U.S. patent application number 10/760148 was filed with the patent office on 2004-10-14 for controlled dissolution crosslinked protein crystals.
This patent application is currently assigned to Altus Biologics Inc.. Invention is credited to Khalaf, Nazer K., Margolin, Alexey L., Persichetti, Rose A., St. Clair, Nancy L..
Application Number | 20040202643 10/760148 |
Document ID | / |
Family ID | 25267485 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040202643 |
Kind Code |
A1 |
Margolin, Alexey L. ; et
al. |
October 14, 2004 |
Controlled dissolution crosslinked protein crystals
Abstract
The present invention relates to crosslinked protein crystals
characterized by the ability to change from insoluble and stable
form to soluble and active form upon a change in the environment of
said crystals, said change being selected from the group consisting
of change in temperature, change in pH, change in chemical
composition, change from concentrate to dilute form, change in
shear force acting upon the crystals and combinations thereof.
According to one embodiment of this invention, such crosslinked
protein crystals are capable of releasing their protein activity at
a controlled rate. This invention also provides methods for
producing such crosslinked protein crystals, methods using them for
protein delivery and methods using them in cleaning agents,
including detergents, pharmaceutical compositions, vaccines,
personal care compositions, including cosmetics, veterinary
compositions, foods, feeds, diagnostics and formulations for
decontamination.
Inventors: |
Margolin, Alexey L.;
(Newton, MA) ; Persichetti, Rose A.; (Stow,
MA) ; St. Clair, Nancy L.; (Durham, NC) ;
Khalaf, Nazer K.; (Worcester, MA) |
Correspondence
Address: |
FISH & NEAVE
1251 AVENUE OF THE AMERICAS
50TH FLOOR
NEW YORK
NY
10020-1105
US
|
Assignee: |
Altus Biologics Inc.
|
Family ID: |
25267485 |
Appl. No.: |
10/760148 |
Filed: |
January 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10760148 |
Jan 16, 2004 |
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09631241 |
Aug 2, 2000 |
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09631241 |
Aug 2, 2000 |
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08834661 |
Apr 11, 1997 |
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6140475 |
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Current U.S.
Class: |
424/94.1 ;
702/19 |
Current CPC
Class: |
Y10S 530/81 20130101;
A61Q 5/02 20130101; C12N 9/96 20130101; A61P 31/12 20180101; A23K
20/147 20160501; C12Y 304/21062 20130101; C07K 14/765 20130101;
C12N 9/54 20130101; C12Y 503/01005 20130101; C12N 9/20 20130101;
C11D 3/3719 20130101; C11D 3/38 20130101; C12N 9/92 20130101; C07K
2299/00 20130101; A61Q 19/00 20130101; C11D 3/386 20130101; C30B
29/58 20130101; A61K 8/64 20130101; C12Y 304/24027 20130101; C12N
11/00 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/094.1 ;
702/019 |
International
Class: |
A61K 038/43; G06F
019/00; G01N 033/48; G01N 033/50 |
Claims
1. A crosslinked protein crystal, said protein crystal being
capable of change from insoluble and stable form to soluble and
active form upon a change in the environment surrounding said
crystal, said change being selected from the group consisting of:
change in temperature, change in pH, change in chemical
composition, change from concentrate to dilute form, change in
shear force acting upon the crystal and combinations thereof.
2. The crosslinked protein crystal according to claim 1, wherein
said change from concentrate to dilute form comprises a change in
solute concentration.
3. The crosslinked protein crystal according to claim 2, wherein
said change in solute concentration comprises an increase or
decrease in salt concentration.
4. The crosslinked protein crystal according to claim 3, wherein
said change in solute concentration comprises a decrease in salt
concentration.
5. The crosslinked protein crystal according to claim 2, wherein
said change in solute concentration comprises an increase or
decrease in water concentration.
6. The crosslinked protein crystal according to claim 5, wherein
said change in solute concentration comprises an increase in water
concentration.
7. The crosslinked protein crystal according to claim 2, wherein
said change in solute concentration comprises an increase or
decrease in organic solvent concentration.
8. The crosslinked protein crystal according to claim 2, wherein
said change in solute concentration comprises a decrease in
detergent concentration.
9. The crosslinked protein crystal according to claim 2, wherein
said change in solute concentration comprises a decrease in protein
concentration.
10. The crosslinked protein crystal according to claim 1, wherein
said change from concentrate to dilute form comprises a change in
concentration of all solutes from about 2-fold to about
10,000-fold.
11. The crosslinked protein crystal according to claim 10, wherein
said change from concentrate to dilute form comprises a change in
concentration of all solutes from about 2-fold to about
700-fold.
12. The crosslinked protein crystal according to claim 1, wherein
said change in pH comprises a change from acidic pH to basic
pH.
13. The crosslinked protein crystal according to claim 1, wherein
said change in pH comprises a change from basic pH to acidic
pH.
14. The crosslinked protein crystal according to claim 1, wherein
said change in temperature comprises an increase or decrease in
temperature.
15. The crosslinked protein crystal according to claim 14, wherein
said change in temperature is an increase in temperature from a low
temperature between about 0.degree. C. and about 20.degree. C. to a
high temperature between about 25.degree. C. and about 70.degree.
C.
16. The crosslinked protein crystal according to claim 1, wherein
said active form of said protein is a form which is active against
macromolecular substrates.
17. A crosslinked protein crystal, said protein crystal having a
half-life of activity under storage conditions which is greater
than at least 2 times that of the soluble form of the protein that
is crystallized to form said crystal that is crosslinked and
activity similar to that of the soluble form of the protein under
conditions of use.
18. A crosslinked protein crystal, said protein crystal being
capable of releasing its protein activity at a controlled rate upon
exposure to a change in the environment surrounding said crystal,
said change being selected from the group consisting of change in
pH, change in solute concentration, change in temperature, change
in chemical composition, change in shear force acting upon the
crystals and combinations thereof.
19. The crosslinked protein crystal according to claim 18, wherein
said controlled rate of releasing protein activity is determined by
a factor selected from the group consisting of: the degree of
crosslinking of said crosslinked protein crystal, the length of
time of exposure of protein crystal to the crosslinker, the rate of
addition of the crosslinking agent to said protein crystal, the
nature of the crosslinker, the chain length of the crosslinker, the
surface area of said crosslinked protein crystal, the size of said
crosslinked protein crystal, the shape of said crosslinked protein
crystal and combinations thereof.
20. The crosslinked protein crystal according to claim 18, wherein
said crystal has a protein activity release rate of between about
0.1% per day and about 100% per day.
21. The crosslinked protein crystal according to claim 18, wherein
said crystal has a protein activity release rate between about
0.01% per hour and about 100% per hour.
22. The crosslinked protein crystal according to claim 18, wherein
said crystal has a protein activity release rate between about 1%
per minute and about 50% per minute.
23. The crosslinked protein crystal according to any one of claims
1, 17 or 18, said protein crystal being substantially insoluble and
stable in a composition under storage conditions and substantially
soluble and active under conditions of use of said composition.
24. The crosslinked protein crystal according to claim 23, wherein
said composition is selected from the group consisting of cleaning
agents, detergents, personal care compositions, cosmetics,
pharmaceuticals, veterinary compounds, vaccines, foods, feeds,
diagnostics and formulations for decontamination.
25. The crosslinked protein crystal according to claim 24, wherein
said detergent is selected from the group consisting of powdered
detergents, liquid detergents, bleaches, household cleaners, hard
surface cleaners, industrial cleaners, carpet shampoos and
upholstery shampoos.
26. The crosslinked protein crystal according to claim 24, wherein
said cosmetic is selected from the group consisting of creams,
emulsions, lotions, foams, washes, gels, compacts, mousses,
sunscreens, slurries, powders, sprays, foams, pastes, ointments,
salves, balms, shampoos, sunscreens and drops.
27. The crosslinked protein crystal according to any one of claims
1, 17 or 18, wherein said protein is an enzyme.
28. The crosslinked protein crystal according to claim 27, wherein
said enzyme is selected from the group consisting of hydrolases,
isomerases, lyases, ligases, transferases and oxidoreductases.
29. The crosslinked protein crystal according to claim 28, wherein
said enzyme is selected from the group consisting of proteases,
amylases, cellulases, lipases and oxidases.
30. The crosslinked protein crystal according to any one of claims
1, 17 or 18, wherein said protein is selected from the group
consisting of therapeutic proteins, cleaning agent proteins,
personal care proteins, veterinary proteins, food proteins, feed
proteins, diagnostic proteins and decontamination proteins.
31. The crosslinked protein crystal according to any one of claims
1, 17 or 18, wherein said protein is selected from the group
consisting of hormones, antibodies, inhibitors, growth factors,
trophic factors, cytokines, lymphokines, toxoids, growth hormones,
nerve growth hormones, bone morphogenic proteins, and
nutrients.
32. The crosslinked protein crystal according to any one of claims
1, 17 or 18, wherein said protein is selected from the group
consisting of insulin, amylin, erythropoietin, Factor VIII, TPA,
dornase-.alpha., .alpha.-1-antitrypsin, urease, fertility hormones,
FSH, LSH, postridical hormones, tetanus toxoid and diptheria
toxoid.
33. The crosslinked protein crystal according to any one of claims
1, 17 or 18, said crystal having a longest dimension of between
about 0.01 .mu.m and about 500 .mu.m.
34. The crosslinked protein crystal according to any one of claims
1, 20 or 21, said crystal having a longest dimension of between
about 0.1 .mu.m and about 50 .mu.m.
35. The crosslinked protein crystal according to any one of claims
1, 17 or 18, said crystal having a shape selected from the group
consisting of: spheres, needles, rods, plates, rhomboids, cubes,
bipyramids and prisms.
36. A composition comprising a crosslinked protein crystal
according to any one of claims 1, 17 or 18, said composition being
selected from the group consisting of cleaning agents, detergents,
personal care compositions, cosmetics, pharmaceuticals, veterinary
compounds, vaccines, foods, feeds, diagnostics and formulations for
decontamination.
37. The composition according to claim 36, wherein said detergent
is selected from the group consisting of powdered detergents,
liquid detergents, bleaches, household cleaners, hard surface
cleaners, industrial cleaners, carpet shampoos and upholstery
shampoos.
38. The composition according to claim 36, wherein said cosmetic is
selected from the group consisting of creams, emulsions, lotions,
foams, washes, gels, compacts, sunscreens, slurries, powders,
sprays, foams, pastes, ointments, salves, balms, shampoos,
sunscreens and drops.
39. A protein delivery system, said system comprising crosslinked
protein crystals according to any one of claims 1, 17 or 18.
40. The protein delivery system according to claim 39, wherein said
protein is selected from the group consisting of: detergent
enzymes, cosmetic proteins, pharmaceutical proteins, agricultural
proteins, vaccine proteins and decontamination proteins.
41. The protein delivery system according to claim 40, said protein
delivery system being a microparticulate protein delivery
system.
42. The protein delivery system according to claim 41, wherein said
microparticulate protein delivery system comprises crosslinked
protein crystals having a longest dimension between about 0.01
.mu.m and about 500 .mu.m.
43. The protein delivery system according to claim 42, wherein said
microparticulate protein delivery system comprises crosslinked
protein crystals having a longest dimension of between about 0.1
.mu.m and about 50 .mu.m.
44. The protein delivery system according to claim 41, wherein said
microparticulate protein delivery system comprises crosslinked
protein crystals having a shape selected from the group consisting
of: spheres, needles, rods, plates, rhomboids, cubes, bipyramids
and prisms.
45. A detergent formulation comprising a crosslinked protein
crystal according to any one of claims 1, 17 or 18.
46. A controlled release formulation comprising a crosslinked
protein crystal according to any one of claims 1, 17 or 18.
47. A pharmaceutical controlled release formulation comprising a
crosslinked protein crystal according to any one of claims 1, 17 or
18.
48. A pharmaceutical controlled release formulation comprising a
crosslinked protein crystal, said crystal being substantially
insoluble under storage conditions and capable of releasing its
protein activity in vivo at a controlled rate.
49. The pharmaceutical controlled release formulation according to
claim 47, said pharmaceutical being capable of administration by
parenteral or non-parenteral routes.
50. The pharmaceutical controlled release formulation according to
claim 49, said pharmaceutical being capable of administration by
oral, pulmonary, nasal, aural, anal, dermal, ocular, intravenous,
intramuscular, intraarterial, intraperitoneal, mucosal, sublingual,
subcutaneous or intracranial route.
51. The pharmaceutical controlled release formulation according to
claim 47, wherein said pharmaceutical is capable of administration
by oral route and said crosslinked protein crystal is substantially
insoluble under gastric pH conditions and substantially soluble
under small intestine pH conditions.
52. A vaccine comprising a crosslinked protein crystal according to
any one of claims 1, 17 or 18.
53. A formulation comprising a crosslinked protein crystal
according to any one of claims 1, 17 or 18, said formulation being
selected from the group consisting of tablets, liposomes, granules,
spheres, microspheres, microparticles and capsules.
54. A method for producing crosslinked protein crystals comprising
the step of reacting protein crystals with a first crosslinking
agent, or a first crosslinking agent and at least a second
crosslinking agent, under conditions sufficient to induce
crosslinking of said crystals to the extent that the resulting
crosslinked crystals are characterized by the ability to change
from insoluble and stable form to soluble and active form upon a
change in their environment, said change being selected from the
group consisting of change in temperature, change in pH, change in
chemical composition, change from concentrate to dilute form,
change in shear force acting upon the crystals and combinations
thereof.
55. A method for producing crosslinked protein crystals comprising
the step of reacting protein crystals with a first crosslinking
agent, or a first crosslinking agent and at least a second
crosslinking agent, under conditions sufficient to induce
crosslinking of said crystals to the extent that the resulting
crosslinked crystals are characterized by a half-life of activity
under storage conditions which is greater than at least 2 times
that of the soluble form of the protein that is crystallized to
form said crystals that are crosslinked and activity similar to
that of the soluble form of the protein under conditions of
use.
56. A method for producing crosslinked protein crystals comprising
the step of reacting protein crystals with a first crosslinking
agent, or a first crosslinking agent and at least a second
crosslinking agent, under conditions sufficient to induce
crosslinking of said crystals to the extent that the resulting
crosslinked crystals are characterized by being capable of
releasing their protein activity at a controlled rate upon exposure
to a change in their environment, said change being selected from
the group consisting of change in pH, change in soluble
concentration, change in temperature, change in chemical
composition, change in shear force acting upon the crystals and
combinations thereof.
57-85 (canceled)
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to crosslinked protein
crystals characterized by the ability to change from insoluble and
stable form to soluble and active form upon a change in the
environment surrounding said crystals, said change being selected
from the group consisting of change in temperature, change in pH,
change in chemical composition, change from concentrate to dilute
form, change in shear force acting upon the crystals and
combinations thereof. According to one embodiment of this
invention, such crosslinked protein crystals are capable of
releasing their protein activity at a controlled rate. This
invention also provides methods for producing such crosslinked
protein crystals, methods using them for protein delivery and
methods using them in cleaning agents, including detergents,
pharmaceutical compositions, vaccines, personal care compositions,
including cosmetics, veterinary compositions, foods, feeds,
diagnostics and formulations for decontamination.
BACKGROUND OF THE INVENTION
[0002] Proteins are used in a wide range of applications in the
fields of industrial chemistry, pharmaceuticals, veterinary
products, cosmetics and other consumer products, foods, feeds,
diagnostics and decontamination. At times, such uses have been
limited by constraints inherent in proteins themselves or imposed
by the environment or media in which they are used. Such
constraints may result in poor stability of the proteins,
variability of performance or high cost. In order for proteins to
realize their full potential in the fields in which they are used,
they must be able to function without excessive intervention by
their surrounding environment. In the past, environmental elements
have often posed barriers to the widespread use of proteins.
[0003] Various approaches have been employed to overcome these
barriers. However, these approaches have incurred either loss of
protein activity or the additional expense of protein stabilizing
carriers or formulations.
[0004] One unique approach to overcoming barriers to the widespread
use of proteins is crosslinked enzyme crystal ("CLEC.TM.)
technology [N. L. St. Clair and M. A., Navia, J. Am. Chem. Soc.,
11.4, pp. 4314-16 (1992)]. Crosslinked enzyme crystals retain their
activity in environments that are normally incompatible with enzyme
function. Such environments include prolonged exposure to proteases
and other protein digestion agents, high temperature or extreme pH.
In such environments, crosslinked enzyme crystals remain insoluble
and stable.
[0005] Protein solubility, leading to controlled release or
dissolution of protein, is important in many industrial fields.
Such industries include those concerning cleaning agents, including
detergents, pharmaceuticals, consumer and personal care products,
veterinary products, foods, feeds, diagnostics and decontamination.
Various approaches to controlled release have been proposed. These
include encapsulation, such as that described in U.S. Pat. Nos.
4,579,779 and 5,500,223. Other approaches include the use of
mechanical or electrical feed devices and osmotic pumps.
[0006] Controlled release in the pharmaceutical field has been
addressed by various means. U.S. Pat. No. 5,569,467 refers to the
use of sustained release microparticles comprising a biocompatible
polymer and a pharmaceutical agent, which is released as the
polymer degrades. U.S. Pat. No. 5,603,956 refers to solid, slow
release pharmaceutical dosage units comprising crosslinked amylase,
alpha amylase and a pharmaceutical agent. U.S. Pat. No. 4,606,909
refers to oral, controlled-release multiple unit formulations in
which homogeneous cores containing particles of sparingly soluble
active ingredients are coated with a pH-sensitive erodable coating.
U.S. Pat. No. 5,593,697 refers to pharmaceutical or veterinary
implants comprising a biologically active material, an excipient
comprising at least one water soluble material and at least one
water insoluble material and a polymer film coating adapted to
rupture at a predetermined period of time after implant.
[0007] The objective of controlled release of proteins, however,
must be balanced with the fact that the protein itself may not be
stable under storage conditions. Protein stability may also be
adversely affected by other components of the formulation in which
it is contained. For example, heavy duty liquid detergents
constitute hostile environments for component enzymes. Such
problems have been approached through the use of mutant subtilisin
proteases, which are said to have improved oxidative stability. See
U.S. Pat. No. 4,760,025 and PCT patent application WO89/06279.
Proteins, the enzymes most widely used in detergents, catalyze
their own decomposition. Strategies such as the addition of
protease inhibitors (e.g., borate with glycols) or the lowering of
water activity have been only partially effective.
[0008] Another approach, described in U.S. Pat. No. 5,385,959, is
encapsulation of degradation-sensitive detergent components in
capsules of composite emulsion polymers, which permit dilution
release thereof. U.S. Pat. No. 5,286,404 refers to a liquid
detergent composition said to have improved enzyme solubility while
preserving enzyme activity. The improvement is attributed to
chemical modification of free primary amino groups in an enzyme
solution via aldehyde treatment, acylation or alkylation.
[0009] Despite such progress in protein technology generally, the
need still exists for proteins which are stable under conditions of
storage, while active under conditions of use.
DISCLOSURE OF THE INVENTION
[0010] The present invention relates to crosslinked protein
crystals characterized by the ability to change from insoluble and
stable form to soluble and active form upon a change in the
environment surrounding said crystals, said change being selected
from the group consisting of change in temperature, change in pH,
change in chemical composition, change from concentrate to dilute
form, change in shear force acting upon the crystals and
combinations thereof. According to one embodiment of this
invention, such crosslinked protein crystals are capable of
releasing their protein activity at a controlled rate.
[0011] Advantageously, the crosslinked protein crystals of this
invention are insoluble and stable under storage conditions and
soluble and active under conditions of use.
[0012] This invention also provides cleaning agents, including
detergents, pharmaceutical compositions, vaccines, personal care
compositions, including cosmetics, veterinary compositions, foods,
feeds, diagnostics and formulations for decontamination.
Additionally, this invention includes methods for producing such
crosslinked protein crystals and methods for protein delivery using
them.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a graph representing the stability of various
enzymes in Ciba detergent # 16 at 40.degree. C.
[0014] FIG. 2 is a graph representing the washing performance of
liquid detergent formulations, including a formulation containing
crosslinked subtilisin crystals according to the present invention,
on fabric soiled with blood, milk and carbon black.
[0015] FIG. 3 is a graph representing the washing performance of
liquid detergent formulations, including a formulation containing
crosslinked subtilisin crystals according to the present invention,
after storage at 30.degree. C., on fabric soiled with cocoa.
[0016] FIG. 4 is a graph representing the washing performance of
liquid detergent formulations, including a formulation containing
crosslinked subtilisin crystals according to the present invention,
after storage at 40.degree. C., on fabric soiled with cocoa.
[0017] FIG. 5 is a graph representing the washing performance of
liquid detergent formulations, including a formulation containing
crosslinked subtilisin crystals according to the present invention,
after storage at 30.degree. C., on fabric soiled with blood, milk
and carbon black.
[0018] FIG. 6 is a graph representing the washing performance of
liquid detergent formulations, including a formulation containing
crosslinked subtilisin crystals according to the present invention,
after storage at 40.degree. C., on fabric soiled with blood, milk
and carbon black.
[0019] FIG. 7 is a graph representing the washing performance of
liquid detergent formulations, including a formulation containing
crosslinked subtilisin crystals according to the present invention,
after storage at 30.degree. C., on fabric soiled with blood.
[0020] FIG. 8 is a graph representing the solubility of crosslinked
subtilisin crystals according to the present invention at
30.degree. C.
[0021] FIG. 9 is a graph representing the solubility of crosslinked
subtilisin crystals according to the present invention at
37.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0022] In order that the invention herein described may be more
fully understood, the following detailed description is set forth.
In the description, the following terms or phrases are
employed:
[0023] Aqueous-organic solvent mixture--a mixture comprising n %
organic solvent, where n is between 1 and 99 and m % aqueous, where
m is 100-n.
[0024] Catalytically effective amount--an amount of crosslinked
protein crystals of this invention which is effective to treat,
protect, repair or detoxify the area to which they are applied over
some period of time.
[0025] Change in chemical composition--any change in the chemical
components of the environment surrounding the crosslinked protein
crystals that affects the environment or the crosslinker, including
addition of chemical reagents, chemical changes induced by
application of energy in the form of light, microwave, or radiation
to the environment, chemical events that affect the crosslinker and
combinations thereof.
[0026] Change in shear force acting upon the crystals--any change
in factors of the environment surrounding the crosslinked protein
crystals under conditions of use, such as, changes in mechanical
pressure, both positive and negative, revolution stirring,
centrifugation, tumbling, mechanical agitation and filtration
pumping.
[0027] Controlled dissolution--dissolution of crosslinked protein
crystals or release of the protein constituent of said crystals
that is (1) triggered by a change in the environment surrounding
said crystals, said change being selected from the group consisting
of change in temperature, change in pH, change in chemical
composition, change from concentrate to dilute form, change in
shear force acting upon the crystals and combinations thereof and
(2) controlled by a factor selected from the group consisting of
the degree of crosslinking of said crosslinked protein crystals,
the length of time of exposure of protein crystals to the
crosslinking agent, the rate of addition of crosslinking agent to
said protein crystals, the nature of the crosslinker, the chain
length of the crosslinker, the surface area of said crosslinked
protein crystals, the size of said crosslinked protein crystals,
the shape of said crosslinked protein crystals and combinations
thereof. As used herein, the phrase "controlled dissolution" does
not include leaching.
[0028] Formulations for decontamination--formulations selected from
the group consisting of: formulations for decontamination of
chemical wastes, herbicides, insecticides, pesticides,
environmental hazards and chemical warfare agents.
[0029] Insoluble and stable form of a protein--a form of a protein
which is insoluble in aqueous solvents, organic solvents or
aqueous-organic solvent mixtures and which displays greater
stability than the soluble form of the protein. According to an
alternate embodiment of this invention, the phrase "insoluble and
stable form of a protein" may be a protein which is insoluble in
dry formulations but soluble in wet formulations. In any
embodiment, the crosslinked protein crystals may be active in
insoluble form. And in one embodiment, the crosslinked protein
crystals may be active in insoluble form, then dissolve or are
removed or digested once their function is complete.
[0030] Organic solvents--any solvent of non-aqueous origin.
[0031] Pharmaceutically effective amount--an amount of crosslinked
protein crystals which is effective to treat a condition in an
individual to whom they are administered over some period of
time.
[0032] Prophylactically effective amount--an amount of crosslinked
protein crystals which is effective to prevent a condition in an
individual to whom they are administered over some period of
time.
[0033] Protein--any peptide having a tertiary structure or any
protein.
[0034] Protein activity--an activity selected from the group
consisting of binding, catalysis, activities which generate a
functional response within the environment in which the protein
used, such as induction of immune response and immunogenicity, or
combinations thereof.
[0035] Protein activity release rate--the quantity of protein
dissolved per unit time.
[0036] The crosslinked protein crystals of this invention are
particularly advantageous because they are stable in harsh
environments imposed by the formulations or compositions in which
they are employed or conditions of their storage. At the same time,
these crosslinked protein crystals are capable of (1) change to
soluble and active form (an active form including, in one
embodiment of this invention, a form which is active against
macromolecular substrates) or
[0037] (2) controlled dissolution or release of their activity when
exposed to one or more triggers in their environment. Such triggers
may be selected from the group consisting of change in temperature,
change in pH, change in chemical composition, change from
concentrate to dilute form, change in shear force acting upon the
crystals and combinations thereof. Controlled dissolution or
release of activity of crosslinked protein crystals according to
this invention may also be triggered over a change in time.
[0038] Specific examples of such triggers include an increase or
decrease in temperature, for example, an increase in temperature
from a low temperature between about 0.degree. C. and about
20.degree. C. to a high temperature between about 25.degree. C. and
about 70.degree. C. Other triggers include a change from acidic pH
to basic pH and a change from basic pH to acidic pH. Examples of
triggers of change from concentrate to dilute form include, for
example, a change in solute concentration, a change in
concentration of all solutes from about 2-fold to about
10,000-fold, a change in concentration of all solutes from about
2-fold to about 700-fold, an increase or decrease in salt
concentration, an increase or decrease in water concentration, an
increase or decrease in organic solvent concentration, a decrease
in protein concentration and a decrease in detergent
concentration.
[0039] Additional triggers involve changes in chemical composition
of the environment surrounding the crosslinked protein crystals
that affect the environment or the crosslinker itself. Such changes
include, for example, addition of chemical reagents, increase or
decrease in organic solvent concentration, chemical events that
affect the crosslinker, chemical changes induced by application of
energy, including light, microwave or radiation. As explained
above, any of these triggers may act in combination or in sequence
with one or more of the other triggers.
[0040] Controlled dissolution of crosslinked protein crystals
according to the present invention may also be effected by a change
in time sufficient to permit a protein activity release rate
between about 0.1% per day and about 100% per day, a change in time
sufficient to permit a protein activity release rate between about
0.01% per hour and about 100% per hour and a change in time
sufficient to permit a protein activity release rate between about
1% per minute and about 50% per minute.
[0041] Crosslinked protein crystals according to this invention,
therefore, include those capable of releasing their protein
activity at a controlled rate upon exposure to a change in their
environment, said change being selected from the group consisting
of change in pH, change in solute concentration, change in
temperature, change in chemical composition, change in shear force
acting upon the crystals and combinations thereof. Said controlled
rate of releasing protein activity may be determined by a factor
selected from the group consisting of the degree of crosslinking of
the crosslinked protein crystals, the length of time of exposure of
protein crystals to the crosslinking agent, the rate of addition of
crosslinking agent to the protein crystals, the nature of the
crosslinker, the chain length of the crosslinker, the surface area
of the crosslinked protein crystals, the size of the crosslinked
protein crystals, the shape of the crosslinked protein crystals and
combinations thereof.
[0042] As a result of their crystalline nature, the crosslinked
protein crystals of this invention achieve uniformity across the
entire crosslinked crystal volume. This uniformity is maintained by
the intermolecular contacts and chemical crosslinks between the
protein molecules constituting the crystal lattice. The protein
molecules maintain a uniform distance from each other, forming
well-defined stable pores within the crosslinked protein crystals
that facilitate access of substrate to the protein, as well as
removal of product. In these crosslinked protein crystals, the
lattice interactions, when fixed by chemical crosslinks, are
particularly important in providing stability and preventing
denaturation, especially in storage, under conditions including
harsh environments created by components of compositions in which
the crystals are used. At the same time, the protein crystals are
crosslinked in such a way that they dissolve or release their
protein activity upon exposure to a trigger in their environment
encountered under conditions of use. Thus, they may be
substantially insoluble and stable in a composition under storage
conditions and substantially soluble and active under conditions of
use of said composition.
[0043] Factors contributing to the release rate of protein activity
of crosslinked protein crystals according to this invention include
the degree of crosslinking of the crosslinked protein crystals, the
length of time of exposure of protein crystals to the crosslinking
agent, the rate of addition of crosslinking agent to the protein
crystals, the length of time of exposure of protein crystals to the
crosslinking agent, the nature of the crosslinker, the chain length
of the crosslinker, the surface area of the crosslinked protein
crystals, the size of the crosslinked protein crystals, the shape
of the crosslinked protein crystals and combinations thereof.
[0044] In addition to their activity, crosslinked protein crystals
according to this invention are particularly stable and insoluble
under storage conditions, including the attendant storage
temperature, storage pH, storage time, storage concentrate form,
storage involving little or no shear force acting upon the
crystals, or combinations thereof. Advantageously, these
crosslinked protein crystals are soluble and active under
conditions of use, including conditions involving change in
temperature, change in pH, change in chemical composition, change
from concentrate to dilute form, change in shear force acting upon
the crystals and combinations thereof. Such properties make the
crosslinked protein crystals of this invention particularly useful
for delivery of cleaning agents, including detergents,
pharmaceuticals, personal care agents or compositions, including
cosmetics, vaccines, veterinary compositions, foods, feeds,
diagnostics and formulations for decontamination.
[0045] According to one embodiment, the crosslinked protein
crystals of this invention are characterized by a half-life of
activity under storage conditions which is greater than at least 2
times that of the soluble form of the protein that is crystallized
to form the crystals that are crosslinked and activity similar to
that of the soluble form of the protein under conditions of
use.
[0046] The protein constituent of the crosslinked protein crystals
of this invention may be any protein, including, for example,
therapeutic proteins, prophylactic proteins, including antibodies,
cleaning agent proteins, including detergent proteins, personal
care proteins, including cosmetic proteins, veterinary proteins,
food proteins, feed proteins, diagnostic proteins and
decontamination proteins. Included among such proteins are enzymes,
such as, for example, hydrolases, isomerases, lyases, ligases,
transferases and oxidoreductases. Examples of hydrolases include
thermolysin, elastase, esterase, lipase, nitrilase, amylase,
pectinase, subtilinase, hydantoinase, asparaginase, urease,
subtilisin and other proteases and lysozyme. Examples of lyases
include aldolases and hydroxynitrile lyase. Examples of
oxidoreductases include peroxidase, laccase, glucose oxidase,
alcohol dehydrogenase and other dehydrogenases. Other enzymes which
may be crystallized and crosslinked include cellulases and
oxidases.
[0047] Examples of therapeutic or prophylactic proteins include
hormones, antibodies, inhibitors, growth factors, trophic factors,
cytokines, lymphokines, toxoids, erythropoietin, Factor VIII,
insulin, amylin, TPA, dornase-.alpha., .alpha.-1-antitripsin, human
growth hormones, nerve growth hormones, bone morphogenic proteins,
urease, toxoids, fertility hormones, FSH, LSH, postridical
hormones, tetanus toxoid, diptheria toxoid, vitamins and
nutrients.
[0048] According to one embodiment of this invention, crosslinked
protein crystals are characterized by activity which is similar to
that of their soluble or uncrosslinked crystallized counterparts
under conditions of use. Advantageously however, the crosslinked
protein crystals of this invention display improved stability under
storage conditions, as compared to their soluble or uncrosslinked
crystallized counterpart proteins.
[0049] The crosslinked protein crystals of this invention may be
used in any of a number of chemical processes. Such processes
include industrial and research-scale processes, such as organic
synthesis of specialty chemicals and pharmaceuticals. Enzymatic
conversion processes include oxidations, reductions, additions,
including esterifications and transesterifications, hydrolyses,
eliminations, rearrangements, and asymmetric conversions, including
stereoselective, stereospecific and regioselective reactions.
[0050] Thus, crosslinked protein crystals according to this
invention may be advantageously used instead of conventional
soluble or immobilized proteins in cleaning agents, including
detergents, pharmaceuticals, veterinary compounds, personal care
compositions, including cosmetics, foods, feeds, vaccines, pulp,
paper and textile processing, diagnostics and formulations for
decontamination.
[0051] Crosslinked protein crystals according to this invention may
also be used in various environmental applications. They may be
used in place of conventional soluble or immobilized proteins for
environmental purposes, such wide area decontamination of
environmental hazards.
[0052] Alternatively, the crosslinked protein crystals of this
invention may be used in cleaning agents, selected from the group
consisting of detergents, such as powdered detergents and liquid
detergents, bleaches, household cleaners, hard surface cleaners,
industrial cleaners and carpet and upholstery shampoos.
[0053] Cleaning agents containing crosslinked protein crystals
according to the present invention may also comprise compounds
conventionally included in such agents. See, for example, Soaps and
Detergents. A Theoretical and Practical Review, Louis Spitz (Ed.),
AOCS Press (Champlain, Ill.) (1996). Such compounds include
anionic, non-ionic, cationic or zwitterionic surfactants, or
mixtures thereof.
[0054] Anionic surfactants are exemplified by alkyl sulfates, alkyl
ether sulfates, alkyl sulfonates, alkylaryl sulfonates, olefin
sulfonates, alkyl ether phosphates, alkyl ether phosphates, fatty
acid salts, soaps, isothionates and sulfonated unsaturated esters
and acids.
[0055] Non-ionic surfactants are exemplified by products of
condensation of an organic aliphatic or alkyl aromatic hydrophobic
compound with an alkylene oxide, alkyl polyglucosides and sugar
esters.
[0056] Cationic surfactants are exemplified by quarternary ammonium
salts of tertiary alkyl amines, amino amides, amino esters or
imidazolines containing al least one long chain (C.sub.8-C.sub.22)
aliphatic group or an alkyl-aryl group, wherein alkyl comprises
about 4 to 12 carbon atoms and aryl is preferably a phenylene
group.
[0057] Zwitterionic surfactants are exemplified by derivatives of
quarternary ammonium, quarternary phosphonium or tertiary sulfonium
compounds, derivatives of secondary and tertiary amines and
derivatives of heterocyclic secondary and tertiary amines.
[0058] And crosslinked protein crystals according to this invention
may be used as ingredients in personal care compositions, including
cosmetics, such as creams, lotions, emulsions, foams, washes,
compacts, gels, mousses, slurries, powders, sprays, pastes,
ointments, salves, balms, drops, shampoos, and sunscreens. In
topical creams and lotions, for example, they may be used as
humectants or for skin protection, softening, bleaching, cleaning,
deproteinization, lipid removal, moisturizing, decoloration,
coloration or detoxification. They may also be used as
anti-oxidants in cosmetics.
[0059] According to this invention, any individual, including
humans and other mammals, may be treated in a pharmaceutically
acceptable manner with a pharmaceutically effective or a
catalytically effective amount of crosslinked protein crystals for
a period of time sufficient to treat a condition in the individual
to whom they are administered over some period of time.
Alternatively, individuals may receive a prophylactically effective
or a catalytically effective amount of crosslinked protein crystals
of this invention which is effective to prevent a condition in the
individual to whom they are administered over some period of
time.
[0060] Such crosslinked protein crystals may be administered alone,
as part of a pharmaceutical, personal care or veterinary
preparation or as part of a prophylactic preparation, such as a
vaccine, with or without adjuvant. They may be administered by
parenteral or non-parenteral route. For example, they may be
administered by oral, pulmonary, nasal, aural, anal, dermal,
ocular, intravenous, intramuscular, intraarterial, intraperitoneal,
mucosal, sublingual, subcutaneous, or intracranial route. In either
pharmaceutical, personal care or veterinary applications,
crosslinked protein crystals may be topically administered to any
epithelial surface. Such epithelial surfaces include oral, ocular,
aural, anal and nasal surfaces, to treat, protect, repair or
detoxify the area to which they are applied.
[0061] The present invention also includes controlled release
formulations comprising crosslinked protein crystals according to
this invention. In such formulations, the crosslinked protein
crystals are substantially insoluble under storage conditions and
capable of releasing their protein activity in vivo at a controlled
rate. For example, a pharmaceutical controlled release formulation
according to this invention, administered by oral route, is
characterized in that the component crosslinked protein crystals
are substantially insoluble under gastric pH conditions and
substantially soluble under small intestine pH conditions.
Alternatively, for these and other uses according to this
invention, the crosslinked protein crystals may be active in the
insoluble form and then dissolve and are removed or digested once
their function is complete.
[0062] Pharmaceutical, personal care, veterinary or prophylactic
compositions comprising crosslinked protein crystals according to
this invention may also be selected from the group consisting of
tablets, liposomes, granules, spheres, microparticles, microspheres
and capsules.
[0063] For such uses, as well as other uses according to this
invention, crosslinked protein crystals may be formulated into
tablets. Such tablets constitute a liquid-free, dust-free form of
crosslinked protein crystal storage which are easily handled and
retain acceptable levels of activity.
[0064] Alternatively, the crosslinked protein crystals may be in a
variety of conventional depot forms employed for administration to
provide reactive compositions. These include, for example, solid,
semi-solid and liquid dosage forms, such as liquid solutions or
suspensions, gels, creams, balms, emulsions, lotions, slurries,
powders, sprays, foams, pastes, ointments, salves, balms and
drops.
[0065] Compositions or formulations comprising the crosslinked
protein crystals of this invention may also comprise any
conventional carrier or adjuvant used in pharmaceuticals, personal
care compositions or veterinary formulations. These carriers and
adjuvants include, for example, Freund's adjuvant, ion exchangers,
alumina, aluminum stearate, lecithin, buffer substances, such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium, trisilicate, cellulose-based substances and polyethylene
glycol. Adjuvants for topical or gel base forms may include, for
example, sodium carboxymethylcellulose, polyacrylates,
polyoxyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wood wax alcohols.
[0066] According to one embodiment of this invention, crosslinked
protein crystals may be combined with any conventional materials
used for controlled release administration. Such materials include,
for example, coatings, shells and films, such as enteric coatings
and polymer coatings and films.
[0067] The most effective mode of administration and dosage regimen
of formulations or compositions comprising crosslinked protein
crystals of this invention will depend on the effect desired,
previous therapy, if any, the individual's health status or status
of the condition itself and response to the crosslinked protein
crystals and the judgment of the treating physician or clinician.
The crosslinked protein crystals may be administered in any dosage
form acceptable for pharmaceuticals, personal care compositions or
veterinary formulations, at one time or over a series of
treatments.
[0068] The amount of the crosslinked protein crystals that may be
combined with carrier materials to produce a single dosage form
will vary depending upon the particular mode of administration,
formulation, dose level or dose frequency. A typical preparation
will contain between about 0.01% and about 99%, preferably between
about 1% and about 50%, crosslinked protein crystals (w/w).
[0069] Upon improvement of the individual's condition, a
maintenance dose of crosslinked protein crystals may be
administered, if necessary. Subsequently, the dosage or frequency
of administration, or both, may be reduced as a function of the
symptoms, to a level at which the improved condition is retained.
When the condition has been alleviated to the desired level,
treatment should cease. Individuals may, however, require
intermittent treatment on a long-term basis upon any recurrence of
the condition or symptoms thereof.
[0070] An alternate embodiment of the present invention includes
protein delivery systems comprising the crosslinked protein
crystals disclosed herein. Such a system may be used to deliver
proteins such as those included in cleaning agents, such as
detergents, personal care products, such as cosmetics,
pharmaceuticals, veterinary compositions, vaccines, foods, feeds,
diagnostics and formulations for decontamination. Protein delivery
systems of this invention, which may be formulations or devices,
such as implantable devices, may be microparticulate protein
delivery systems, wherein the crosslinked protein crystals have a
longest dimension between about 0.01 .mu.m and about 500 .mu.m,
alternatively between about 0.1 .mu.m and about 50 .mu.m. The
crosslinked protein crystal components of such systems may have a
shape selected from the group consisting of: spheres, needles,
rods, plates, such as hexagons and squares, rhomboids, cubes,
bipryamids and prisms. Advantageously, the crosslinked crystal form
of the proteins of this invention allow loading of up to between
about 50% and about 90% protein per unit of weight.
[0071] One example of a protected protein system according to this
invention is suitable for storage in a medium such as a liquid
detergent, prior to use. The crosslinked protein crystal components
of such a system are insoluble under storage conditions in said
medium--which typically causes degradation of the soluble form of
the protein that is crystallized to form said crystal that is
crosslinked--and soluble under conditions of use.
[0072] According to the present invention, preparation of
crosslinked protein crystals includes the steps of crystallizing
and crosslinking the protein. This may be carried out as
illustrated below.
[0073] Preparation of Crosslinked Protein Crystals--Protein
Crystallization
[0074] Protein crystals are grown by controlled crystallization of
protein out of aqueous solution or aqueous solution-containing
organic solvents. Conditions to be controlled include, for example,
the rate of evaporation of solvent, the presence of appropriate
co-solutes and buffers, pH and temperature. A comprehensive review
of the various factors affecting the crystallization of proteins
has been published by McPherson, Methods Enzymol., 114, pp. 112-20
(1985).
[0075] McPherson and Gilliland, J. Crystal Growth, 90, pp. 51-59
(1988) have compiled comprehensive lists of proteins and nucleic
acids that have been crystallized, as well as the conditions under
which they were crystallized. A compendium of crystals and
crystallization recipes, as well as a repository of coordinates of
solved protein and nucleic acid structures, is maintained by the
Protein Data Bank at the Brookhaven National Laboratory
[http//www.pdb.bnl.gov; Bernstein et al., J. Mol. Biol., 112, pp.
535-42 (1977)]. These references can be used to determine the
conditions necessary for crystallization of a protein, as a prelude
to the formation of an appropriate crosslinked protein crystal, and
can guide the crystallization strategy for other proteins.
Alternatively, an intelligent trial and error search strategy can,
in most instances, produce suitable crystallization conditions for
many proteins, provided that an acceptable level of purity can be
achieved for them [see, e.g., C. W. Carter, Jr. and C. W. Carter,
J. Biol. Chem., 254, pp. 12219-23 (1979)].
[0076] For use in crosslinked protein crystals according to this
invention, the large single crystals which are needed for X-ray
diffraction analysis are not required. Microcrystalline showers are
suitable.
[0077] For example, the crosslinked protein crystals may have a
longest dimension between about 0.01 .mu.m and about 500 .mu.m,
alternatively, between 0.1 .mu.m and about 50 .mu.m. They may also
have a shape selected from the group consisting of: spheres,
needles, rods, plates, such as hexagons and squares, rhomboids,
cubes, bipryamids and prisms.
[0078] In general, crystals are produced by combining the protein
to be crystallized with an appropriate aqueous solvent or aqueous
solvent containing appropriate crystallization agents, such as
salts or organic solvents. The solvent is combined with the protein
and subjected to agitation at a temperature determined
experimentally to be appropriate for the induction of
crystallization and acceptable for the maintenance of protein
activity and stability. The solvent can optionally include
co-solutes, such as divalent cations, co-factors or chaotropes, as
well as buffer species to control pH. The need for co-solutes and
their concentrations are determined experimentally to facilitate
crystallization. In an industrial-scale process, the controlled
precipitation leading to crystallization can best be carried out by
the simple combination of protein, precipitant, co-solutes and,
optionally, buffers in a batch process. Alternative laboratory
crystallization methods, such as dialysis or vapor diffusion, can
also be adopted. McPherson, supra and Gilliland, supra, include a
comprehensive list of suitable conditions in their reviews of the
crystallization literature. Occasionally, incompatibility between
the crosslinking agent and the crystallization medium might require
exchanging the crystals into a more suitable solvent system.
[0079] Many of the proteins for which crystallization conditions
have already been described, may be used to prepare crosslinked
protein crystals according to this invention. It should be noted,
however, that the conditions reported in most of the above-cited
references have been optimized to yield, in most instances, a few
large, diffraction quality crystals. Accordingly, it will be
appreciated by those of skill in the art that some degree of
adjustment of these conditions to provide a high yielding process
for the large scale production of the smaller crystals used in
making crosslinked protein crystals may be necessary.
[0080] Preparation of Crosslinked Protein Crystals--Crosslinking of
Protein Crystals
[0081] Once protein crystals have been grown in a suitable medium
they can be crosslinked. Crosslinking results in stabilization of
the crystal lattice by introducing covalent links between the
constituent protein molecules of the crystal. This makes possible
transfer of the protein into an alternate environment that might
otherwise be incompatible with the existence of the crystal lattice
or even with the existence of intact protein.
[0082] Advantageously, crosslinking according to the present
invention is carried out in such a way that, under conditions of
storage, the crosslinking interactions prevent the constituent
protein molecules in the crystal from going back into solution,
effectively insolubilizing or immobilizing the protein molecules
into microcrystalline particles. Upon exposure to a trigger in the
environment surrounding the crosslinked protein crystals, such as
under conditions of use rather than storage, the protein molecules
dissolve, releasing their protein activity. The rate of dissolution
is controlled by one or more of the following factors: the degree
of crosslinking, the length of time of exposure of protein crystals
to the crosslinking agent, the rate of addition of crosslinking
agent to the protein crystals, the nature of the crosslinker, the
chain length of the crosslinker, the surface area of the
crosslinked protein crystals, the size of the crosslinked protein
crystals, the shape of the crosslinked protein crystals and
combinations thereof.
[0083] Crosslinking can be achieved using one or a combination of a
wide variety of multifunctional reagents, at the same time (in
parallel) or in sequence, including bifunctional reagents. Upon
exposure to a trigger in the surrounding environment, or over a
given period of time, the crosslinks between protein crystals
crosslinked with such multifunctional crosslinking agents lessen or
weaken, leading to protein dissolution or release of activity.
Alternatively, the crosslinks may break at the point of attachment,
leading to protein dissolution or release of activity. Such
crosslinking agents include glutaraldehyde, succinaldehyde,
octanedialdehyde and glyoxal. Additional multifunctional
crosslinking agents include halo-triazines, e.g., cyanuric
chloride; halo-pyrimidines, e.g., 2,4,6-trichloro/bromo-pyrimidine;
anhydrides or halides of aliphatic or aromatic mono- or
di-carboxylic acids, e.g., maleic anhydride, (meth)acryloyl
chloride, chloroacetyl chloride; N-methylol compounds, e.g.,
N-methylol-chloro acetamide; di-isocyanates or di-isothiocyanates,
e.g., phenylene-1,4-di-isocyanate and aziridines. Other
crosslinking agents include epoxides, such as, for example,
di-epoxides, tri-epoxides and tetra-epoxides. According to a
preferred embodiment of this invention, the crosslinking agent is
glutaraldehyde, used alone or in sequence with an epoxide. For a
representative listing of other available crosslinking reagents
see, for example, the 1996 catalog of the Pierce Chemical Company.
Such multifunctional crosslinking agents may also be used, at the
same time (in parallel) or in sequence, with reversible
crosslinking agents, such as those described below.
[0084] According to an alternate embodiment of this invention,
crosslinking may be carried out using reversible crosslinkers, in
parallel or in sequence. The resulting crosslinked protein crystals
are characterized by a reactive multi-functional linker, into which
a trigger is incorporated as a separate group. The reactive
functionality is involved in linking together reactive amino acid
side chains in a protein and the trigger consists of a bond that
can be broken by altering one or more conditions in the surrounding
environment (e.g., pH, temperature, or thermodynamic water
activity). This is illustrated diagrammatically as:
X-Y-Z+2 AA residues-->AA.sub.1-X-Y-Z-AA.sub.2
change in environment-->AA.sub.1-X+Y-Z-AA.sub.2
[0085] where X and Z are groups with reactive functionality
[0086] where Y is a trigger
[0087] where AA.sub.1 and AA.sub.2 represent reactive amino acid
residues on the same protein or on two different proteins. The bond
between the crosslinking agent and the protein may be a covalent or
ionic bond, or a hydrogen bond. The change in surrounding
environment results in breaking of the trigger bond and dissolution
of the protein. Thus, the crosslinks between protein crystals
crosslinked with such reversible crosslinking agents break, leading
to protein crystal dissolution or release of activity.
[0088] Alternatively, the reactive functionality of the crosslinker
and the trigger may be the same, as in:
X-Z+2AA residues-->AA.sub.1-X-Z-AA.sub.2
change in environment-->AA.sub.1+X-Z-AA.sub.2.
[0089] The crosslinker may be homofunctional (X.dbd.Y) or
heterofunctional (X is not equal to Y). The reactive functionality
X and Y may be, but not limited to the following functional groups
(where R, R', R", and R'" may be alkyl, aryl or hydrogen
groups):
[0090] I. Reactive acyl donors are exemplified by: carboxylate
esters RCOOR', amides RCONHR', Acyl azides RCON.sub.3,
carbodiimides R--N.dbd.C.dbd.N--R', N-hydroxyimide esters,
RCO--O--NR', imidoesters R--C.dbd.NH2.sup.+(OR'), anhydrides
RCO--O--COR', carbonates RO--CO--O--R', urethanes RNHCONHR', acid
halides RCOHal (where Hal=a halogen), acyl hydrazides RCONNR'R",
O-acylisoureas RCO--O--C.dbd.NR'(--NR"R'"),
[0091] II. Reactive carbonyl groups are exemplified by: aldehydes
RCHO and ketones RCOR', acetals RCO(H.sub.2)R', ketals
RR'CO.sub.2R'R". Reactive carbonyl containing functional groups
known to those well skilled in the art of protein immobilization
and crosslinking are described in the literature [Pierce Catalog
and Handbook, Pierce Chemical Company, Rockford, Ill. (1994); S. S.
Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC
Press, Boca Raton, Fla. (1991)].
[0092] III. Alkyl or aryl donors are exemplified by: alkyl or aryl
halides R-Hal, azides R--N.sub.3, sulfate esters RSO.sub.3R',
phosphate esters RPO(OR'.sub.3), alkyloxonium salts R.sub.3O+,
sulfonium R.sub.3S+, nitrate esters RONO.sub.2, Michael acceptors
RCR'.dbd.CR'"COR", aryl fluorides ArF, isonitriles RN+.ident.C--,
haloamines R.sub.2N-Hal, alkenes and alkynes.
[0093] IV. Sulfur containing groups are exemplified by disulfides
RSSR', sulfhydryls RSH, epoxides R.sub.2C.sup.OCR'.sub.2.
[0094] V. Salts are exemplified by alkyl or aryl ammonium salts
R.sub.4N+, carboxylate RCOO--, sulfate ROSO.sub.3--, phosphate
ROPO.sub.3" and amines R.sub.3N.
[0095] The table below includes examples of triggers, organized by
release mechanism. In the table, R.dbd. is a multifunctional
crosslinking agent that can be an alkyl, aryl, or other chains with
activating groups that can react with the protein to be
crosslinked. Those reactive groups can be any variety of groups
such as those susceptible to nucleophilic, free radical or
electrophilic displacement including halides, aldehydes,
carbonates, urethanes, xanthanes, epoxides among others.
1 Release Trigger Examples Conditions 1. Acid Labile R--O--R
H.sup.+ or Lewis Linkers e.g. Thp, MOM, Acidic catalysts Acetal,
ketal Aldol, Michael adducts, esters 2. Base Labile R'OCO2--R'
Variety of basic Linkers Carbonates media R'O--CONR.sub.2
Carbamates R.sub.2'NCONR.sub.2 Urethanes Aldol, Michael adducts,
esters 3. Fluoride R--OSiR.sub.3 Aqueous F.sup.- Labile Linkers
Various Si containing linkers 4. Enzyme RCOOR, RCONR.sub.2' Free
lipases, Labile Linkers amidases, esterases 5. Reduction Disulfide
H.sub.2 catalyst; Labile Linkers linkers that Hydrides cleave via
Hydrogenolysis Reductive Elimination R'--S--S--R 6. Oxidation
R--OSiR.sub.3 Oxidizing Labile Linkers Glycols R-- agents: e.g.
CH(OH)--CH(OH)--R' H.sub.2O.sub.2, NaOCl, IO.sub.4.sup.- Metal
based oxidizers, other hypervalent oxidents 7. Thio-labile
R'--S--S--R Thiols, e.g., linkers Cys, DTT, mercaptoethanol 8.
Heavy Metal Various Allyl Transition metal Labile Linkers Ethers
based reagents ROCH.sub.2CH.dbd.CHR (Pd, Ir, Hg, Ag, Alkyl, Acyl
Cu, Tl, Rh) Allyl ester Pd(0) catalysts 9. Photolabile
O-nitrobenzyl light (hv) Linkers (ONB) DESYL groups in linker 10.
Free Thiohydroxamate Free radical Radical Labile ester initiator
Linkers (Barton ester) 11. Metal- Iron (III) Metal removal chelate
linked diphenanthroline e.g. by chelation or precipitation 12.
Thermally Peroxides Increase in Labile Linkers R--OO--R temperature
13. "Safety Methylthioethyl Base; amines, Catch" Labile (Mte)
others Linkers Dithianes
[0096] Additional examples of reversible crosslinkers are described
in T. W. Green, Protective Groups in Organic Synthesis, John Wiley
& Sons (Eds.) (1981). Any variety of strategies used for
reversible protecting groups can be incorporated into a crosslinker
suitable for producing crosslinked protein crystals capable of
reversible, controlled solubilization. Various approaches are
listed, in Waldmann's review of this subject, in Anaewante Chemie
Inl. Ed. Engl., 35, p. 2056 (1996).
[0097] Other types of reversible crosslinkers are disulfide
bond-containing crosslinkers. The trigger breaking crosslinks
formed by such crosslinkers is the addition of reducing agent, such
as cysteine, to the environment of the crosslinked protein
crystals.
[0098] Disulfide crosslinkers are described in the Pierce Catalog
and Handbook (1994-1995).
[0099] Examples of such crosslinkers include:
[0100] Homobifunctional (Symmetric)
[0101] DSS--Dithiobis(succinimidylpropionate), also know as
Lomant's Reagent
[0102] DTSSP--3-3'-Dithiobis(sulfosuccinimidylpropionate), water
soluble version of DSP
[0103] DTBP--Dimethyl 3,3'-dithiobispropionimidate.HCl
[0104] BASED--Bis-(.beta.-[4-azidosalicylamido]ethyl)disulfide
[0105]
DPDPB--1,4-Di-(3'-[2'-pyridyldithio]-propionamido)butane.
[0106] Heterobifunctional (Asymmetric)
[0107] SPDP--N-Succinimidyl-3-(2-pyridyldithio)propionate
[0108]
LC-SPDP--Succinimidyl-6-(3-[2-pyridyldithio]propionate)hexanoate
[0109]
Sulfo-LC-SPDP--Sulfosuccinimidyl-6-(3-[2-pyridyldlthio]propionate)h-
exanoate, water soluble version of LC-SPDP
[0110]
APDP--N-(4-[p-azidosalicylamido]butyl)-3'-(2'-pyridyldithio)propion-
amide
[0111] SADP--N-Succinimidyl(4-azidophenyl)1,3'-dithiopropionate
[0112] Sulfo-SADP--Sulfosuccinimidyl(4-azidophenyl)
1,3'-dithiopropionate, water soluble version of SADP
[0113]
SAED--Sulfosuccinimidyl-2-(7-azido-4-methycoumarin-3-acetamide)ethy-
l-1,3'dichiopropionate
[0114]
SAND--Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'-dith-
iopropionate
[0115]
SASD--Sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopro-
pionate
[0116] SMPB--Succinimidyl-4-(p-maleimidophenyl)butyrate
[0117]
Sulfo-SMPB--Sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate
[0118]
SMPT--4-Succinimidyloxycarbonyl-methyl-.alpha.-(2-pyridylthio)tolue-
ne
[0119]
Sulfo-LC-SMPT--Sulfosuccinimidyl-6-(.alpha.-methyl-.alpha.-(2-pyrid-
ylthio)toluamido)hexanoate.
[0120] In order that this invention may be better understood, the
following examples are set forth. These examples are for the
purpose of illustration only and are not to be construed as
limiting the scope of the invention in any manner.
EXAMPLES
Example 1
Preparation of Crosslinked Subtilisin Crystals
[0121] Crystallization of Subtilisin
[0122] One volume of Alcalase 2.5 L (Novo Nordisk Bioindustrials,
Franklinton, N.C.) was added to 2 volumes of a solution of 15%
sodium sulfate (pH 5.5) prepared at 30-35.degree. C. The
crystallization solution was seeded with {fraction
(1/2,000)}-{fraction (1/500)} volume seeds (30 mg/ml slurry of
crystals in 15% sodium sulfate (pH 5.5), pH supported at 5.5 by
adding NaOH. The seeded crystallization solution was incubated at
30-35.degree. C., stirring by magnetic stirrer overnight. This
yielded 60-80% (by activity) crystal rods, 10-50 .mu.m, in length,
1-3 .mu.m in width, after 24-48 hours.
Example 2
Crosslinking of Subtilisin Crystals
[0123] Subtilisin crystals were crosslinked using one of a variety
of crosslinkers, including: glutaraldehyde, glyoxal,
succinaldehyde, octanedialdehyde and epoxides.
[0124] Glutaraldehyde Crosslinking
[0125] Glutaraldehyde ("GA") (supplied as 50% in aqueous by Aldrich
Chemical Co.) was diluted in deionized water at 4.degree. C. in the
various amounts listed in Table I below. For each ml of subtilisin
crystals (27 mg/ml) in 15% sodium sulfate, 10 .mu.l of the diluted
glutaraldehyde was added to the slurry while shaking on a vortex at
low speed (for amounts less than 5 ml) or stirring with an overhead
stirrer at medium speed (for amounts 25 ml-500 ml). After mixing
for the allotted crosslinking time, the samples were centrifuged
for 20 seconds at maximum speed, the supernatant was discarded and
replaced with 15% sodium sulfate. This "washing" was repeated a
total of 5 times. The final resuspension was effected with 900
.mu.l of 15% sodium sulfate.
2TABLE I Glutaraldehyde Crosslinking Crosslinking % GA-final GA(ml)
H.sub.2O(ml) time (min) 0.0076 1.0 64.96 60 0.0189 1.0 25.46 39
0.02 1.0 24.0 39, 81 0.05 1.0 9.00 15, 60, 89 0.08 1.0 5.25 39, 81
0.10 1.0 4.00 60, 81 0.125 1.0 3.00 3, 10, 17, 39 0.15 1.0 2.33 81,
120 0.20 1.0 1.50 19, 60, 120 0.231 1.0 1.16 10, 39, 120 0.3 1.0
0.67 60 0.5 1.0 0 60
[0126] Glyoxal Crosslinking
[0127] Glyoxal (supplied as 40% in aqueous by Aldrich Chemical Co.)
("GY") was added to the crystal suspension to give a final
concentration of 0.01-1.0%. For each ml of subtilisin crystals (27
mg/ml) in 15% sodium sulfate 0.25 .mu.l to 25 ml (0.01 to 1%) of
the glyoxal was added to the slurry, while magnetically stirring at
ambient temperature. After stirring for 1 hour, the crosslinked
crystals were centrifuged and washed, as described for
glutaraldehyde crosslinking.
[0128] Octanedialdehyde Crosslinking
[0129] Octanedialdehyde ("OA") (100% as supplied by DSM Chemie
Linz), in the amounts shown in Table II below, was added undiluted
to 1 ml of subtilisin crystal slurry (27 mg/ml in 15% sodium
sulfate) while magnetically stirring at ambient temperature.
Stirring was continued for the specified time of minutes or hours
before the crosslinked crystals were centrifuged and washed, as
described for glutaraldehyde crosslinking.
3TABLE II Octanedialdehyde Crosslinking % OA - final OA (.mu.l)
Crosslinking time 0.05 0.5 16 h 0.1 1.0 16 h 0.2 2.0 16 h 0.25 2.5
16 h 0.5 5.0 16 h 1.0 10.0 30 m, 1 h, 3 h, 16 h
[0130] Succinaldehyde Crosslinking
[0131] Succinaldehyde ("SA")(40% as supplied by DSM Chemie Linz)
was added undiluted, in the amounts shown in Table III below, to 1
ml of subtilisin crystal slurry (27 mg/ml in 15% sodium sulfate)
while magnetically stirring at ambient temperature. Stirring was
continued for the specified time of minutes or hours before the
crosslinked crystals were centrifuged and washed, as described for
glutaraldehyde crosslinking.
4TABLE III Succinaldehyde Crosslinking % SA - final SA (.mu.l)
Crosslinking time 1.0 25 30 m, 1 h, 3 h
[0132] Epichlorohydrin Crosslinking
[0133] A 10 .mu.l aliquot of epichlorohydrin ("EP") (99%, Sigma
Chemical Co., St. Louis, Mo.) was added undiluted to 1 ml of
subtilisin crystal slurry (27 mg/ml in 15% sodium sulfate) while
stirring at ambient temperature. Stirring was continued for the
specified time of minutes or hours before the crosslinked crystals
were centrifuged and washed, as described for glutaraldehyde
crosslinking.
[0134] Epoxide Crosslinking
[0135] General Procedure
[0136] Crosslinking of subtilisin was carried out individually
using one of a variety of epoxides. These included:
[0137] 1) General Name--DENACOL
[0138] a) DENACOL EX-411
[0139] b) DENACOL EX-421
[0140] c) DENACOL EX-614
[0141] d) DENACOL EX-201
[0142] e) DENACOL EX-202; all obtained from Nagase American
Corporation.
[0143] 2) Obtained from Tokyo Kasei Inc. America:
[0144] a) Neopentyl Glycol diglycidyl Ether (N448) ("NP")
[0145] b) Ethylene Glycol diglycidyl Ether (EO342)("EG").
[0146] The concentration of the epoxide was varied between 0.01 and
4.0% and the crosslinking time was varied from 1 hour to 72 hours.
The procedure for addition to and removal of crosslinker from
enzyme was as described above for glutaraldehyde crosslinking.
[0147] Subsequent crosslinking with glutaraldehyde (0.01 to 0.2%)
for (1 hour to 5 hours) yielded strongly crosslinked enzyme
crystals, insoluble in water, but active in the azocasein
assay.
[0148] A sample of 1 ml of subtilisin crystal slurry (27 mg/ml in
15% sodium sulfate) was mixed by vortexing at low speed to assure a
uniform suspension of crystals. Epoxide (10% solution in DMF) was
added to the crystal slurry in the amounts specified in Table IV,
and the mixture was shaken at ambient temperature. After the
allotted time between 1 and 72 hours at ambient temperature,
glutaraldehyde (10% in DMF) was added to the epoxide/crystal
mixture and stirring was continued at ambient temperature for the
time specified in Table IV. The resulting crosslinked enzyme
crystals were washed 2.times. with 1% (NH.sub.4).sub.2SO.sub.4/10
mM CaCl.sub.2 then 3.times. with water and finally 1.times. with 1%
(NH.sub.4).sub.2SO.sub.4/10 mM CaCl.sub.2 before resuspending in 1%
(NH.sub.4).sub.2SO.sub.4/10M CaCl.sub.2.
5TABLE IV Epoxide/Glutaraldehyde Crosslinking Epoxide
Glutaraldehyde Epoxide Epoxide Crosslinking Glutaraldehyde
Crosslinking Name Amount Time Amount Time EX-411 0.01-4% 1-72 h
0.01-0.1% 0.5-2 h EX-421 0.01-4% 1-72 h 0.01-0.1% 0.5-2 h EX-614
0.01-4% 1-72 h 0.01-0.1% 0.5-2 h EX-201 0.01-4% 1-72 h 0.01-0.1%
0.5-2 h EX-202 0.01-4% 1-72 h 0.01-0.1% 0.5-2 h NP 0.01-4% 1-72 h
0.01-0.1% 0.5-2 h (N448) EG 0.01-4% 1-72 h 0.01-0.1% 0.5-2 h
(EO342)
[0149] Large Scale Preparation of A Preferred Epoxide Sample
[0150] Prior to crosslinking, a sample of 380 ml of crystalline
subtilisin in 15% sodium sulfate (27 mg/ml) was mixed by overhead
stirring at ambient temperature for 5 minutes to assure a uniform
suspension of crystals. Neopentyl glycol diglycidyl ether (3.838 ml
of a 10% solution in DMF) was added to the crystal slurry and the
mixture was stirred at ambient temperature. After 5 hours at
ambient temperature, 3.838 ml of glutaraldehyde (10% in DMF) was
added to the epoxide/crystal mixture and stirring was continued at
ambient temperature for 1.5 hours. The resulting crosslinked enzyme
crystals were washed 2.times. with 1% (NH.sub.4).sub.2SO.sub.4/10
mM CaCl.sub.2 then 3.times. with water and finally 1.times. with 1%
(NH.sub.4).sub.2SO.sub.4/10 mM CaCl.sub.2, before resuspending in
1% (NH.sub.4).sub.2SO.sub.4/10 mM CaCl.sub.2'
Example 3
Activity Assay
[0151] In order to test the activity of crosslinked protein
crystals according to this invention, as well as other enzyme
samples, we developed the following azocasein assay.
[0152] Materials:
[0153] 2.0M Tris Buffer. 500 ppm CaCl.sub.2
[0154] 0.2M Tris Buffer. 50 ppm CaCl.sub.2
[0155] 50% urea
[0156] Azocasein
[0157] 5% trichloroacetic acid ("TCA")
[0158] Alcalase (2.5 L)
[0159] ChiroCLEC-BL.TM. (crosslinked subtilisin crystals, available
from Altus Biologics, Inc., Cambridge, Mass.)
[0160] The assay was carried out, preparing azocasein just prior to
use, by dissolving 600 mg azocasein with 10 ml of 50% urea and
vortexing lightly to complete the dissolution. Then 10 ml 2.0M Tris
was added and vortexed to mix, increasing the volume to 100 ml by
adding deionized water.
[0161] The stock solutions of the enzyme to be assayed in 0.2M Tris
were prepared, to provide 50 .mu.l aliquots to be assayed, as
follows:
[0162] Without detergent:
[0163] 0.03 mg/ml Alcalase (soluble, uncrosslinked subtilisin
Carlsberg 80.3 mg/ml) 3.0 mg/ml ChiroCLEC-BL>.
[0164] With 120 .mu.l detergent/ml solution:
[0165] 0.03 mg/ml Alcalase
[0166] 3.0 mg/ml ChiroCLEC-BL.TM..
[0167] We added 50 .mu.l aliquots of enzyme to 150 .mu.l of 0.2M
Tris and placed the mixtures in 5 ml test tubes with micro-stir
bars. We then warmed both the test tubes and the azocasein at
40.degree. C. for 1 minute using a metal heating block. After that,
we added 1 ml of the azocasein to each tube and stirred at
40.degree. C. for 15 minutes using the heating block at stir speed
4. We then added 2 ml TCA to each tube, mixing by vortex, and
placed the tubes in an ice bath immediately, allowing the samples
to stand at 0.degree. C. for 20 minutes. We microfuged the samples
for 5 minutes at maximum rpm and microfiltered, if necessary. We
measured absorbance of the expressed activity in
abs.multidot.units/mg protein.multidot.min supernatant at
.lambda.390. In this assay, all measurements were done in
triplicate. Controls were void of enzyme but contained detergent if
it was present in the assay. This time=0 assay was repeated at
time=15 minutes and other times, if necessary.
[0168] The detergents used in the various assays included Tide,
Wisk and Ciba-Geigy detergents #15, #16 and #44 ("Ciba
detergents"). Ciba detergent #15 constitutes a typical European
detergent formulation--liquid (aqueous) detergent on the basis of
15% alkylbenzene sulfonate, 14% fatty alcohol ethoxylate and 10%
fatty acid salt (soap). Ciba detergent #16 constitutes a typical
United States detergent formulation--liquid (aqueous) detergent on
the basis of 7.5% alkyl benzene sulfonate, 10% fatty alcohol
ethoxylate and 17% alkyl either sulfate. Ciba detergent #44
constitutes a typical compact detergent formulation--liquid
(aqueous) detergent on the basis of 6% fatty alcohol ethoxylate,
23% alkyl ether sulfate and 10% sodium citrate. Ciba detergents
#15, #16 and #44 may be obtained upon request from Ciba Specialty
Chemicals Corp., Division Consumer Care Chemicals, Greensboro,
N.C.
[0169] We prepared assay stock solutions from dilution stocks, and
carried out the assays, as follows.
[0170] Activity Assay--200.times. Dilution--Crosslinked Subtilisin
Crystals and Crystalline Subtilisin in Heavy Duty Liquid Detergent
(Ciba #15, Ciba #44, Tide and Wisk)
[0171] Stocks A, B, C and D were prepared in 10 ml neoprene tubes
as follows.
[0172] Stock A: Crosslinked Subtilisin Crystals Prepared According
to this Invention (.about.27 mg/ml)
[0173] We centrifuged 37 .mu.l slurry of crosslinked subtilisin
crystals (equal to 1 mg crosslinked enzyme crystals) to remove
supernatant, added 1 ml detergent and vortexed to mix. A 50 .mu.l
aliquot of the resulting mixture was added to 9.95 ml water, to a
final concentration of 5 .mu.g/ml.
[0174] Stock B: Uncrosslinked Subtilisin Crystals (.about.27
mg/ml)
[0175] We centrifuged 37 .mu.l slurry of subtilisin crystals (equal
to 1 mg enzyme crystals) to remove supernatant, added 1 ml
detergent and vortexed to mix. A 50 .mu.l aliquot of the resulting
mixture was added to 9.95 ml water, to a final concentration of 5
.mu.g/ml.
[0176] Stock C: Alcalase
[0177] We added 18.75 .mu.l Alcalase (80.3 mg/ml) to 3 ml detergent
and vortexed to mix. A 50 .mu.l aliquot of the resulting mixture
was added to 9.95 ml water, to a final concentration of 2.5
.mu.g/ml.
[0178] Stock D: Detergent
[0179] A 50 .mu.l aliquot of detergent was added to 9.95 ml
water.
[0180] Azocasein stock (6 mg/ml) was prepared as described above.
Upon dilution of Stock A and B to 5 .mu.g/ml, the t=0 assay was set
up immediately and carried out as described above, except that the
amount of stock sample used was 200 .mu.l, instead of 50 .mu.l+150
.mu.l 0.2M Tris. While the tubes were heating for 1 minute at
40.degree. C., two additional samples of 2 ml each of Stocks A, B
and C were placed in 1.5 ml microcentrifuge tubes and heated to
52.degree. C. while shaking for further testing after 5 minutes and
15 minutes dilution with heating.
[0181] Activity Assay--670.times. Dilution--Crosslinked Subtilisin
Crystals and Crystalline Subtilisin in Detergent Concentrate (Ciba
#16)
[0182] Stocks A, B, C and D were prepared in 10 ml neoprene tubes
as follows.
[0183] Stock A: Crosslinked Subtilisin Crystals Prepared According
to this Invention (.about.27 mg/ml)
[0184] We centrifuged 124 .mu.l slurry of crosslinked subtilisin
crystals (equal to 3.35 mg crosslinked enzyme crystals) to remove
supernatant, added 1 ml detergent and vortexed to mix. A 50 .mu.l
aliquot of the resulting mixture was added to 33.45 ml water, to a
final concentration of 5 .mu.g/ml.
[0185] Stock B: Uncrosslinked Subtilisin Crystals (.about.27
mg/ml)
[0186] We centrifuged 124 .mu.l slurry of subtilisin crystals
(equal to 3.35 mg enzyme crystals) to remove supernatant, added 1
ml detergent and vortexed to mix. A 50 .mu.l aliquot of the
resulting mixture was added to 33.45 ml water, to a final
concentration of 5 .mu.g/ml.
[0187] Stock C: Alcalase
[0188] We added 167 .mu.l Alcalase (80.3 mg/ml) to 8 ml detergent
and vortexed to mix. A 50 .mu.l aliquot of the resulting mixture
was added to 33.45 ml water, to a final concentration of 2.5
.mu.g/ml.
[0189] Stock D: Detergent
[0190] A 50 .mu.l aliquot of detergent was added to 33.45 ml
water.
[0191] Azocasein stock (6 mg/ml) was prepared as described above.
The t=0 assay was set up immediately and carried out as described
above, except that the amount of stock sample used was 200 .mu.l,
instead of 50 .mu.l, plus 150 .mu.l 0.2M Tris buffer. While the
tubes were heating for 1 minute at 40.degree. C., two additional
samples of 2 ml each of Stocks A, B and C were placed in Eppendorf
tubes and heated to 40.degree. C. while shaking for further testing
after 5 minutes and 15 minutes dilution with heating.
Example 4
Stability Study
[0192] In order to test the stability of crosslinked enzyme
crystals according to this invention, as well as other enzyme
samples, we developed the following assays.
[0193] Azocasein Assay--Stability Study 52.degree. C.
[0194] First, we prepared stock solutions of the enzyme samples in
detergent in 2 ml Eppendorf tubes with screw caps. After incubating
the mixtures in a water bath at 52.degree. C. for the appropriate
times, we added 1.47 ml of 0.2M Tris buffer to one of each enzyme
sample tube, and mixed well. To assay for activity after the
appropriate time of incubation followed by dilution, we removed a
50 .mu.l aliquot from each tube and assayed as described below. The
remaining samples of enzyme/detergent stocks were placed in a water
bath at 52.degree. C., with further aliquots being removed for
assay at specific times.
[0195] The assay was performed by adding 50 .mu.l enzyme sample to
150 .mu.l 0.2M Tris buffer and heating to 40.degree. C. for 1
minute. At a constant 40.degree. C. temperature, we then added 1.0
ml azocasein stock (as described in Example 1) to each sample,
stirring for 15 minutes using a heating block at stir speed 4. We
then added 2 ml TCA to each tube, mixing by vortex, and placed the
tubes in an ice bath immediately, allowing the samples to stand at
0.degree. C. for 20 minutes. We microfuged the samples for 5
minutes at maximum rpm and microfiltered, if necessary. We measured
absorbance of the supernatant at .lambda.390 and expressed activity
as abs.multidot.units/mg protein.multidot.min. In this assay, all
measurements were done in triplicate. Controls were void of enzyme
but contained detergent if it was present in the assay.
[0196] In order to assess activity as part of stability studies
carried out at 52.degree. C., we prepared assay stock solutions
from dilution stocks, and carried out the assays, as follows.
[0197] For Alcalase
[0198] Stocks:
[0199] Stock A: Alcalase (80.3 mg/ml) in commercial detergent (Tide
or Wisk, deactivated by heating at 70.degree. C. for 4
hours)--final concentration=0.25 mg/ml. The stock was prepared by
adding 31.2 .mu.l Alcalase to 9.97 ml detergent. A 200 .mu.l
aliquot of the resulting mixture was placed in each of several 2 ml
Eppendorf tubes (3.times. for t=0, 30 and others).
[0200] Stock B: Alcalase (80.3 mg/ml) in Ciba detergent (Ciba #15,
Ciba #16 or Ciba #44)--final concentration=0.25 mg/ml. The stock
was prepared by adding 31.2 .mu.l Alcalase to 9.97 ml detergent. A
200 .mu.laliquot of the resulting mixture was placed in each of
several 2 ml Eppendorf tubes (3.times. for t=0, 1 hour and 4-6
hours).
[0201] Stock C: Commercial detergent (Tide or Wisk, deactivated by
heating at 70.degree. C. for 4 hours) 3.times. (200 .mu.l of the
above in 2 ml Eppendorf tubes).
[0202] Stock D: Ciba detergent (Ciba #15, Ciba #16 or Ciba #44)
3.times. (200 .mu.l of the above in 2 ml Eppendorf tubes).
[0203] The t=0 assay was performed immediately after 1.47 ml of 0.2
M Tris was added to one of each of tubes containing Stocks A-D and
the contents mixed well. Remaining samples of Stocks A-D were
placed in a water bath and heated to 52.degree. C. Otherwise, the
assays were carried out as described above.
[0204] For Crosslinked Subtilisin Crystals and Crystalline
Subtilisin
[0205] Stocks A, B, C and D were prepared in 2 ml Eppendorf tubes
with screw caps as follows.
[0206] Stock A: ChiroCLEC-BL.TM. in commercial detergent (denatured
Tide or Wisk)--final concentration=25 mg/ml. The stock was prepared
by centrifuging 3.12 ml enzyme slurry to remove water and then
diluting the enzyme to 10 ml with detergent. A 200 .mu.l aliquot of
the resulting mixture was placed in each of several 2 ml Eppendorf
tubes (4.times. for t=0, 24 hours, 48 hours and 72 hours).
[0207] Stock B: ChiroCLEC-BL.TM. in Ciba detergent (Ciba #15, Ciba
#16 or Ciba #44)--final concentration=25 mg/ml. The stock was
prepared by centrifuging 3.12 ml enzyme slurry to remove water and
then diluting the enzyme to 10 ml with detergent. A 200 .mu.l
aliquot of the resulting mixture was placed in each of several 2 ml
Eppendorf tubes (4.times. for t=0, 24 hours, 48 hours and 72 hours
at 52.degree. C.).
[0208] Stock C: Commercial detergent (Tide or Wisk, deactivated by
heating at 70.degree. C. for 4 hours) 4.times. (200 .mu.l of the
above in 2 ml Eppendorf tubes).
[0209] Stock D: Ciba-Geigy detergent (Ciba #15, #16 or #44,
depending on which detergent was chosen for Stock B) 4.times. (200
.mu.l of the above in 2 ml Eppendorf tubes).
[0210] The t=0 assay was set up immediately after 1.47 ml of 0.2M
Tris was added to one of each of tubes containing Stocks A-D and
the contents mixed well. Remaining samples of Stocks A-D were
placed in a water bath and heated to 52.degree. C. Otherwise, the
assays were carried out as described above.
[0211] For Crosslinked Subtilisin Crystals and Crystalline
Subtilisin
[0212] Stocks A, B and C were prepared in 2 ml Eppendorf tubes with
screw caps as follows.
[0213] Stock A: Uncrosslinked subtilisin crystals (.about.27 mg/ml)
in Ciba detergent (Ciba #15, #16 or #44)--final concentration=1
mg/ml. The stock was prepared by centrifuging 50 .mu.l crystal
slurry to remove supernatant, then adding 1.35 ml detergent (Ciba
#15, Ciba #16 or Ciba #44) to a final concentration of 1 mg/ml. An
80 .mu.l aliquot of the resulting mixture was placed in each of
several 2.0 ml Eppendorf tubes (3.times. for t=0, 15 minutes and
others).
[0214] Stock B: Crosslinked subtilisin crystals according to this
invention (.about.27 mg/ml) in Ciba detergent (Ciba #15, #16 or
#44)--final concentration=.about.1 mg/ml. The stock was prepared by
centrifuging 50 .mu.l crystal slurry to remove supernatant, then
adding 1.35 ml detergent, to a final concentration of 1 mg/ml. An
80 .mu.l aliquot of the resulting mixture was placed in each of
several 2.0 ml Eppendorf tubes (3.times. for t=0, 15 minutes and
others).
[0215] Stock C: Ciba detergent (Ciba #15, #16 or #44)-3.times. (80
.mu.l of the above in 2.0 ml tubes).
[0216] The t=0 assay was set up immediately after 1.8 ml of water
was added to one of each of tubes containing Stocks A-C and the
contents mixed well. Remaining samples of Stocks A-C were placed in
a water bath and heated to 52.degree. C. Otherwise, the assays were
carried out as described above, except for the addition of 150
.mu.l 0.2 M Tris buffer instead of 200 .mu.l.
[0217] Azocasein Assay--Stability Study 40.degree. C.
[0218] First, we prepared stock solutions of the enzyme samples in
detergent in 2 ml Eppendorf tubes with screw caps. To assay
stability at t=0, we added 1.8 ml of deionized water to a 25 .mu.l
of each sample and mixed well. We removed a 25 .mu.l aliquot from
each tube and assayed as described below. The remaining samples of
enzyme/detergent stocks were placed in a water bath at 40.degree.
C., with further aliquots being removed for assay at specific
times.
[0219] The assay was performed by adding 25 .mu.l of the diluted
enzyme sample to 175 .mu.l 0.2M Tris buffer and heating to
40.degree. C. for 1 minute. At a constant 40.degree. C.
temperature, we then added 1.0 ml azocasein stock (as described in
Example 0.3) to each sample, stirring for 15 minutes using a
heating block at stir speed 4. We then added 2 ml TCA to each tube,
mixing by vortex, and placed the tubes in an ice bath immediately,
allowing the samples to stand at 0.degree. C. for 20 minutes. We
microfuged the samples for 5 minutes at maximum rpm and
microfiltered, if necessary. We measured absorbance of the
supernatant at .lambda.390 and expressed activity as
abs.multidot.units/mg protein.multidot.min. In this assay, all
measurements were done in triplicate. Controls were void of enzyme
but contained detergent if it was present in the assay.
[0220] In order to assess activity as part of stability studies
carried out at 40.degree. C., we prepared assay stock solutions
from dilution stocks, and carried out the assays, as follows.
[0221] Stock A: Crosslinked Subtilisin Crystals According to this
Invention (.about.27 mg/ml)
[0222] We centrifuged 124 .mu.l slurry of crosslinked subtilisin
crystals (equal to 3.35 mg crosslinked enzyme crystals) to remove
supernatant, added 1 ml detergent and vortexed to mix, to a final
concentration of 3.35 mg/ml.
[0223] Stock B: Uncrosslinked Subtilisin Crystals (.about.27
mg/ml)
[0224] We centrifuged 124 .mu.l slurry of subtilisin crystals
(equal to 3.35 mg enzyme crystals) to remove supernatant, added 1
ml detergent and vortexed to mix, to a final concentration of 3.35
mg/ml.
[0225] Stock C: Alcalase
[0226] We added 20.9 .mu.l Alcalase (80.3 mg/ml) to 1 ml Ciba
detergent (Ciba #15, Ciba #16 or Ciba #44) and commercial detergent
(Tide or Wisk, denatured by heating at 70.degree. C. for 4 hours)
and vortexed to mix, to a final concentration of 1.67 mg/ml.
[0227] Stock D: Detergent
[0228] One ml of commercial detergent (Tide or Wisk, deactivated by
heating at 70.degree. C. for 4 hours) and Ciba detergents #15, #16
and #44. A 25 .mu.l aliquot of each stock was added to 1.8 ml of
deionized water and mixed well. A further 25 .mu.l aliquot of the
diluted stock was added to each reaction tube.
[0229] The t=0 assay was performed immediately after 175 .mu.l of
0.2M Tris was added to each tube containing 25 .mu.l of the various
stock samples. Remaining samples of Stocks A-D were placed in a
water bath and heated to 0.40.degree. C. Otherwise, the assays were
carried out as described above.
Example 5
Dissolution Studies
[0230] We also assessed the characteristics of crosslinked enzyme
crystals according to this invention, as well as other enzyme
samples, with respect to dissolution in concentrate and upon
dilution, as detailed below. Stock solutions were prepared and
diluted as described above. The resulting dispersions were heated
at 40.degree. C. and analyzed under a microscope at 250.times. for
dissolution progress.
Example 6
Results of Activity and Stability Assays
[0231] Crosslinked enzyme crystals of subtilisin, as described
above, as well as soluble enzymes and other commercial enzymes,
alone and in the presence of commercial detergents, were tested for
activity in the azocasein assay, as described above. Catalyst
concentrations for equivalent activities were determined for
Alcalase, ChiroCLEC-BL.TM. Wisk with active protease and Tide with
active protease:
6 ChiroCLEC-BL .TM.: 150 .mu.g/6 .mu.l detergent 0.4-0.5 absorbance
units Alcalase: 15 .mu.g/6 .mu.l detergent 0.5-0.6 absorbance units
Tide: 6 .mu.l detergent approximately 0.6 absorbance units Wisk: 6
.mu.l detergent approximately 0.6 absorbance units.
[0232] The dilution studies (discussed supra) were started by
assessing the activities of Alcalase and uncrosslinked crystals of
Alcalase in Ciba detergents #15 and #16. Initial activities were
comparable and losses of up to -50% were seen after 15 minutes at
52.degree. C.
[0233] Table V summarizes the stability of samples of Alcalase
(0.25 mg/ml) and ChiroCLEC-BL.TM. (25 mg/ml) in denatured Wisk or
Tide detergent, or in Ciba detergents #15 and #16 at 52.degree. C.
Activity was measured by the azocasein assay.
7TABLE V Stability of Subtilisin in Detergents at 52.degree. C.
Alcalase T.sub.1/2 ChiroCLEC-BL .TM. T.sub.1/2 Detergent at
52.degree. C. at 52.degree. C. #15 less than 15 min >>100
hours #16 less than 15 min >>100 hours Tide (denatured) 16
hours >>100 hours Wisk (denatured) 60-70 hours >>100
hours
[0234] We also assessed the stability of various enzymes in Ciba
detergent #15 at 52.degree. C. The results are depicted in Table VI
below:
8TABLE VI Stability of Subtilisin in Ciba Detergent #15 at
52.degree. C. Activity 15 min T.sub.1/2 in Initial dilute
concentrate Catalyst Activity at 52.degree. C. 52.degree. C.
Alcalase 36 14 .about.15 min Alcalase 33 13 .about.15 min OA 34 18
.about.15 min 0.1%, 16 h OA 23 17 .about.30 min 1%, 1 h OA 10 14
.about.30 min 1%, 3 h GA 27 16 .about.40 min 0.05%, 30 min GA 35 15
.about.40 min 0.05%, 10 min
[0235] All of the crosslinked crystals prepared as described in the
table above which had half-lives in detergent concentrate of
.about.30 minutes or more also had good solubility profiles.
[0236] In addition, we assessed the stability of various enzymes in
Ciba detergent #15 versus Ciba detergent #16 at 40.degree. C. The
results are depicted in the Table VII below:
9TABLE VII Stability of Subtilisin in Ciba Detergent #15 vs. #16 at
40.degree. C. T.sub.1/2 in #15 T.sub.1/2 in #16 Initial concentrate
concentrate Catalyst Activity at 40.degree. C. at 40.degree. C.
Alcalase 33 10 h 2.5 h GA 27 7 h .about.10 h 0.05%, 30 min OA 32 9
h 8 h 0.1%, 16 h OA 15 12 h 16 h 0.2%, 16 h
[0237] We also assessed the effects of crosslinking time on
activity and stability of the resulting crosslinked enzyme
crystals. These results are summarized in the tables below. In
Table VIII, an asterisk indicates values measured by incubating 25
.mu.l in a 2 ml tube and "Xs" denotes uncrosslinked protein
crystals.
[0238] In preparing the crosslinked protein crystals described in
Tables VIII, 1.times. and X, the protein crystals were crystallized
as described in Example 1 and crosslinked with glutaraldehyde as
described in Example 2, using the crosslinking times and
glutaraldehyde concentrations set forth in that example, or those
specified in the tables.
10TABLE VIII Crosslinking Time vs. Concentration of Glutaraldehyde
on Stability of Subtilisin in Ciba Detergent #16 at 40.degree. C.
Activity Activity Stability, Crosslinking abs/mg/min abs/mg/min 18
h GA (%) Time t = 0 t = 18 h % of Xs, t = 0 (Xs) 0 0 33.6 1.1 3.3
(Xs) 0 0 31.7 2.6* 8.2* 0.0189 10.0 28.5 5.2 16.5 0.0189 10.0 31.3
5.7 17.8 0.0189 39.3 14.1 5.4 17.0 0.0189 39.3 14.3 5.0 15.8 0.05
5.0 20.7 3.8 12.0 0.05 15.0 16.4 7.0 22.1 0.05 18.6 19.6 8.7 27.4
0.05 18.6 17.8 9.8 30.9 0.05 60.0 0 13.5 42.6 0.05 60.0 3.0 14.7
46.4 0.125 3.0 18.3 9.1 28.7 0.125 3.0 15.4 9.0 28.4 0.125 10.0 7.9
14.9 46.9 0.125 10.0 9.5 14.8 46.6 0.125 10.0 7.9 14.7 46.4 0.125
10.0 9.5 13.4 42.1 0.125 10.0 9.4 12.2 38.5 0.125 10.0 8.1 12.2
38.5 0.125 10.0 8.3 16.0 50.5 0.125 17.0 5.4 14.0 44.2 0.125 17.0
6.4 15.3 48.3 0.125 39.3 2.1 3.3 10.4 0.125 39.3 1.0 5.8 18.3 0.125
39.3 1.1 4.4 13.9 0.125 39.3 1.7 4.5 14.3 0.125 39.3 1.6 5.7 18.0
0.125 39.3 0.9 3.3 10.4 0.125 68.6 1.3 3.1 9.9 0.2 5.0 10.4 12.1
38.2 0.2 15.0 2.5 9.0 28.4 0.2 18.6 1.8 6.9 21.8 0.2 18.6 0.8 3.1
9.8 0.2 60.0 0.4 1.3 4.1 0.2 60.0 1.4 1.4 4.4 0.231 10.0 2.7 13.0
41.0 0.231 10.0 4.8 11.7 37.0 0.231 39.3 0.5 1.1 3.5 Alcalase 28.0
3.1* 9.8* Alcalase 32.9 0.2 1.0
[0239] In Table IX, an asterisk indicates that crystals were
crushed during crosslinking and dash marks indicate that no
measurements were taken at those points. All samples were prepared
at 1 ml (27 mg) scale.
11TABLE IX Activity of Glutaraldehyde Crosslinked Subtilisin in
Ciba Detergent #16 at 40.degree. C. Cross- linking Activity at
40.degree. C. time (abs/mg/min) GA (%) (min) t = 0 18 h 39 h 63 h
80 h 90 h 6 days (Xs) 0 0 33.6 1.1 -- -- -- -- -- 0.0076 60 14.1
3.0 -- -- -- -- -- 0.02 39 12.0 7.7 -- -- -- -- -- 0.02 80 6.9 11.5
-- -- -- 0 -- 0.02* 80 12.2 26.2 10.7 3 -- -- -- 0.05* 31 13.5 26.3
9.8 5.3 -- -- -- 0.05 60 4.9 17.7 7.9 2.4 -- -- 1.0 0.05* 60 8.7
27.3 12.5 7.3 -- -- -- 0.05 89 1.3 12.1 -- -- -- 3.7 -- 0.08 39 2.2
12.6 -- -- -- 5.3 -- 0.08 81 0.8 3.9 -- -- 5.8 -- 3.9 0.08* 81 9.8
-- -- -- 10.2 -- -- 0.125 3 16.4 9.7 1.7 0.3 -- -- -- 0.125 10 9.4
14.8 9.4 1.9 -- -- -- 0.125 17 6.4 15.3 11.5 8.5 4.6 -- -- 0.2 5
10.4 12.7 5.1 0.3 -- -- -- 0.23 10 4.8 11.7 10.0 8.5 5.2 -- --
Alcalase 30.5 1.6
[0240]
12TABLE X Conditions for Larger Scale Crosslinked Enzyme Crystal
Preparation - Stability of Glutaraldehyde Crosslinked Subtilisin in
Ciba Detergent #16 at 40.degree. C. Crosslinking time Stability at
40.degree. C. GA (%) (min) (abs/mg/min) t = 0 18 h (Xs) 0 33.9 -- t
= 0 16 h 38 h 59 h 110 h *0.05 60 23.2 16.8 5.9 1.9 5.6 *0.08 80
14.4 12.2 4.1 2.4 -- *0.1 80 8.0 13.6 5.5 3.1 1.4 *0.125 60 9.3
20.9 13.6 -- 2.3 *0.15 80 3.8 11.2 7.4 5.2 2.8 *0.231 60 5.2 9.9
9.3 6.6 8.3 *1.0 60 1.3 2.5 2.2 1.1 2.4 t = 0 24 h 48 h 72 h 120 h
168 h 264 h .sctn. 0.25 120 4.8 9.5 7.7 6.7 5.7 4.3 4.4 .sctn. 0.20
120 2.6 9.5 8.6 8.7 5.6 -- 4.5 .sctn. 0.15 120 5.2 14.3 9.6 5.6 3.7
-- 1.2 .sctn. 0.1-NP/ 5 h/1.5 h 9.6 10.2 6.3 -- -- 5.7 0.1 GA
[0241] In Table X, an asterisk indicates that crosslinkings were
carried out at a 1-2 g scale, .sctn. indicates that crosslinkings
were carried out at a 10 g scale on previously crushed crystals and
dash marks indicate that no measurements were taken at those
points.
[0242] FIG. 1 graphically depicts the stability of 10 g scale
preparations of crosslinked subtilisin crystals according to this
invention in Ciba detergent #16 at 40.degree. C. In FIG. 1, "Altus
I" represents crystals crosslinked with 0.25% glutaraldehyde for 2
hours; "Altus II" represents crystals crosslinked with 0.20%
glutaraldehyde for 2 hours; "Altus III" represents crystals
crosslinked with 0.15% glutaraldehyde for 2 hours and "Altus IV"
represents crystals crosslinked with 0.1% neopentyl glycol
diglycidyl ether for 5 hours, followed by 0.1% glutaraldehyde for
1.5 hours. All the crosslinked samples were crushed prior to
crosslinking using a Brinkman Polytron Homogenizer, then prepared
on a 10 g scale and monitored by the azocasein assay over one week
at 40.degree. C.
Example 7
Results of Dissolution Study
[0243] The dissolution study demonstrated whether various
crosslinked enzyme crystals dissolve in concentrate and the extent
to which they dissolve upon dilution under conditions of use, for
example under wash conditions. Representative results of this test
are included in the tables below, in which "+" indicates that the
sample dissolved, "-" indicates that the sample did not dissolve,
"-/+" indicates that the sample dissolved somewhat (1 mg/ml in
detergent liquid). In the tables, "GP" denotes crystals crosslinked
as described infra for GA crosslinking using, instead, ultrapure
glutaraldehyde (supplied as an 8% aqueous solution by the Sigma
Chemical Co.) which was not diluted prior to addition to the
protein crystals.
13TABLE XI Detergent Liquid Incubation Study - Dissolution Study -
Concentrate 14 h at 40.degree. C. Catalyst Ciba #15 Ciba #16 Ciba
#44 Tide OA - -/+ + - 1%, 16 h OA - - + - 0.5%, 16 h OA - + + -
0.1%, 16 h OA - -/+ + - 0.2%, 16 h GA - -/+ + - 0.5%, 1 h GA - - -
- 0.9%, 1h GA - - + - 0.7%, 1 h EP -/+ + + - 1.0%, 20 min GP - -/+
+ - 0.08%, 20 min CLECBL .TM. - - - - Crystals + + + - (uncross-
linked)
[0244]
14TABLE XII Detergent Liquid Incubation Study - Dissolution Study -
200 fold Dilution 20 minutes at 52.degree. C. Catalyst Ciba #15
Ciba #16 Ciba #44 Tide OA -/+ -/+ + - 1%, 16 h OA - + + - 0.5%, 16
h OA - + + + 0.1%, 16 h OA -/+ + + - 0.2%, 16 h GA - -/+ + - 0.5%,
1 h GA - + + - 0.9%, 1 h GA - - + - 0.7%, 1 h EP + + + + 1.0%, 20
min GP - + + - 0.08%, 20 min CLECBL .TM. - - - - Crystals + + + +
(uncross- linked)
[0245] As demonstrated in the tables above, crosslinked enzyme
crystals according to this invention are essentially insoluble in
concentrated detergent and essentially soluble in diluted detergent
under wash conditions.
Example 8
Summary of Properties of Crosslinked Enzyme Crystals of this
Invention
[0246] Table XIII below summarizes the overall
stability/instability, activity and dissolution properties in Ciba
detergent #15 of crosslinked subtilisin crystals prepared according
to this invention using dialdehydes.
15TABLE XIII Cross- Solubility Solubility Activity Stability linker
in Ciba #15 on Dilution (t = 0) at 52.degree. C. Glyoxal low
dissolve at high low 52.degree. C. Succini- low dissolve at 17-66%
of ND maldehyde 52.degree. C.; Alcalase partially dissolve at
25.degree. C. Glutaral- very low dissolve at 1-100% of low
52.degree. C. dehyde 52.degree. C.; Alcalase moderate partially to
40.degree. C. fully dissolve at 25.degree. C. Octane- very low %
dissolve at 30-66% of low 52.degree. C. dialdehyde 52.degree. C.;
Alcalase moderate partially to 40.degree. C. fully dissolve at
25.degree. C.
[0247] As demonstrated in Table XIII above, the crosslinked enzyme
crystals of the present invention are insoluble and, therefore,
stable under storage conditions, while quickly releasing their
activity under conditions of use. Advantageously, the crosslinked
enzyme crystals of this invention exhibit activity similar to their
soluble or uncrosslinked crystallized counterparts under conditions
of use, while displaying 5-6 fold improved stability, as well as
favorable dissolution properties.
Example 9
Effect of Change of Chemical Composition on Crosslinked Subtilisin
Crystals
[0248] We crystallized subtilisin as described in Example 1 and
crosslinked the resulting crystals as described in Example 2, using
GA 1%/1 hour. When 100 .mu.L (2.2 mg) of the resulting crosslinked
subtilisin crystals was suspended in 1.5 mL of 33.3% of
acetonitrile/phosphate buffer (0.3 M, pH 7.5), the crystals were
completely dissolved after 45 minutes at 40.degree. C.
[0249] Using similar conditions, suspending the crosslinked
subtilisin crystals in 1.2 mL of 16.7% acetonitrile/buffer, the
crystals were completely dissolved after 5 hours.
16 Activity (U) in 100% ACN/16.7% ACN/33.3% time buffer Buffer
Buffer 0 27.3 27.3 27.3 1.5 h 27.3 -- 7.4* 3.3 h 27.3 25.5 7.0 h
27.3 24.0** *crystals were completely dissolved at this time.
**crystals were not completely dissolved.
[0250] Assay: 0.2 mmol (75.8 mg) of TAME in 2.5 mL phosphate buffer
was incubated with each crosslinked subtilisin crystal sample
(equal to 0.044 mg enzyme crystals) suspension (or solution) at
room temperature. One unit hydrolyzed 1.0 mmole of TAME per min.
from per mg crosslinked crystals.
[0251] The results above illustrate the trigger of addition of
organic solvent to the environment of crosslinked protein crystals
of this invention.
Example 10
Wash Performance of Detergents Containing Crosslinked Subtilisin
Crystals
[0252] We assessed the activity and storage stability of
crosslinked enzyme crystals of this invention in liquid detergent,
using a washing assay designed to test the ability of the detergent
to remove stains from a fabric.
Washing Assay
[0253] Preparation of Fabric
[0254] Cloth samples of the same size and weight were cut from the
same bolt:
[0255] 5 g of soiled test cloth and
[0256] 5 g of cotton ballast with no soil (Ciba No. 1-3005).
[0257] Prior to washing the samples, we measured the light
intensity (=lightness) remitted by the soiled fabric samples (as
described below).
[0258] Preparation of Detergent Solution
[0259] The sample of liquid detergent to be tested was heated in a
flask for two hours at 20.degree. C. The sample was then
homogenized by vigorous shaking and 0.8 g of the detergent was
removed from the flask and added to 200 ml of tap water (20.degree.
C.) in a metallic beaker. The aqueous detergent solution was
stirred for 60 seconds.
[0260] Washing
[0261] A sample of soiled test cloth and a sample of unsoiled
ballast were placed together into the beaker containing the aqueous
detergent solution. The beaker was closed tightly and immediately
inserted into a pre-heated (40.degree. C.) washing machine
(Unitest, manufactured by Hereus, Switzerland). During the washing
process, the beaker was rotated constantly in a water bath heated
to 40.degree. C. As a result, the contents of the beaker
continuously warmed, up to a temperature of 40.degree. C.
[0262] Exactly 20 minutes after the fabric was placed in the
detergent solution, washing was stopped and the washed fabric was
immediately removed from the detergent solution and rinsed for 30
seconds with cold tap water (13-15.degree. C.). The wet fabric was
centrifuged and ironed to remove wrinkles and dried at the same
time.
[0263] Measurement of Washing Performance
[0264] Each sample of the washed and dried fabric was examined for
stain removal by remission measurements (lightness Y) between 460
and 700 nm using a Spectraflash 500 (Datacolor). A cut off filter
was used to eliminate potential interference by contamination with
UV-absorbing materials. The lightness value of each test cloth was
measured 5.times. and an average calculated.
[0265] With increasing washing performance, the lightness of the
fabric increases Washing performance is thus defined as a
difference in lightness, .DELTA.Y:
.DELTA.Y=Lightness of fabric after washing-Lightness of fabric
before washing
Example 11
Effect of Concentration of Crosslinked Subtilisin Crystals on
Washing Performance of Detergents Containing them
[0266] Washing performance of crosslinked enzyme crystals according
to this invention was examined as a function of their concentration
in the liquid detergent, using the materials described below.
[0267] Test fabric: EMPA (Eidgen{overscore (o)}ssische
Materialpr{overscore (u)}fungs und Forschungsanstalt, St. Gallen,
Switzerland) #116 soiled with a combination of blood, milk and
carbon black.
[0268] Liquid detergent: Ciba detergent #16.
[0269] Enzyme:
[0270] Crosslinked enzyme crystals; sample Altus IV (as described
in Example 6)
[0271] Uncrosslinked enzyme (Alcalase).
[0272] Concentration of Enzyme in Liquid Detergent:
[0273] enzyme concentrations were between 0.05 and 0.9 w % (dry
matter weight). Table XIV provides further details.
17 TABLE XIV Weight of enzyme Dry matter Liquid suspension (g)
weight of Detergent Enzyme w % Alcalase Altus IV enzyme g Ciba #16
g 0.05 0.106 0.0053 10 0.05 0.130 0.0056 10 0.1 0.207 0.0104 10 0.1
0.240 0.0104 10 0.3 0.599 0.0301 10 0.3 0.683 0.0297 10 0.5 0.492
0.0247 5 0.5 0.580 0.0252 5 0.9 0.886 0.0445 5 0.9 1.046 0.0455
5
[0274] Preparation of Liquid Detergent with Enzyme
[0275] Specific aliquots of the suspension of enzyme crystals (see
Table XIV) were added to a flask and centrifuged to separate the
crystals from the liquid. The liquid was discarded and the crystals
were suspended and homogenized in the liquid detergent (for
quantities see Table XIV). The resulting preparations were used in
the washing tests.
[0276] Washing tests to evaluate the performance of the enzyme
detergent formulations were carried out as described in the assay
above. The results of the study are depicted in FIG. 2. The figure
demonstrates that at enzyme concentrations .gtoreq.0.1 w %, the
washing effect of Ciba liquid detergent #16 formulated with Altus
IV exceeds that of the formulation with uncrosslinked Alcalase. The
efficacy of both crosslinked and uncrosslinked enzymes was reduced
at enzyme concentrations below 0.1 w %.
Example 12
Storage Stability and Washing Performance of Detergents Containing
Crosslinked Subtilisin Crystals
[0277] Detergents formulated with crosslinked and uncrosslinked
enzymes were stored at a constant temperature, in order to examine
enzyme stability in concentrated liquid detergent. The detergent
formulations (150 g each) were prepared by the same procedure as
the samples in Example 10.
[0278] Liquid detergent: Ciba detergent #16.
[0279] Enzyme:
[0280] Crosslinked enzyme crystals: sample Altus IV (Example 6)
[0281] Uncrosslinked enzyme (Alcalase)
18 Enzyme concentration: 0.3 w % (dry matter) in liquid
detergent.
[0282] Storage Temperature for Stability Studies:
[0283] All samples were stored at 30.degree. C. for between 0 and 7
days. After 7 days, the samples were divided after 7 days into two
equal portions, in order to study stability at elevated
temperature. One portion continued to be stored at 30.degree. C.,
while the other was stored at 40.degree. C.
[0284] Test fabric: Three different soiled fabrics were used. All
of them were standard test materials available from EMPA:
[0285] EMPA #112: cocoa soiled fabric
[0286] EMPA #116: blood, milk and carbon black soiled fabric
[0287] EMPA #111: blood soiled fabric.
[0288] Washing Performance on Cocoa Soiled Fabric
[0289] Washing performance of various enzyme formulated liquid
detergents was studied with respect to removal of cocoa stains from
a cocoa soiled test fabric, using the washing assay described
above. Storage stability was determined by assessing washing
performance periodically during the detergent storage time, thus
monitoring the impact of storage temperature on enzyme performance
in the liquid detergent. In this assay, the effectiveness of the
liquid detergent decreases as enzyme stability degrades. The
results of this assay, shown in FIGS. 3 and 4, are discussed
below.
[0290] Storage Stability at 30.degree. C.
[0291] As demonstrated in FIG. 3, both Alcalase and Altus IV
formulated detergents exhibited an improved performance after 2
days of storage (compared to initial values). However, as storage
time increased, the performance of the Alcalase formulation
decreased continuously over time, while the Altus IV formulated
detergent exhibited no degradation, even after 28 days of
storage.
[0292] Storage Stability at 40.degree. C.
[0293] As demonstrated in FIG. 4, when the temperature was raised
from 30 to 40.degree. C., Alcalase formulated detergent lost
activity within 2 days, while the Altus IV formulated detergent
degraded slightly, while removing the cocoa soil from the test
fabric significantly, even after 21 days of storage at 40.degree.
C.
[0294] Washing Performance on Fabric Soiled by a Combination of
Blood, Milk and Carbon Black (EMPA #116 Test Fabric)
[0295] The experimental conditions and detergents were the same
(except the stained fabric) as for washing of cocoa stains. The
results of the washing tests are depicted in FIGS. 5 and 6.
[0296] Storage Stability at 30.degree. C.
[0297] FIG. 5 clearly illustrates the decay of washing performance
of the Alcalase formulated detergent after 2 days of storage at
30.degree. C. However, liquid detergent containing Altus IV enzyme
maintained its original washing performance, even after 28 days of
storage.
[0298] Storage Stability at 40.degree. C.
[0299] As demonstrated in FIG. 6, when the storage temperature was
raised from 30 to 40.degree. C., Alcalase formulated detergent lost
nearly all of its washing performance within 2 days. In contrast,
detergent containing Altus IV retained its washing power for an
additional 14 days.
[0300] Washing Performance on Fabric Soiled with Blood
[0301] Washing performance on blood stains was tested with enzyme
containing detergents stored at 30.degree. C. The detergent
composition, washing conditions were the same as in washing of
cocoa stains. The results of the washing test are illustrated in
FIG. 7.
[0302] The assays show that the washing effect on blood stain by
Ciba #16 liquid detergent formulated with Alcalase was low in
comparison to detergent without enzyme. On the other hand, the
Altus IV formulation was more active in washing conditions and more
stable in storage.
[0303] Storage Stability at 30.degree. C.
[0304] The washing effect of Alcalase formulated detergent
decreased rapidly with storage time, whereas Altus IV formulated
detergent retained almost completely its full capacity after 28
days of storage.
Example 13
Solubility of Crosslinked Subtilisin Crystals at 30.degree. C. and
37.degree. C.
[0305] We studied the solubility of various subtilisin crystals,
which had been crosslinked with glutaraldehyde (GA),
octanedialdehyde (OA), neopentyl glycol diglycidyl ether (NP)
followed by glutaraldehyde, or DENACOL EX-411 (411) followed by
glutaraldehyde.
[0306] In 1.5 ml Eppendorf tubes, samples of uncrosslinked
subtilisin crystals and crosslinked subtilisin crystal slurry,
equal to 37.5 mg of enzyme, were microfuged at 5,000 rpm for 5 min
and the supernatant liquid was removed. A 1.5 ml aliquot of PBS
buffer (0.01 M phosphate, 0.0027 M potassium chloride, 0.137 M
sodium chloride, pH 7.4) was added to each sample, bringing the
concentration of subtilisin to 25 mg/ml. The samples were
transferred to 2 ml glass vials with screw caps and magnetic stir
bars then were incubated at 30.degree. C. or at 37.degree. C.
Samples were studied for dissolution by periodically removing 50
.mu.l of the slurry, microfuging at 13,000 rpm for 5 mins, removing
20 .mu.l of the aliquot and placing it in 980 .mu.l of deionized
water, then measuring UV absorbance at 280 nm.
[0307] The following samples were studied:
19 Crosslinker Crosslinker Concentration Crosslinking Time GA 1.0%
1.5 h GA 0.25% 2 h GA 0.2% 2 h GA 0.15% 2 h NP/GA 0.1%/0.1% 5 h/1.5
h 411/GA 0.015%/0.035% 16 h/1 h OA 0.2% 16 h OA 0.1% 16 h OA 0.05%
16 h
[0308] The solubility profiles of the samples, shown in FIGS. 8 and
9, illustrate different rates of dissolution for the crosslinked
crystals.
Example 14
Reversible Crosslinkers--Disulfide Crosslinked Subtilisin
Crystals
[0309] We prepared subtilisin crystals (30-40 .mu.m average, 27
mg/ml in Na.sub.2SO.sub.4) as previously described for subtilisin
crystallization.
[0310] We then crosslinked the crystals using one of the following
crosslinkers:
[0311] 1) Dimethyl 3,3'-dithiobispropionimidate.HCl--(DTBP)
(Pierce)
[0312] 2) Dithiobis(succinimidylpropionate)--(DSP)(Pierce)
[0313] 3) 3,3'-Dithiobis (sulfosuccinimidylpropionate)--(DTSSP)
(Pierce).
[0314] Crosslinking was carried out in 15 ml neoprene screw cap
tubes by placing 740 .mu.l of subtilisin crystal slurry (20 mg) in
9.26 ml of buffer (25 mM NaCO.sub.3/50 mM NaHCO.sub.3, pH 8.0). One
crosslinker was added to each tube as follows: A) 93 mg DTBP (30
mM) B) 100 mg DTSSP (16 mM) C) 120 mg DSP (30 mM).
[0315] The tubes were tumbled at ambient temperature (24-26.degree.
C.) until all samples were determined to be insoluble in 32 mM NaOH
(5 days)-100 .mu.l sample in 300 .mu.l NaOH. Uncrosslinked samples
were readily soluble in 32 mM NaOH at the same concentrations.
Crosslinking was stopped by the addition of 1 ml of 1 M Tris, pH
7.5. The samples were centrifuged at 3,000 rpm for 5 minutes, the
supernatant removed and replaced by 5 ml of 100 mM Tris, pH 7.5.
Centrifugation at 3,000 rpm, for 5 min, followed by replacement of
supernatant with 5 ml of 100 mM Tris (pH 7.5) was repeated
3.times..
Example 15
Dissolution of Disulfide Bond-Containing Crosslinked Subtilisin
Crystals
[0316] A 200 mM solution of cysteine was prepared by dissolving 121
mg cysteine in 5 ml 100 mM Tris (pH 7.5). A 400 .mu.l aliquot of
the cysteine solution was added to 3.times.750 .mu.l vials. A 400
.mu.l aliquot of 100 mM Tris (pH 7.5) was added to another
3.times.750 .mu.l vials. Each crosslinked sample (100 .mu.l) was
added to one vial containing cysteine and one vial without
cysteine. All samples were incubated at 37.degree. C. and monitored
for dissolution of crosslinked enzyme crystals (direct visual and
microscopic observation).
[0317] After incubation for 3 hrs at 37.degree. C., the DTBP sample
appeared to be fully soluble in the presence of cysteine and
insoluble in its absence. The DTSSP sample appeared to be nearly
fully soluble in the presence of cysteine and insoluble in its
absence. The DSP sample was barely soluble in the presence of
cysteine and insoluble in its absence.
Example 16
Crystallization of Candida Rugosa Lipase
[0318] A 5 kg aliquot of Candida rugosa lipase ("CRL") in powder
form (Meito) was mixed with 5 kg celite and dissolved in 102 L
distilled deionized water and the volume brought to 200 L with the
deionized water. The suspension was mixed in an Air Drive Lightning
Mixer for 2 hours at room temperature and then filtered through a
0.5 micron filter to remove celite. The mixture was then
ultrafiltered and concentrated to 14 L (469 g) using a 3K hollow
fiber filter membrane cartridge. Solid calcium acetate was added to
a concentration of 5 mM Ca(CH.sub.3COO).sub.2. The pH was adjusted
to pH 5.5 with concentrated acetic acid as necessary. A 7 litre
aliquot was crystallized by either addition of 1.75 litres of
2-methyl-2,4-pentanediol ("MPD") or by addition of 3.5 litres of a
30% solution of PEG-8000. The resulting solution was mixed and
crystallization allowed to proceed overnight at ambient temperature
for about 17-20 hrs. The crystal yield was about 70%.
[0319] Recrystallization
[0320] The Candida rugosa lipase crystals were solubilized by the
addition of 50 mM sodium phosphate (pH 5.2). Soluble protein
concentration of the crystallization solution was adjusted to 20
mg/ml. MPD was added gradually with stirring over a 6-hour period,
to a final concentration of 25%. The resulting solution was mixed
and crystallization allowed to proceed at ambient temperature for
20 hours.
Example 17
Crystallization of Candida Rugosa Lipase
[0321] Candida rugosa lipase crystals prepared as described in
Example 16, prior to the solubilization and recrystallization
steps, were solubilized by the addition of 50 mM sodium acetate (pH
6.5). Soluble protein concentration of the crystallization solution
was adjusted to 20 mg/ml. MPD was added gradually with stirring
over a 6-hour period to a final concentration of 20%. The resulting
solution was mixed and crystallization allowed to proceed at
ambient temperature for 20 hours.
Example 18
Crosslinking of Candida Rugosa Lipase Crystals
[0322] Candida rugosa lipase crystals, prepared as described in
Example 16, were crosslinked by addition of untreated neat
glutaraldehyde (Sigma) by adding 2 ml of 20% glutaraldehyde
stepwise in a 40.5 ml volume over one hour to 8 ml of stirred
lipase crystals (25 mg/ml), at ambient temperature. The final
crosslinker concentration was-4.0%. Crosslinking was allowed to
proceed over 24 hours. Crystals were recovered by low speed
centrifugation and washed with 25% MPD in 50 mM sodium phosphate
(pH 5.2).
Example 19
Crosslinking of Candida Rugosa Lipase Crystals
[0323] Candida rugosa lipase crystals, prepared as described in
Example 16, were crosslinked by addition of untreated neat
glutaraldehyde by adding 2 ml of 20% glutaraldehyde gradually over
a one hour period. Crystals were crosslinked and processed as
described in Example 18.
Example 20
Crosslinking of Candida Rugosa Lipase Crystals
[0324] Candida rugosa lipase crystals, prepared as described in
Example 16, were crosslinked as described in in Example 19, except
that the reaction was allowed to proceed for 24 hours. The crystals
were then processed as described in Example 18.
Example 21
Crosslinking of Candida Rugosa Lipase Crystals
[0325] Candida rugosa lipase crystals, prepared as described in
Example 17, were crosslinked by addition of glutaraldehyde to a
final concentration of 4.0%. Crosslinking was allowed to proceed
for 3 hours. The crystals were processed as described in Example
18.
Example 22
Crosslinking of Candida Rugosa Lipase Crystals
[0326] Candida rugosa lipase crystals, prepared as described in
Example 17, were crosslinked in neat glutaraldehyde at a
concentration of 6.5% for 1 hour. Crosslinking and processing were
performed as described in Example 18.
Example 23
Crosslinking of Candida Rugosa Lipase Crystals
[0327] Candida rugosa lipase crystals, prepared as described in
Example 17, were crosslinked in neat glutaraldehyde at a
concentration of 6.0% for 1 hour. Crosslinking and processing were
performed as described in Example 18.
Example 24
pH Controlled Solubility of Crosslinked Candida Rugosa Lipase
Crystals
[0328] Solubility of various crosslinked Candida rugosa lipase
crystals was studied following an increase in pH from 6.5 to 9.0.
The crystals were incubated at 1 mg/ml in 50 mM sodium phosphate
(pH 9) containing 25% MPD. Aliquots were removed after 3 hour and
24 hour incubation at 25.degree. C. with stirring. Activity and
soluble protein concentration were measured as described in Example
25. The results are described in the table below.
20 Time (hr) Crosslinked 3 24 Crystal [Prot.] [Prot.] Preparation
Activity (U) (mg/ml) Activity (U) (mg/ml) Example 18 7.5 0.47 20 1
Example 19 10.8 0.60 11.3 0.63 Example 20 7.5 0.42 8.8 0.49
Example 25
pH Solubility of Crosslinked Candida Rugosa Lipase Crystals
[0329] Solubility of various crosslinked Candida rugosa lipase
crystals was studied following an increase in pH from 5.2 to 7.5.
The crystals were incubated at 1 mg/ml in 50 mM sodium phosphate
(pH 7.5) containing 25% MPD. Aliquots were removed after 3 hour and
24 hour incubation at 25.degree. C. with stirring. Insoluble
material was removed by filtration (0.25 micron). Activity in
solution was measured spectrophotometrically by monitoring the
hydrolysis of para nitrophenyl acetate (Fluka) at 400 nm. Substrate
concentration was 1 mM. The assay was performed at 25.degree. C. in
a 1 ml volume of 50 mM sodium acetate (pH 6.5). Soluble protein
concentration was measured by absorbance at 280 mm. Results are
presented in the table below.
21 Time (hr) Crosslinked 3 24 Crystal [Prot.] [Prot.] Preparation
Activity (U) (mg/ml) Activity (U) (mg/ml) Example 21 2.4 0.12 15
0.91 Example 22 10.0 0.63 15 1.0 Example 23 2.5 0.17 11 0.69
Example 26
Crystallization of Human Serum Albumin
[0330] Human serum albumin ("HSA") was purchased from Sigma
Chemical Company as a lyophilized powder. We added 10 grams of
protein powder to a 75 ml stirred solution of 100 mM phosphate
buffer pH 5.5 at 4.degree. C. Final protein concentration was 120
mg/ml (determined from OD.sub.280, extinction coefficient for serum
albumin was assumed to equal 1). Saturated ammonium sulfate
solution (767 g/l) prepared in deionized water was added to the
protein solution to a final concentration of 50% saturation (350
g/l). The crystallization solution was "seeded" with 1 ml of
albumin crystals (50 mg/ml) in 50% ammonium sulfate (pH 5.5). Seed
crystals were prepared by washing a sample of crystals free of
precipitate with a solution of 50% saturated ammonium sulfate in
100 mM phosphate buffer (pH 5.5). The seeded crystallization
solution was incubated at 4.degree. C. overnight on a vigorously
rotating platform. Crystal rods (20.mu.) appeared in the solution
overnight (16 hr).
Example 27
Crosslinking of Human Serum Albumin Crystals
[0331] We crosslinked human serum albumin crystals, prepared as
described in Example 18, at 4.degree. C. in a 10 ml stirred
solution of crystals and mother liquor containing 50% saturated
ammonium sulfate, as described above. The crystals, which were not
washed prior to crosslinking, were crosslinked with glutaraldehyde
as supplied by the manufacturer (Sigma). Glutaraldehyde ("GA")
(20%) was added to the stirred crystallization solution in. 4 equal
volumes (62.5 .mu.l) at 15 minute intervals to a final
concentration of 0.5% (250 .mu.l GA). The crystals were then
incubated at 4.degree. C. Aliquots were removed at incubation times
0, 30 min, 60 min and 4 hours incubation. Crosslinked albumin
crystals were collected by low speed centrifugation and washed
repeatedly with pH 7.5, 100 mM Tris HCl, 4.degree. C. Washing was
stopped when the crystals could be centrifuged at high speed
without aggregation.
Example 28
Crosslinking of Human Serum Albumin Crystals
[0332] We crosslinked human serum albumin crystals as described in
Example 27 above, with one modification; glutaraldehyde (20%) was
added to the crystallization solution in 4 equal volumes (131.3
.mu.l) at 15 minute intervals to a final concentration of 1% (525
.mu.l GA).
Example 29
Solubility of Human Serum Albumin Crystals Crosslinked in 0.5% GA,
Time: 0 Minutes Incubation. Dissolution Induced by Elevated
Temperature
[0333] Human serum albumin, crystallized as described in Example 26
and crosslinked for 0 minutes in 0.5% glutaraldehyde, as described
in Example 27, was assayed for solubility by incubating the
crystals (20 mg/ml) with stirring, in phosphate buffered saline
solution (pH 7.5) at room temperature ("RT") or at 37.degree. C.
Aliquots were removed for assay at times 0.5, 1, 4 and 24 hours.
Insoluble material was removed from the solution by centrifugation
and the soluble protein concentration was measured
spectrophotometrically at 280 nm, as indicated in Table XV.
22 TABLE XV Soluble Protein (mg/ml) Time (hr) RT 37.degree. C. 0.5
0.3 1.5 1.0 3 5 4.0 4 12.5 24.0 17 18.5
Example 30
Solubility of Human Serum Albumin Crystals Crosslinked in 0.5% GA,
Time: 30 Minutes Incubation. Dissolution Induced by Elevated
Temperature
[0334] Human serum albumin, crystallized as described in Example 26
and crosslinked in 0.5% glutaraldehyde, as described in Example 27,
was assayed for solubility by incubating the crystals (20 mg/ml) in
phosphate buffered saline solution (pH 7.5) at room temperature or
at 37.degree. C. Aliquots were removed for assay at times 0.5, 1, 4
and 24 hours. Insoluble material was removed from the solution by
centrifugation and the soluble protein concentration was measured
spectrophotometrically at 280 nm, as indicated in Table XVI.
23 TABLE XVI Soluble Protein (mg/ml) Time (hr) RT 37.degree. C. 0.5
1.5 4 1.0 3 5.5 4.0 7 10 24.0 13.5 17.5
Example 31
Solubility of Human Serum Albumin Crystals Crosslinked in 0.5% GA,
Time: 60 Minutes Incubation. Dissolution Induced by Elevated
Temperature
[0335] Human serum albumin, crystallized as described in Example 26
and crosslinked with 0.5% glutaraldehyde, as described in Example
27, was assayed for solubility by incubating the crystals (20
mg/ml) in phosphate buffered saline solution (pH 7.5) at room
temperature or at 37.degree. C. Aliquots were removed for assay at
times 0.5, 1, 4 and 24 hours. Insoluble material was removed from
the solution by centrifugation and the soluble protein
concentration was measured spectrophotometrically at 280 nm, as
indicated in Table XVII.
24 TABLE XVII Soluble Protein (mg/ml) Time (hr) RT 37.degree. C.
0.5 0 0.4 1.0 0 0.6 4.0 0 3 24.0 8 17
Example 32
Solubility of Human Serum Albumin Crystals Crosslinked in 0.5% GA,
Time: 240 Minutes Incubation. Dissolution Induced by Elevated
Temperature
[0336] Human serum albumin, crystallized as described in Example 26
and crosslinked with 0.5% glutaraldehyde, as described in Example
27, was assayed for solubility by incubating the crystals (20
mg/ml) in phosphate buffered saline solution (pH 7.5) at room
temperature or at 37.degree. C. Aliquots were removed for assay at
times 0.5, 1, 4 and 24 hours. Insoluble material was removed from
the solution by centrifugation and the soluble protein
concentration was measured spectrophotometrically at 280 nm, as
indicated in Table XVIII.
25 TABLE XVIII Soluble Protein (mg/ml) Time (hr) RT 37.degree. C.
0.5 0 0 1.0 0.5 0 4.0 3.5 3 24.0 8.5 14.5
Example 33
Solubility of Human Serum Albumin Crystals Crosslinked in 1.0% GA,
Time: 0 Minutes Incubation. Dissolution Induced by Elevated
Temperature
[0337] Human serum albumin, crystallized as described in Example 26
and crosslinked as described in Example 27, was assayed for
solubility by incubating the crystals (20 mg/ml) in phosphate
buffered saline solution (pH 7.5) at room temperature or at
37.degree. C. Aliquots were removed for assay at times 0.5, 1, 4
and 24 hours. Insoluble material was removed from the solution by
centrifugation and the soluble protein concentration was measured
spectrophotometrically at 280 nm, as indicated in Table XIX.
26 TABLE XIX Soluble Protein (mg/ml) Time (hr) RT 37.degree. C. 0.5
1 2 1.0 3 7 4.0 10.5 16 24.0 19 18.5
Example 34
Solubility of Human Serum Albumin Crystals Crosslinked in 1.0% GA,
Time: 30 Minutes Incubation. Dissolution Induced by Elevated
Temperature
[0338] Human serum albumin, crystallized as described in Example 26
and crosslinked as described in Example 27, was assayed for
solubility by incubating the crystals (20 mg/ml) in phosphate
buffered saline solution (pH 7.5) at room temperature or at
37.degree. C. Aliquots were removed for assay at times 0.5, 1, 4
and 24 hours. Insoluble material was removed from the solution by
centrifugation and the soluble protein concentration was measured
spectrophotometrically at 280 nm, as indicated in Table XX.
27 TABLE XX Soluble Protein (mg/ml) Time (hr) RT 37.degree. C. 0.5
0 0 1.0 0 2 4.0 4.5 7 24.0 8 13
Example 35
Solubility of Human Serum Albumin Crystals Crosslinked in 1.0% GA
Time: 60 Minutes Incubation. Dissolution Induced by Elevated
Temperature
[0339] Human serum albumin, crystallized as described in Example 26
and crosslinked as described in Example 27, was assayed for
solubility by incubating the crystals (20 mg/ml) in phosphate
buffered saline solution (pH 7.5) at room temperature or at
37.degree. C. Aliquots were removed for assay at times 0.5, 1, 4
and 24 hours. Insoluble material was removed from the solution by
centrifugation and the soluble protein concentration was measured
spectrophotometrically at 280 nm, as indicated in Table XXI.
28 TABLE XXI Soluble Protein (mg/ml) Time (hr) RT 37.degree. C. 0.5
0 0.5 1.0 0 1.5 4.0 1 4 24.0 9 13.5
Example 35
Solubility of Human Serum Albumin Crystals Crosslinked in 1.0% GA,
Time: 240 Minutes Incubation. Dissolution Induced by Elevated
Temperature
[0340] Human serum albumin, crystallized as described in Example 26
and crosslinked as described in Example 27, was assayed for
solubility by incubating the crystals (20 mg/ml) in phosphate
buffered saline solution (pH 7.5) at room temperature or at
37.degree. C. Aliquots were removed for assay at times 0.5, 1, 4
and 24 hours. Insoluble material was removed from the solution by
centrifugation and the soluble protein concentration was measured
spectrophotometrically at 280 nm, as indicated in Table XXII.
29 TABLE XXII Soluble Protein (mg/ml) Time (hr) RT 37.degree. C.
0.5 0 0 1.0 0 0 4.0 0 2 24.0 6 10.3
Example 36
Crystallization of Thermolysin
[0341] Thermolysin was purchased from Diawa (Japan) as a
lyophilized powder. Fifteen grams of protein powder were added to a
100 ml stirred solution of 10 mM calcium acetate (pH 11) at ambient
temperature. The pH was maintained at 11 by addition of 2 N NaOH,
until the thermolysin was completely solubilized. The pH was then
adjusted to pH 7.5 by addition of 2 N acetic acid. Crystallization
was allowed to proceed overnight at 4.degree. C. Final protein
concentration was 40 mg/ml (determined from OD.sub.280, extinction
coefficient for thermolysin was assumed to equal 1.8). Crystals
were recovered by centrifugation and recrystallized to obtain a
more uniform crystal size. Recrystallization was performed in a
manner nearly identical to that described for the initial
crystallization. Crystals (40 mg/ml protein) were dissolved by
addition of base at room temperature. The pH of the crystallization
solution was adjusted to 6.5 and crystallization was permitted to
proceed at ambient temperature. Crystal rods (50.mu.) appeared in
the solution overnight (16 hr).
Example 37
Crosslinking of Thermolysin Crystals
[0342] Thermolysin crystals, prepared as described in Example 36,
were suspended (50 mg/ml) in a 50 mM solution of sodium acetate (pH
6.5). Crystals were crosslinked with glutaraldehyde as supplied by
the manufacturer (Sigma). Ten milliliters of glutaraldehyde (10%)
were added gradually over a 1 hour period with stirring to a 10 ml
suspension of crystals. After all of the glutaraldehyde was added,
the crystallization solution incubated at ambient temperature.
Aliquots were removed at incubation times 0.5, 1 and 3 hr.
Crosslinked crystals were collected by low speed centrifugation and
washed exhaustively with pH 7.5 50 mM Tris HCl, containing 10 mM
calcium acetate.
Example 38
Solubility of Thermolysin Crystals Crosslinked for 0.5 hr.
Dissolution Induced by Removal of Calcium Ions by EDTA
[0343] Thermolysin, crystallized as described in Example 36 and
crosslinked for 0.5 hr as described in Example 37, was assayed for
solubility by incubating the crystals (1 mg/ml) with stirring, in
10 mM Tris HCl (pH 7.2) containing 1 mM EDTA (Sigma) 40.degree. C.
One ml aliquots were removed for assay at times 0.5, 3 and 24
hours. Insoluble crystals were removed from the solution by
filtration. One ml of 500 mM calcium acetate (pH 7.2) was added to
each aliquot. Soluble protein concentration was measured
spectrophotometrically at 280 nm. Enzymatic activity was measured
spectrophotometrically by monitoring the hydrolysis of a dipeptide
substrate, FAGLA (Feder). Substrate concentration was 1.67 mM. One
unit is defined as the amount of enzyme required to hydrolyze 1
mmole of substrate in one minute at pH 7.2, 40.degree. C. The
activity of soluble thermolysin was 27 U/mg protein. Data is
presented in Table XXIII.
30TABLE XXIII Soluble Protein Activity Time (hr) (% of Max) (% of
Max) 0.5 80 30 3.0 103 97 24.0 100 91
Example 39
Solubility of Thermolysin Crystals Crosslinked for 1 hr.
Dissolution Induced by Removal of Calcium Ions by EDTA
[0344] Thermolysin, crystallized as described in Example 36 and
crosslinked for 1 hr as described in Example 37, was assayed for
solubility by incubating the crystals (1 mg/ml) with stirring, in
10 mM Tris HCl (pH 7.2) containing 1 mM EDTA 40.degree. C. One ml
aliquots were removed for assay at times 0.5, 3 and 24 hours.
Insoluble crystals were removed from the solution by filtration.
One ml of 500 mM calcium acetate (pH 7.2) was added to each
aliquot. Soluble protein concentration was measured
spectrophotometrically at 280 nm. Enzymatic activity was measured
spectrophotometrically by monitoring the hydrolysis of a dipeptide
substrate, FAGLA (Feder). Substrate concentration was 1.67 mM. One
unit is defined as the amount of enzyme required to hydrolyze 1
.mu.mole of substrate in one minute at pH 7.2, 40.degree. C. The
activity of soluble thermolysin was 27 U/mg protein. Data is
presented in Table XXIV.
31TABLE XXIV Soluble Protein Activity Time (hr) (% of Max) (% of
Max) 0.5 7 11 3.0 24 29 24.0 104 87
Example 40
Solubility of Thermolysin Crystals Crosslinked for 3 hr.
Dissolution Induced by Removal of Calcium Ions by EDTA
[0345] Thermolysin, crystallized as described in Example 36 and
crosslinked for 3 hr as described in Example 37, was assayed for
solubility by incubating the crystals (1 mg/ml) with stirring, in
10 mM Tris HCl (pH 7.2) containing 1 mM EDTA 40.degree. C. One ml
aliquots were removed for assay at times 0.5, 3 and 24 hours.
Insoluble crystals were removed from the solution by filtration.
One ml of 500 mM calcium acetate (pH 7.2) was added to each
aliquot. Soluble protein concentration was measured
spectrophotometrically at 280 nm. Enzymatic activity was measured
spectrophotometrically by monitoring the hydrolysis of a dipeptide
substrate, FAGLA (Feder). Substrate concentration was 1.67 mM. One
unit is defined as the amount of enzyme required to hydrolyze 1
.mu.mole of substrate in one minute at pH 7.2, 40.degree. C. The
activity of soluble thermolysin was 27 U/mg protein. Data is
presented in Table XXV.
32TABLE XXV Soluble Protein Activity Time (hr) (% of Max) (% of
Max) 0.5 2 0 3.0 2 0 24.0 100 73
Example 41
Solubility of Thermolysin Crystals Crosslinked for 3 hr.
Dissolution Induced by Removal of Calcium Ions by Dilution
[0346] Thermolysin crystals, prepared as described in Example 36
and crosslinked for 3 hr as described in Example 37, were washed
free of calcium containing buffer and assayed for solubility by
incubating the crystals (1 mg/ml) with stirring in deionized water.
One ml aliquots were removed for assay at times 0.5, 3 and 24
hours. Insoluble crystals were removed from the solution by
filtration. One ml of 500 mM calcium acetate (pH 7.2) was added to
each aliquot. Soluble protein concentration was measured
spectrophotometrically at 280 nm. Enzymatic activity was measured
spectrophotometrically by monitoring the hydrolysis of a dipeptide
substrate, FAGLA (Feder). Substrate concentration was 1.67 mM. One
unit is defined as the amount of enzyme required to hydrolyze 1
.mu.mole of substrate in one minute at (pH 7.2), 40.degree. C. The
activity of soluble thermolysin was 27 U/mg protein. Data is
presented in Table XXVI.
33TABLE XXVI Soluble Protein Activity Time (hr) (% of Max) (% of
Max) 0.5 0 0 3.0 0 7 24.0 111 81
Example 42
Crystallization of Glucose Isomerase
[0347] Glucose isomerase ("GA") was supplied by Cultor (Finland) as
a crystal slurry. The enzyme was recrystallized by solubilizing a
50 ml volume of the crystal slurry at 50.degree. C. with stirring
for 15 minutes. The solution was clarified by filtration and
allowed to cool slowly at room temperature. Fifty micron crystals
appeared within 5 hours. Crystals were recovered by low speed
centrifugation and washed with 166 mM magnesium sulfate.
Example 43
Crosslinking of Glucose Isomerase Crystals
[0348] Five hundred milligrams of glucose isomerase crystals,
prepared as described in Example 42, were suspended in a 50 ml
solution of 166 mM magnesium sulfate. The crystals were crosslinked
with glutaraldehyde as supplied by the manufacturer (Sigma). Five
milliliters of glutaraldehyde (10%) were added gradually over a 1
hour period with stirring to the 50 ml suspension. After all of the
glutaraldehyde was added, the crystallization solution incubated at
ambient temperature. Aliquots were removed at incubation times 1, 3
and 24 hr. Crosslinked crystals were collected by low speed
centrifugation and washed exhaustively with 50 mM Tris HCl (pH
7.0).
Example 44
Solubility of Glucose Isomerase Crystals Crosslinked for 1 hr.
Dissolution Induced by Removal of Calcium Ions by Dilution at
50.degree. C.
[0349] Glucose isomerase crystals, prepared as described in Example
42 and crosslinked for 1 hr as described in Example 43, assayed for
solubility by incubating the crystals (1 mg/ml) with stirring in
deionized water. One ml aliquots were removed for assay at times 1,
3 and 24 hours. Soluble protein concentration was measured
spectrophotometrically at 280 nm (OD280) (extinction coefficient
for GI was assumed to equal 1). Enzymatic activity was measured
spectrophotometrically by monitoring the conversion of fructose to
glucose.
[0350] Glucose concentration was quantitated spectrophotometrically
using a coupled enzyme assay containing hexokinase and
glucose-6-phosphate dehydrogenase. The dehydrogenase uses NADP as a
cofactor and the amount of NADPH formed in the reaction is
stoichiometric with the concentration of substrate (glucose). The
assay was purchased as a kit from Boehringer Mannheim and was used
according to the manufacturer's instructions. One unit is defined
as the amount of enzyme required to convert 1 .mu.mole fructose to
glucose in one minute at pH 7.0, 60.degree. C. The activity of
soluble glucose isomerase was 51 U/mg protein. Data is presented in
Table XXVII.
34TABLE XXVII Soluble Protein Activity Time (hr) (% of Max) (% of
Max) 0.5 8 0 3.0 52 31 24.0 100 57
Example 45
Solubility of Glucose Isomerase Crystals Crosslinked for 3 hr.
Dissolution Induced by Removal of Calcium Ions by Dilution at
50.degree. C.
[0351] Glucose isomerase crystals, prepared as described in Example
42 and crosslinked for 3 hr as described in Example 43, were
assayed for solubility by incubating the crystals (1 mg/ml) with
stirring in deionized water. One ml aliquots were removed for assay
at times 1, 3 and 24 hours. Soluble protein concentration was
measured spectrophotometrically at 280 nm (OD280) (extinction
coefficient for GI was assumed to equal 1). Enzymatic activity was
measured spectrophotometrically by monitoring the conversion of
fructose to glucose.
[0352] Glucose concentration was quantitated spectrophotometrically
using the coupled enzyme assay containing hexokinase and
glucose-6-phosphate dehydrogenase, as described in Example 44. Data
is presented in Table XXVIII.
35TABLE XXVIII Soluble Protein Activity Time (hr) (% of Max) (% of
Max) 0.5 2 0 3.0 10 6.5 24.0 86 43
Example 46
Solubility of Glucose Isomerase Crystals Crosslinked for 24 hr.
Dissolution Induced by Removal of Calcium Ions by Dilution at
50.degree. C.
[0353] Glucose isomerase crystals, prepared as described in Example
42 and crosslinked for 1 hr as described in Example 43, were
assayed for solubility by incubating the crystals (1 mg/ml) with
stirring in deionized water. One ml aliquots were removed for assay
at times 1, 3 and 24 hours. Soluble protein concentration was
measured spectrophotometrically at 280 nm (OD280) (extinction
coefficient for glucose isomerase was assumed to equal 1).
Enzymatic activity was measured spectrophotometrically by
monitoring the conversion of fructose to glucose.
[0354] Glucose concentration was quantitated spectrophotometrically
using the coupled enzyme assay containing hexokinase and
glucose-6-phosphate dehydrogenase, as described in Example 44. Data
is presented in Table XXIX.
36TABLE XXIX Soluble Protein Activity Time (hr) (% of Max) (% of
Max) 0.5 2 0 3.0 24 5 24.0 83 61
Example 47
Preparation of Tablets Containing Crosslinked Protein Crystals
According to this Invention
[0355] Tablets containing crosslinked protein crystals according to
this invention may be prepared as follows. A suspension of
crosslinked protein crystals is placed in 0.1 M sodium acetate, 20
mM calcium chloride and buffer (pH 7) and dried at 35.degree. C.
The resulting dried material may be mixed with sorbitol 50:50 by
weight and granulated with Eudragit NE 30D (a neutral copolymer
based on ethyl- and methylacrylate) or Eudagit RL 30D (an
ammonio-methacrylate copolymer). The granules are dried (for
example, for 16 hours at 40.degree. C.) and compressed to round
tablets of about 5 mm diameter and weight of about 125 mg. The
content of crosslinked protein crystals in such solid preparations
is about 45% by weight. If the above-described preparation is made
without using sorbitol, the resulting tablets contain about 63% by
weight crosslinked protein crystals.
[0356] When introduced into water or aqueous buffer (such as the
above-described acetate buffer) all the tablets disintegrate in a
matter of 10 minutes under mild shaking at room temperature)
producing particles less than 100 .mu.m in size, the majority in
the range of 4-10 .mu.m. Microscopic examination reveals
polymer-free singular protein crystals, as the predominant species.
The slurry obtained by disrupting the tablets is assayed
titrimetrically using hydrolysis of N(.alpha.)-p-tosyl L-arginine
methyl ester (TAME) at 25.degree. C. (pH 8). Activity corresponding
to between about 50% and 80% of activity of an equal amount of
crosslinked protein crystals (counting the indicated weight of the
crosslinked crystals, rather than of the whole tablets)
results.
[0357] While we have hereinbefore described a number of embodiments
of this invention, it is apparent that our basic constructions can
be altered to provide other embodiments which utilize the processes
and compositions of this invention. Therefore, it will be
appreciated that the scope of this invention is to be defined by
the claims appended hereto rather than by the specific embodiments
which have been presented hereinbefore by way of example.
* * * * *