-weight ratio of the neutral
protease versus all proteases present in the composition
(NP/(NP+C)[w/w]) is in the range of about 1 to about 25. [0075] 10.
The composition according to item 9, characterized in that
NP/(NP+C) [w/w] is in the range of about 2 to about 22. [0076] 11.
The composition according to item 9, characterized in that
NP/(NP+C) [w/w] is about 2. [0077] 12. The composition according to
item 9, characterized in that NP/(NP+C) [w/w] is about 3. [0078]
13. The composition according to item 9, characterized in that
NP/(NP+C) [w/w] is about 22. [0079] 14. The composition according
to any of the items 1 to 13, characterized in that 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. [0080] 15. The composition according to item 14,
characterized in that the (NP+C)/NaCl[w/w] is in the range of about
0.15 to about 3. [0081] 16. The composition according to item 14,
characterized in that the (NP+C)/NaCl[w/w] is in the range of about
0.18 to about 2. [0082] 17. The composition according to item 14,
characterized in that the (NP+C)/NaCl[w/w] is about 0.18. [0083]
18. The composition according to item 14, characterized in that the
(NP+C)/NaCl[w/w] is about 1.3. [0084] 19. The composition according
to item 14, characterized in that the (NP+C)/NaCl[w/w] is about 2.
[0085] 20. The composition according to any of the items 1 to 19,
characterized in that the weight-by-weight ratio of all proteases
present in the composition and calcium chloride hexahydrate
((NP+C)/CaCl.sub.2[w/w]) is in the range of about 10 to about 500.
[0086] 21. The composition according to item 20, characterized in
that (NP+C)/CaCl.sub.2[w/w] is in the range of about 15 to about
470. [0087] 22. The composition according to item 20, characterized
in that (NP+C)/CaCl.sub.2[w/w] is about 17.5. [0088] 23. The
composition according to item 20, characterized in that
(NP+C)/CaCl.sub.2[w/w] is about 240. [0089] 24. The composition
according to item 20, characterized in that (NP+C)/CaCl.sub.2[w/w]
is about 470. [0090] 25. The composition according to any of the
items 1 to 24, characterized in that the weight-by-weight ratio of
all proteases present in the composition and the buffer salt
((NP+C)/buffer salt [w/w]) is in the range of about 0.05 to about
2. [0091] 26. The composition according to item 25, characterized
in that (NP+C)/buffer [w/w] is in the range of about 0.1 to about
1. [0092] 27. The composition according to item 25, characterized
in that (NP+C)/buffer [w/w] is about 0.1. [0093] 28. The
composition according to item 25, characterized in that
(NP+C)/buffer [w/w] is about 0.5. [0094] 29. The composition
according to item 25, characterized in that (NP+C)/buffer [w/w] is
about 1. [0095] 30. The composition according to any of the items
25 to 29, characterized in that the buffer salt is selected from
the group consisting of BES
(N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Tris
(2-Amino-2-(hydroxymethyl)propane-1,3-diol), BisTris
(Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane), BisTris
propane (1,3-bis(tris(hydroxymethyl)methylamino)propane), HEPES
(N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid), MES
(2-(N-morpholino)ethanesulfonic acid), MOPS
(3-(N-morpholino)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
(Triethanolamine), and Tricine
(N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine. [0096] 31. An
aqueous solution comprising water and, in dissolved form, a
composition according to any of the items 1 to 30. [0097] 32. The
aqueous solution according to item 31, characterized in that the pH
of the solution is in the range of pH 7 to pH 8, and more preferred
pH 7.5. [0098] 33. The aqueous solution according to any of the
items 31 and 32, characterized in that the concentration of
proteases in the solution is in the range of 0.1 mg/ml to 100
mg/ml. [0099] 34. The aqueous solution according to item 33,
characterized in that the concentration of proteases in the
solution is in the range of about 1 mg/ml to about 75 mg/ml. [0100]
35. The aqueous solution according to item 34, characterized in
that the concentration of proteases in the solution is in the range
of about 5 mg/ml to about 50 mg/ml. [0101] 36. Use of an aqueous
solution according to any of the items 31 to 35 for treating a
biological tissue. [0102] 37. A kit comprising package material and
one or more containers containing a solid composition according to
any of the items 1 to 30. [0103] 38. The kit according to item 37,
characterized in that the one or more containers are sealed,
thereby protecting the composition in the containers from moisture.
[0104] 39. A method to prepare a solution with proteases,
comprising the step of contacting a composition according to any of
the items 1 to 30 with water. [0105] 40. The method according to
item 39, characterized in that measured amounts of the composition
and water are mixed, in order to yield a concentration of proteases
in solution, said concentration being in the range of about 5 mg/ml
to about 50 mg/ml. [0106] 41. The method according to any of the
items 39 and 40, characterized in that a homogeneous solution is
obtained within a period in the range of less than about 1 min to 3
min, or within the period of less than 3 min, counted from the
moment the composition is contacted with water. [0107] 42. The
method according to any of the items 40 and 41, characterized in
that the concentration of proteases is in the range of about 5
mg/ml to about 30 mg/ml, and a homogeneous solution is obtained
within a period of less than about 1 min, counted from the moment
the composition is contacted with water. [0108] 43. The method
according to any of the items 40 and 41, characterized in that the
concentration of proteases is in the range of about 15 mg/ml to
about 50 mg/ml, and a homogeneous solution is obtained within a
period of about 3 min or shorter than 3 min, counted from the
moment the composition is contacted with water.
[0109] The figures and following examples are provided to aid the
understanding of the present invention, the true scope of which is
set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
Example 1: Materials, General Conditions, and Procedures
Applied
[0110] If not stated otherwise, all aqueous solutions were kept and
used at temperatures between 2.degree. C. and 8.degree. C.
[0111] 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.sub.2, 5 mM HEPES, pH 7.5.
[0112] The major parts of a freeze drying (lyophilization)
procedure include freezing, primary drying (sublimation), and
secondary drying (desorption). One objective of 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.
[0113] 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:
TABLE-US-00001 time No. Step (hh:mm) temp. pressure comment 1.
loading of 00:03 2.degree. C. ambient enzyme pressure solution 2.
Freezing 00:30 -50.degree. C. ambient temperature pressure may
fluctuate by .+-.4.degree. C. 3. Freezing 04:00 -50.degree. C.
ambient pressure 4. Evacuating 00:20 -50.degree. C. 0.01 mbar 5.
primary drying 00:40 -50.degree. C. 0.01 mbar
[0114] In the present freeze drying processes, three different
regimens for the initial secondary drying were used. One step
selected from either 6a, 6b, or 6c was performed.
TABLE-US-00002 time No. Step (hh:mm) temp. pressure comment 6a.
initial 14:00 -8.degree. C. 0.01 mbar secondary drying 6b. initial
20:00 -8.degree. C. 0.01 mbar secondary drying 6c. initial 10:00
22.degree. C. 0.01 mbar temperature secondary may fluctuate drying
by .+-.3.degree. C.
[0115] Following the initial secondary drying step the following
regimen was applied. The partial aeration steps 8. and 10 were
optional.
TABLE-US-00003 time No. Step (hh:mm) temp. pressure comment 7.
secondary drying 06:00 22.degree. C. 0.01 mbar 8. partial aeration
(test) 00:02 22.degree. C. 0.17 mbar 9. secondary drying 03:30
22.degree. C. 0.01 mbar 10. partial aeration (test) 00:02
22.degree. C. 0.05 mbar 11. aeration with N.sub.2 00:01 22.degree.
C. 700 mbar 12. incubation 00:05 22.degree. C. 700 mbar 13.
aeration with N.sub.2 00:01 22.degree. C. ambient pressure 14.
unloading 00:03 4.degree. C. ambient pressure 15. storage 4.degree.
C. ambient pressure
Example 2: Preparation of Crystallized Thermolysin and
Solubilization of Crystallized Thermolysin
[0116] 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. For a crystallization step, 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.
[0117] An exemplary lyophilizate of crystallized thermolysin had a
protein content of about 66.1% [w/w] and was used in blending
experiments.
[0118] Before blending, the crystallized thermolysin lyophilizate
was suspended in aqueous HEPES buffer (1 mM CaCl.sub.2, 5 mM HEPES,
pH 7.5). In order to dissolve the protein, about 7.2 mM NaOH were
added (as 0.1 N NaOH) to raise the pH to a value of about pH 11.
Subsequently, 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.sub.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. Notably, a clear solution was obtained which, however, was
not stable. It was observed that the resulting solution became
turbid after about 30 min due to beginning precipitation of
thermolysin. Only small amounts of thermolysin solution could be
prepared using the above method. As a further disadvantage, the
small volume of thermolysin solution had to be used instantly in
the blending process in which enzyme in homogeneous solution is
required. Because the blending process takes a certain amount of
time it has to be assured that the thermolysin enzyme solution is
homogeneous during the whole process.
Example 3: Preparation of a Stabilized Solution Containing
Thermolysin
[0119] In view of the shortcomings described in Example 2, a more
favorable method for providing thermolysin in homogeneous solution
was developed. A further preparation containing crude thermolysin
(EC 3.4.24.27) is available from Daiwa Kasei K. K. (Japan) under
the trade name THERMOASE. The THERMOASE quality of thermolysin used
here was a lyophilisate. The protein content of "crude thermolysin"
in the lyophilizate was about 33% [w/w]. About 65% [w/w] of the
lyophilisate was NaCl. The remaining amount of about 2% [w/w] was
Na.sub.2SO.sub.4. "Crude thermolysin" in the present context is a
protein mixture consisting of [0120] (i) about 70% substantially
undegraded (intact) thermolysin, [0121] (ii) about 24% of
thermolysin degradation products which retain proteolytic activity
(to different degrees), and [0122] (iii) about 6% of
proteolytically inactive fragments and further impurities.
[0123] A volume of 6.5 l of an aqueous buffer containing 5 mM
CaCl.sub.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.sub.2, 20 mM HEPES, pH 7.5, and mixing by
stirring. A homogeneous solution was obtained. Taking into account
that about 65% [w/w] of the lyophilisate consists of NaCl, 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.
[0124] The solution of 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.
[0125] Diafiltration was performed using a filter with an exclusion
limit of about 10 kDa and against an aqueous buffer containing 5 mM
CaCl.sub.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.
[0126] Diafiltrated thermolysin was either used directly in
blending processes, or the solution was aliquoted and aliquots were
frozen and stored at -20.degree. C. Frozen aliquots were thawed
before use and remained stable afterwards for 6 hours or more.
Example 4: Preparation of a Lyophilized Thermolysin-Containing
Blend of Proteases (Blend 1) with a Low Amount of Thermolysin
[0127] 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.
TABLE-US-00004 TABLE 1 concentration vol. of stock concentration
amount in stock solution in in final substance (absolute) solution
mixture mixture collagenase I 26,565 mg 55 mg/ml 483 ml 29.3 mg/ml
collagenase II 17,435 mg 55 mg/ml 317 ml 19.2 mg/ml thermolysin
1,265 mg .sup.$ 13.2 mg/ml 96.6 ml 1.4 mg/ml Protein conc. total
45,265 mg 49.9 mg/ml NaAc total 2.6 mM CaAc total 3 mM CaCl.sub.2
total 0.8 mM NaOH 0.1N 9 ml 1 mM HEPES total 5.2 mM
HEPES.sup..dagger-dbl. 0.5M 1.5 ml HEPES.sup..sctn. buffer --
Volume total 907.1 ml pH 7.5 .sup..dagger-dbl.HEPES solution, not
alkali-titrated; for neutralization .sup..sctn.HEPES buffer (1 mM
CaCl.sub.2, 5 mM HEPES, pH 7.5) for volume adjustment, if necessary
.sup.$ corresponding to 1,914 mg of lyophilized crystals (i.e.,
including solid Calcium acetate, see Example 2)
Example 5: Preparation of a Lyophilized Thermolysin-Containing
Blend of Proteases (Blend 2) with a Medium Amount of
Thermolysin
[0128] 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.
TABLE-US-00005 TABLE 2 concentration volume of stock concentration
amount in stock solution in in final substance (absolute) solution
mixture mixture collagenase I 8,580 mg 55 mg/ml 156 ml 8.2 mg/ml
collagenase II 5,610 mg 55 mg/ml 102 ml 5.4 mg/ml thermolysin 5,585
mg .sup.$ 13.1 mg/ml 422 ml 5.3 mg/ml Protein total 19,775 mg 18.9
mg/ml NaAc total 9.9 mM CaAc total 11.6 mM CaCl.sub.2 total 0.5 mM
NaOH 0.1N 75 ml 7.2 mM HEPES total 5.9 mM HEPES.sup..dagger-dbl.
0.5M 7 ml HEPES.sup..sctn. buffer solution 283 ml Volume total
1,045 ml pH 7.5 .sup..dagger-dbl.HEPES solution, not
alkali-titrated; for neutralization .sup..sctn.HEPES buffer (1 mM
CaCl.sub.2, 5 mM HEPES, pH 7.5) for volume adjustment, if necessary
.sup.$ corresponding to 8,449 mg of lyophilized crystals (i.e.,
including solid Calcium acetate, see Example 2)
Example 6: Preparation of a Lyophilized Thermolysin-Containing
Blend of Proteases (Blend 3) with a High Amount of Thermolysin
[0129] 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.
TABLE-US-00006 TABLE 3 concentration volume of stock concentration
amount in stock solution in in final substance (absolute) solution
mixture mixture collagenase I 2,140 mg 55 mg/ml 38.9 ml 5.6 mg/ml
collagenase II 1,422 mg 55 mg/ml 25.85 ml 3.7 mg/ml thermolysin
2,772 mg $ 13.2 mg/ml 211 ml 7.2 mg/ml Protein total 6,334 mg 16.5
mg/ml NaAc total 13.4 mM CaAc total 15.7 mM CaCl2 total 0.4 mM NaOH
0.1N 19 ml 5 mM HEPES total 4.5 mM HEPES.dagger-dbl. 0.5M 2 ml
HEPES.sctn. buffer solution 85.75 ml Volume total 382.5 ml pH 7.5
.dagger-dbl.HEPES solution, not alkali-titrated; for neutralization
.sctn.HEPES buffer (1 mM CaCl.sub.2, 5 mM HEPES, pH 7.5) for volume
adjustment, if necessary $ corresponding to 4,194 mg of lyophilized
crystals (i.e., including solid Calcium acetate, see Example 2)
Example 7: Preparation of an Acetate-Free Thermolysin-Containing
Blend of Proteases (Blend 4)
[0130] 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.
TABLE-US-00007 TABLE 4 concentration volume of stock concentration
amount in stock solution in in final substance (absolute) solution
mixture mixture collagenase I 410 mg 55 mg/ml 7.45 ml 16 mg/ml
collagenase II 275 mg 55 mg/ml 5 ml 10.7 mg/ml
thermolysin.sup..dagger-dbl. 33 mg 2.5 mg/ml 13.2 ml 1.3 mg/ml
Protein total 718 mg 28 mg/ml CaCl.sub.2 total 3.1 mM NaCl 87 mM
HEPES total 12.8 mM Volume total 25.65 ml pH 7.5
.sup..dagger-dbl.the total amount of protein present in the
stabilized solution after diafiltration, see Example 3
Example 8: Preparation of an Acetate-Free Thermolysin-Containing
Blend of Proteases (Blend 5)
[0131] 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.
TABLE-US-00008 TABLE 5 concentration volume of stock concentration
amount in stock solution in in final substance (absolute) solution
mixture mixture collagenase I 660 mg 55 mg/ml 12 ml 2.85 mg/ml
collagenase II 440 mg 55 mg/ml 8 ml 1.9 mg/ml
thermolysin.sup..dagger-dbl. 530 mg 2.5 mg/ml 212 ml 2.3 mg/ml
Protein total 1,630 mg 7.05 mg/ml CaCl.sub.2 total 4.7 mM NaCl 155
mM HEPES total 18.7 mM Volume total 232 ml pH 7.5
.sup..dagger-dbl.the total amount of protein present in the
stabilized solution after diafiltration, see Example 3
Example 9: Preparation of an Acetate-Free Thermolysin-Containing
Blend of Proteases (Blend 6)
[0132] 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.
TABLE-US-00009 TABLE 6 concentration volume of stock concentration
amount in stock solution in in final substance (absolute) solution
mixture mixture collagenase I 660 mg 55 mg/ml 12 ml 1.5 mg/ml
collagenase II 440 mg 55 mg/ml 8 ml 1 mg/ml
thermolysin.sup..dagger-dbl. 1,060 mg 2.5 mg/ml 424 ml 2.4 mg/ml
Protein total 2,160 mg 4.9 mg/ml CaCl.sub.2 total 4.8 mM NaCl 162
mM HEPES total 19.3 mM Volume total 444 ml pH 7.5
.sup..dagger-dbl.the total amount of protein present in the
stabilized solution after diafiltration, see Example 3
Example 10: Solubilization of Lyophilizates
[0133] 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.degree. 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.
TABLE-US-00010 TABLE 7 protein concentration in solution (after
time needed for solubilization), solubilization, Blend # in [mg/ml]
in [min] 1 49.9 3 1 25 3 2 18.9 75 2 7 75 3 16.5 75 3 5.5 75 4 47 3
4 28 <1 5 21 3 5 7 <1 6 17 3 6 5 <1
Example 11: Solubilization of Lyophilizates
[0134] Under the same conditions as described in Example 10,
lyophilized blends in sealed bottles were dissolved in different
amounts of purified water, in order to yield solutions with
different protein concentrations. The time needed to dissolve the
lyophilizates was recorded. Recordings were stopped after 75 min,
even if solubilization was not complete at this point. Completeness
of solubilization was firstly assessed by visual inspection.
Secondly. the turbidity of each obtained solution was measured by
determining its optical density (OD) at a wavelength 600 nm using
standard quartz cuvettes and a photometer. Table 8 indicates the
time intervals needed for dissolving the lyophilizates as well as
the results of the assessments of turbidity.
TABLE-US-00011 TABLE 8 protein concentration in turbidity as
turbidity as solution (after time needed for determined determined
solubilization), solubilization, by OD at by visual Blend # in
[mg/ml] in [min] 600 nm inspection 1 49.9 3 0.013 clear 1 25 3
0.014 clear 2 18.9 75 2.159.sup. opaque, precipitate 2 7 75 0.979
opaque, precipitate 3 16.5 75 2.127.sup. opaque, precipitate 3 5.5
75 1.665 opaque, precipitate 4 47 3 0.033 clear 4 28 <1 0.026
clear 5 21 3 0.078 clear 5 7 <1 0.037 clear 6 17 3 0.088 clear 6
5 <1 0.078 clear .sup. measurement out of range of
proportionality
* * * * *