Note: Descriptions are shown in the official language in which they were submitted.
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' WO 99/55310 PCT/US99/09099 '
STABILIZED PROTEIN CRYSTALS,
FORMULATIONS CONTAINING THEM AND METHODS OF MAKING THEM
TECHNICAL FIELD OF THE INVENTION
This invention relates to methods for the
stabilization, storage and delivery of biologically
active macromolecules, such as proteins, peptides and
nucleic acids. In particular, this invention relates
to protein or nucleic acid crystals, formulations and
compositions comprising them. Methods are provided for
the crystallization of proteins and nucleic acids and
for the preparation of stabilized protein or nucleic
acid crystals for use in dry or slurry formulations.
The crystals, crystal formulations and compositions of
this invention can be reconstituted with a diluent for
the parenteral administration of biologically active
macromolecular components.
The methods of this invention are useful for
preparing crystals of "naked" DNA and RNA sequences
that code for therapeutic or immunogenic proteins and
can be administered parenterally. The dissolving DNA
and RNA molecules, subsequently taken up by the cells
and used to express the protein with the proper
glycosylation pattern, can be either therapeutic or
immunogenic. Alternatively, the present invention is
useful for preparing crystals, crystal formulations and
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compositions of sense and antisense polynucleotides of
RNA or DNA.
The present invention is further directed to
encapsulating proteins, glycoproteins, enzymes,
antibodies, hormones and peptide crystals or crystal
formulations into compositions for biological delivery
to humans and animals. According to this invention,
protein crystals or crystal formulations are
encapsulated within a matrix comprising a polymeric
carrier to form a composition. The formulations and
compositions enhance preservation of the native
biologically active tertiary structure of the proteins
and create a reservoir which can slowly release active
protein where and when it is needed. Such polymeric
carriers include biocompatible and biodegradable
polymers. The biologically active protein is
subsequently released in a controlled manner over a
period of time, as determined by the particular
encapsulation technique, polymer formulation, crystal
geometry, crystal solubility, crystal crosslinking and
formulation conditions used. Methods are provided for
crystallizing proteins, preparing stabilized
formulations using pharmaceutical ingredients or
excipients and optionally encapsulating them in a
polymeric carrier to produce compositions and using
such protein crystal formulations and compositions for
biomedical applications, including delivery of
therapeutic proteins and vaccines. Additional uses for
the protein crystal formulations and compositions of
this invention involve protein delivery in human food,
agricultural feeds, veterinary compositions,
diagnostics, cosmetics and personal care compositions.
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BACKGROUND OF THE INVENTION
Proteins are used in a wide range of
applications in the fields of pharmaceuticals,
veterinary products, cosmetics and other consumer
products, foods, feeds, diagnostics, industrial
chemistry 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
10 poor stability of the proteins, variability of
performance or high cost.
It is imperative that the higher order three-
dimensional architecture or tertiary structure of a
protein be preserved until such time that the
individual protein molecules are required to perform
their unique function. To date, a limiting factor for
use of proteins, particularly in therapeutic regimens,
remains the sensitivity of protein structure to
chemical and physical denaturation encountered during
delivery.
Various approaches have been employed to
overcome these barriers. However, these approaches
often incur either loss of protein activity or the
additional expense of protein stabilizing carriers or
formulations .
One approach to overcoming barriers to the
widespread use of proteins is crosslinked enzyme
crystal ("CLEC'1'""') technology [N.L. St. Clair and M.A.
Navia, J. Am. Chem. Soc., 114, pp. 4314-16 (1992)x.
30 See also PCT patent application PCT/US91/05415.
Crosslinked enzyme crystals retain their activity in
environments that are normally incompatible with enzyme
function. Such environments include prolonged exposure
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to proteases, organic solvents, high temperature or
extremes of pH. In such environments, crosslinked
enzyme crystals remain insoluble, stable and active.
Despite recent progress in protein technology
generally, two problems which are discussed below
continue to limit the use of biological macromolecules
in industry and medicine. The first problem relates to
molecular stability and sensitivity of higher order
tertiary structures to chemical and physical
denaturation during manufacturing and storage. Second,
the field of biological delivery of therapeutic
proteins requires that vehicles be provided which
release native proteins, such as proteins,
glycoproteins, enzymes, antibodies, hormones, nucleic
acids and peptides at a rate that is consistant with
the needs of the particular patient or the disease
process.
Macromolecule Stability
Numerous factors differentiate biological
macromolecules from conventional chemical entities,
such as for example, their size, conformation and
amphiphilic nature. Macromolecules are not only
susceptible to chemical, but also physical degradation.
They are sensitive to a variety of environmental
factors, such as temperature, oxidizing agents, pH,
freezing, shaking and shear stress [Cholewinski, M.,
Luckel, B. and Horn, H., Acta Helv., 71, 405 (1996)].
In considering a macromolecule for drug development,
stability factors must be considered when choosing a
production process.
Maintenance of biological activity during the
development and manufacture of pharmaceutical products
depends on the inherent stability of the macromolecule,
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as well as the stabilization techniques employed. A
range of protein stabilization techniques exist
including:
a) Addition of chemical "stabilizers" to
the aqueous solution or suspension of protein. For
example, United States patent 4,297,344 discloses
stabilization of coagulation factors II and VIII,
antithrombin III and plasminogen against heat by adding
selected. amino acids. United States patent 4,783,441
discloses a method for stabilizing proteins by adding
surface-active substances. United States patent
4,812,557 discloses a method for stabilizing
interleukin-2 using human serum albumin. The drawback
of such methods is that each formulation is specific to
the protein of interest and requires significant
development efforts.
b) Freeze/thaw methods in which the
preparation is mixed with a cryoprotectant and stored
at very low temperatures. However, not all proteins
will survive a freeze/thaw cycle.
c) Cold storage with cryoprotectant
additive, normally glycerol.
d) Storage in the glass form, as described
in United States patent 5,098,893. In this case,
proteins are dissolved in water-soluble or water-
swellable substances which are in amorphous or glassy
state.
e) The most widely used method for the
stabilization of proteins is freeze-drying or
lyophilization [Carpenter, J.F., Pical, M.J., Chang,
B.S. and Randolph, T.W., Pharm. Res., 14:(8) 969
(1997)]. Whenever sufficient protein stability cannot
be achieved in aqueous solution, lyophilization
provides the most viable alternative. One
19-05-2000 ~ 02330476 2000-10-26 ~~~'~)~~ ~ US 009909099
a~: i =i'vWe .. r " r, ~. : e-
J S 99109099
~~sF_~~:-c'~;~~;. 4
ALTUS BIOLOGICS INC., et al. _
~ ~~~w,~,~!~ ~tw~~'P
.. ~ ~.~~ ~~ Ii:vJ
Our Ref.: D 2570 PCT
. ~ ~ ~ ~ ... . ~ . 1.. ~ . ~ . .
~ ~ . . ~ . 1 . .
- 6 - . . ... .. .. ... .. ..
d~.sadvaritae~e of lyophilization is that it requires
sophist~~a~ed processing, is ruse consuming and
expensive Carpenter, J.F., Pical, M.J., Chang, B.S.
and Randolph~ T.W., charm. Res., ~:t8) 969 (1997) and
l~teraturw c~.ted therein] . In add3 tion, .f
lyophil~.xation is not carried out carefully, most
preparat:.op.s are at least Fartial.ly denatured by the
freezing a~d dehydrat~.on steps of the technique. The
result is ~regueatly irreversible «ggregation of a
portzon oi~~protein molecules, rendering a formulation
unacceptab~~ , for parenteral $d~uini~aration_
the vast ma~orit~ of protein formulations
produced~Y~~ the above-described techniques require cold
storage, s metimes as low as -20°C. Exposure to
I5 elevated t~m eratures during shipp~.ng or storage can
P
reszlt in Significant activity losses. Thus, storage
at elevater~, or even amb~.ent temperatures, is not
possible for many proteins.
Proteins, peptides and nucleic acids are
. 20 increas~.ng~.y employed in the pharmaceutical,
diagnost;.c~ food, cosmetic, deterg~:nt and research
industr~.es; There is a great need for alternative
stabilization procedures, ~,hich arE: fast, inexpensive
and appl~.c~.ble to a broad range of biological
25 macromol~c~lles. In particular, stavbilizat:ion
procedures~are needed that do not rely on the excessive
use of excip~-ents, which can inter~'ere with the
functions of those biological macrGmolecules.
'the stability of small iuc>lecule crystalline
30 drugs is such that they can withstand extreme forces
during thy forutulat.ion process (see: United States
patent 5~ 9.0,118) . Forces associated with twilling
nanoparticl.es of crystalline u~aterlal of relat.wely
~.nsoluble drugs ~.nclude: shear stress, turbulent flow,
35 h~.gh impact collas~.ons, cavitat~.on and grinding. Small
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... .. .. ... .. ..
_ 7 _
molecular Crystalline compounds have been recogni2ed as
being much:more stable toward cheru cal degradation than
the corres~ondi.ag amorphous solid (Pical, M.J., Lukes,
A.L., Lang, J.E. and Gaines, J. Phs.rm..Sci., 67, 767
(19?8~].~ Unfortunately, crystals c~f macromolecules,
such as proteins and nucleic acids, present additional
problems aid difficulties not associated with small
molecules..
. for most of this century, science aid
medicz.ne have tried to solve the problem of providing
insulin ~.n a useful form to d.~abet~ es . Attexupt3 havo
been made ~o solve some of the protWems of stability
and biolog~.cal del~.very of that protein. For example,
United Stakes patent 5,506,203 describes the use of
amorphous ~.nsulin combine. with an absorbtz.on enha:~cer.
The solid. state insulin was exclu~.ively amorphous
material, ors shown by a palari2ed Sight microscope.
,~en.sen et a1. co-pxecipitated in.sul~n with an
absorbti4n enhancer for use in res~.iratory tract
delivery.~o~ insulin (See ACT patent application WO
98/42368.. Here, the absorbtion er,hancer was desribed
as a surfactant, such as ,s salt of a fatty acid_or a
bZle salt. Tnsulzn crystals of le;.s than 10
micrometers in da.ameter aid lackin5 zinc were produced
by S. Have~.und (See PCT patent application WO
98/42?49~. Similarly, crystals weze also produced in
the presence of surfactants to enh~ce pulmonary
adm.i.nist.~ation.
OA-A-1 196 864 relates to couepositions
compz-isinc~ crystals of insulin and a biodegradable
matrix of ~ b~.odegradable polymer. The biodegradable
polymer is ~ further defa.zied as any convenient
biodegradable polymer which is solLble in organic
solvents to pexznz.t the formation of a matrix therewz.th.
AMENDED SHEET
19-05-2000 ~ 02330476 2000-10-26
US 009909099
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..
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g _
'~o date, those of skzll in the art recognize
that the q;eatly enhanced stabilit~~ of the crystalline
state obsE~ved for small ~noiecules does not translate
to bzologWal macroiaolecules Iprcal, M.J. and Rigsbee,
D.R., Phar~a. Res., 14:13?9 (1997) ] . &or exaaaple,
agueous ~u$pensions of crystalline insulin are only
slightly m~re stable (to the degree of a factor of two)
- than corresponding suspensions of zvmorphous phase
j8range, J~ , Langkjaer, L. , Havelut~d, S . and Volund,
A., Pharra. : Res., 9:?15 (19°2) ] . Ir. the solid state,
lyophiliTe~l amorphoa3 insulin is fear more stable than
lyophili2e~1 crystalline insulin under all conditions
- investig~t~d jPical, M_J. and Rigsk~ee, p.R., Pharm.
rtes., 19;179 (1997)].
l~tolecular weight has proround effect on all
praperties~of macromolecules, including their
macramalecular volume, hydration, t~iscosity, diffusion,
mobility arid stability. jc~antor, C.R and Schimmel, P.R,
Bi.ophysiga~. Cheu~istry, W.H. Freemar, and Co., New York,
19801. '
WV 96/18417 relates to pr.armaceutical
composrt~.ons comprising a crystalline protein at~d
polyethyyei~e glycol or a pharmaceutically acceptable
vegetable coil. The invenCion is dESCribed by the
authors a~s~unexpectedly high contrclled release
propert~es~that result when crystalline proteins are
suspended ~n a PEG solutic~a or gel, or in a vegetable
oil. The authors spec~.fically claim compositions
co~aprisirg :crystall~.ne proteins selected from the group
consisti~gcof crystalline erythropcietin (EPO),
crystall~n~ granulocyte-colany stiz~ulating factor iG-
CSF), crysllalline insulin, and crystalline granulocyte-
macrophage-colony .st~.mulaxi2g factcr. (GM-CSF) .
WO-A-9 641 873 relates to formulations of
pclynucle~oxide complexes which are stab~.lized with a
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- 8a -
cryoprotec~ compound and lyophxl~.zed. The lyophil~.zed
formulat:.,oiis are then mil i.ed or sieved into a dry
powder formulation which may be usEd to deliver the
polynucleo~~de co~aplex.
~L~L~RY OF TH~_~~IJTIQLT
~pTe have found, sux'prisingly, that biological
iaacromoleci~les which are nat stable when held in
solution at ambient or elevated temperatures can
nevertheless be successfully storey in dry form for
long pc~iaals of time at such tempctatures in
cryszall~.ne form. As a pcact~.cal natter, five aspects
of this d.~~covery are particularly advantageous.
First. crystalli.nity of stored materials is
very impos~.aat, since large scale c:rystalli2ation can
be introduced as a final purification step and/or
concentrat~.on step in clinical manLfacturing processes,
such as ~hQse for manufacturing thFrapeutics and
vaccines. Moreover, large scale cnystalli2ation can
replace 5o~e of the purification steps in the
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manufacturing process. For example, protein
crystallization can streamline the production of
protein formulations making it more affordable.
Second, macromolecular interactions which
occur in solution are prevented or severely reduced in
the crysi~alline state, due to considerable reduction of
all reaction rates. Thus, the crystalline state is
uniquely suited to the storage of mixtures of
biological macromolecules.
Third, solid crystalline preparations can be
easily reconstituted to generate ready to use
parenteral formulations having very high protein
concentration. Such protein concentrations are
considered to be particularly useful where the
formulation is intended for subcutaneous
administration. (See PCT patent application WO
97/04801). For subcutaneous administration, injection
volumes of 1.5 ml or less are well tolerated. Thus,
for proteins that are dosed at 1 mg/kg on a weekly
basis a protein concentration of at least 50 mg/ml is
required and 100-200 mg/ml is preferred. These
concentrations are difficult to achieve in liquid
formulations, due to the aggregation problems. They
can easily be achieved in the crystalline formulations
of this invention.
Fourth, protein crystals also constitute a
particularly advantageous form for pharmaceutical
dosage preparation. The crystals may be used as a
basis for' slow release formulations in vivo. As those
of skill in the art will appreciate, particle size is
of importance for the dissolution of crystals and
.release of activity. It is also known that the rate of
release is more predictable if the crystals have
substantially uniform particle size and do not contain
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amorphous precipitate (see European patent 0 265 214).
Thus, protein crystals may be advantageously used (see
PCT patent application WO 96/40049), on implantable
devices. Implant reservoirs are generally on the order
5 of 25-250 ul. With this volume restriction, a
formulation of high concentration (greater than 10%)
and a minimum amount of suspension vehicle is
preferred. Protein crystals of this invention may be
easily formulated in non-aqueous suspensions in such
high concentrations.
Fifth, another advantage of crystals is that
certain variables can be manipulated to modulate the
release of macromolecules over time. For example,
crystal size, shape, formulation with excipients that
15 effect dissolution, crosslinking, level of crosslinking
and encapsulation into a polymer matrix can all be
manipulated to produce delivery vehicles for biological
molecules.
The present invention overcomes the above-
described obstacles by employing the most stable form
of an active protein, the crystalline form and either
(1) adding ingredients or excipients where necessary to
stabilize dried crystals or (2) encapsulating the
protein crystals or crystal formulations within a
25 polymeric carrier to produce a composition that
contains each crystal and subsequently allows the
release of active protein molecules. Any form of
protein, including glycoproteins, antibodies, enzymes,
hormones or peptides, may be crystallized and
30 stabilized or encapsulated into compositions according
to the methods of this invention. In addition, the
nucleic acids coding for such proteins may be similarly
treated.
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The crystals) may be encapsulated using a
variety of polymeric carriers having unique properties
suitable for delivery to different and specific
environments or for effecting specific functions. The
rate of dissolution of the compositions and, therefore,
delivery of the active protein can be modulated by
varying crystal size, polymer composition, polymer
crosslinking, crystal crosslinking, polymer thickness,
polymer hydrophobicity, polymer crystallinity or
polymer solubility.
The addition of ingredients or excipients to
the crystals of the present invention or the
encapsulation of protein crystals or crystal
formulations results in further stabilization of the
protein constituent.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the relative stability of
the following molecular states of Candida rugosa
lipase: crosslinked amorphous, liquid, crystalline in
20° organic solvent, crosslinked crystalline in 200
organic solvent, crosslinked crystalline without
organic solvent ("Xlinke ppt" denotes crosslinked
precipitate).
Figure 2 depicts the specific activity of
soluble lipase over time at 40 °C.
Figure 3 depicts the shelf stabilities of
lipase crystal formulations dried by method 1 at 40 °C
and 75o humidity.
Figure 4 depicts the shelf stabilities of
lipase crystal formulations dried by method 4 at 40 °C
and 75o humidity.
Figure 5 depicts lipase crystals formulated
with polyethylene oxide at initial time 0.
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Figure 6 depicts lipase crystals formulated
with polyethylene oxide after incubation for 129 days
at 40 °C and 75o humidity.
Figure 7 depicts human serum albumin crystals
formulated with gelatin at initial time 0.
Figure 8 depicts human serum albumin crystals
formulated with gelatin after incubation for 4 days at
50 °C
Figure 9 depicts the specific activity of
soluble Penicillin acylase over time at 55 °C.
Figure 10 depicts the shelf stabilities of
various dried Penicillin acylase crystal formulations
at 55 °C.
Figure 11 depicts the specific activity of
soluble glucose oxidase over time.
Figure 12 depicts the shelf stabilities of
various dried glucose oxidase crystal formulations at
50 °C.
Figure 13 depicts glucose oxidase crystals
formulated with lactitol at initial time 0.
Figure 14 depicts glucose oxidase crystals
formulated with lactitol after incubation for 13 days
at 50 °C.
Figure 15 depicts glucose oxidase crystals
formulated with trehalose at initial time 0.
Figure 16 depicts glucose oxidase crystals
formulated with trehalose after incubation for 13 days
at 50 °C.
Figure 17 depicts encapsulated crosslinked
enzyme crystals of lipase from Candida rugosa.
Figure 18 depicts encapsulated uncrosslinked
enzyme crystals of lipase from Candida rugosa.
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Figure 19 depicts encapsulated crosslinked
enzyme crystals of Penicillin acylase from Escherichia
col i .
Figure 20 depicts encapsulated uncrosslinked
enzyme crystals of Penicillin acylase from Escherichia
coli .
Figure 21 depicts encapsulated crosslinked
enzyme crystals of glucose oxidase from Aspergillus
ni ger .
Figure 22 depicts encapsulated uncrosslinked
enzyme crystals of glucose oxidase from Aspergillus
niger.
Figure 23 depicts an encapsulated aqueous
slurry of uncrosslinked enzyme crystals of lipase from
Pseudomo.nas cepacia.
Figure 24 depicts uncrosslinked enzyme
crystals of lipase from Pseudomonas cepacia.
DETAILED DESCRIPTION OF THE INVENTION
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 are employed:
Amorphous solid -- a non-crystalline solid
form of protein, sometimes referred to as amorphous
precipitate, which has no molecular lattice structure
characteristic of the crystalline solid state.
Anti-sense polynucleotides - RNA or DNA which
codes for RNA, which is complementary to the mRNA of a
gene whose expression is intended to be inhibited.
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Aqueous-organic solvent mixture -- a mixture
comprising n~ organic solvent, where n is between 1 and
99 and ms aqueous, where m is 100-n.
Biocompatible polymers -- polymers that are
non-antigenic (when not used as an adjuvant), non-
carcinogenic, non-toxic and which are not otherwise
inherently incompatible with living organisms.
Examples include: poly (acrylic acid), poly
(cyanoacrylates), poly (amino acids), poly
(anhydrides), poly (depsipeptide), poly (esters) such
as poly (lactic acid) or PLA, poly (lactic-co-glycolic
acid) or PLGA, poly ((3-hydroxybutryate), poly
(caprolactone) and poly (dioxanone); poly (ethylene
glycol), poly ((hydroxypropyl)methacrylamide, poly
[(organo)phosphazene], poly (ortho esters), poly (vinyl
alcohol), poly (vinylpyrrolidone), malefic anhydride-
alkyl vinyl ether copolymers, pluronic polyols,
albumin, alginate, cellulose and cellulose derivatives,
collagen, fibrin, gelatin, hyaluronic acid,
oligosaccarides, glycaminoglycans, sulfated
polysaccarides, blends and copolymers thereof.
Biodegradable polymers -- polymers that
degrade by hydrolysis or solubilization. Degradation
can be heterogenous -- occurring primarily at the
particle surface, or homogenous -- degrading evenly
throughout the polymer matrix, or a combination of such
processes.
Biological macromolecule -- biological
polymers such as proteins, deoxyribonucleic acids (DNA)
and ribonucleic acids (RNA). For the purposes of this
application, biological macromolecules are also
referred to as macromolecules.
Change in chemical composition -- any change
in the chemical components of the environment
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surrounding a protein or nucleic acid crystal or
crystal formulation that affects the stability or rate
of dissolution of the crystal component.
Chance in shear force -- any change in
factors of the environment surrounding a protein or
nucleic acid crystal or crystal formulation under
conditions of use, such as, changes in mechanical
pressure, both positive and negative, revolution
stirring, centrifugation, tumbling, mechanical
agitation and filtration pumping.
Composition -- either uncrosslinked protein
crystals, crosslinked protein crystals, nucleic acid
crystals or formulations containing them, which have
been encapsulated within a polymeric carrier to form
coated particles. As used herein, composition always
refers to encapsulated crystals or formulations.
Controlled dissolution -- dissolution of a
protein or nucleic acid crystal or crystal formulation
or release of the crystalline constituent of said
formulation that is controlled by a factor selected
from the group consisting of the following: the surface
area of said crystal; the size of said crystal; the
shape of said crystal, the concentration of excipient
component; the number and nature of excipient
components; the molecular weight of the excipient
components and combinations thereof.
Co-polymer -- a polymer made with more than
one monomer species.
Crvstal -- one form of the solid state of
matter, which is distinct from a second form -- the
amorphous solid state. Crystals display characteristic
features including a lattice structure, characteristic
shapes and optical properties such as refractive index.
A crystal consists of atoms arranged in a pattern that
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repeats periodically in three dimensions (C. S. Baxrett,
Structure of Metals, 2nd ed., McGraw-Hill, New York,
1952, p.l.). The crystals of the present invention may
be protein, glycoprotein, peptide, antibodies,
therapeutic proteins, or DNA or RNA coding for such
proteins.
Drvina of Protein or Nucleic Acid Crystals --
removal of water, organic solvent or liquid polymer by
means including drying with NZ, air or inert gases,
vacuum oven drying, lyophilization, washing with a
volatile organic solvent followed by evaporation of the
solvent, or evaporation in a fume hood. Typically,
drying is achieved when the crystals become a free
flowing powder. Drying may be carried out by passing a
stream of gas over wet crystals. The gas may be
selected from the group consisting of: nitrogen, argon,
helium, carbon dioxide, air or combinations thereof.
Effective amount -- an amount of a protein or
nucleic acid crystal or crystal formulation or
composition of this invention which is effective to
treat, immunize, boost, protect, repair or detoxify the
subject or area to which it is administered over some
period of time.
Emulsifier -- a surface active agent which
reduces interfacial tension between polymer coated
protein crystals and a solution.
Formulations or (Protein or nucleic acid
crystal formulations) --- a combination of the protein
or nucleic acid crystals of this invention and one or
more ingredients or excipients, including sugars and
biocompatible polymers, Examples of excipients are
described in the Handbook of Pharmaceutical Excipients,
published jointly by the American Pharmaceutical
Association and the Pharmaceutical Society of Great
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Britian. For the purposes of this application,
"formulat:ions" include "crystal formulations".
Furthermore, "formulations" include "protein or nucleic
acid crystal formulations".
Formulations for decontamination --
formulations selected from the group consisting of:
formulations for decontamination of chemical wastes,
herbicides, insecticides, pesticides and environmental
hazards.
Gene therapy -- therapy using formulations
and/or compositions of DNA coding for a protein which
is defective, missing, or insufficiently expressed in
an individual. The crystals are injected into non-
proliferating tissue where the DNA is taken up into the
cells and expressed for a period of one to six months.
The expressed protein serves to temporarily replace or
supplement the endogenous protein. Gene therapy can
also serve to inhibit gene expression by providing
transgenes with the gene orientation reversed relative
to the promoter so that antisense mRNA is produced in
V1V0.
GlyCOprotein -- a protein or peptide
covalently linked to a carbohydrate. The carbohydrate
may be monomeric or composed of oligosaccharides.
Homo-polymer -- a polymer made with a single
monomer species.
Immunotherapeutic -- a protein derived from a
tumor cell, virus or bacteria having a protein activity
of inducing protective immunity to said tumor cell,
virus, or bacteria. An immunotherapeutic may be
administered directly -- as a protein or indirectly --
by injecting DNA or RNA which codes for the protein.
Immunotherapeutics may also be protein or
glycoprotein cytokines or immune cell co-stimulatory
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molecules which stimulate the immune system to reduce
or eliminate said tumor cell, virus or bacteria.
Liauid polymer -- pure liquid phase synthetic
polymers, such as poly-ethylene glycol (PEG), in the
absence of aqueous or organic solvents.
Macromolecules -- proteins, glycoproteins,
peptides, therapeutic proteins, DNA or RNA molecules.
Method of Administration -- protein or
nucleic acid crystals or crystal formulations or
compositions may be appropriate for a variety of modes
of administration. These may include oral, parenteral,
subcutaneous, intravenous, pulmonary, intralesional,
or topical administration, Alternatively, nucleic acid
crystals may be covalently attached to gold particles,
or other carrier beads, for delivery to non-
proliferating tissues such as muscles with a "DNA gun".
Naked DNA -- a nonreplicating, nonintegrating
polynucleotide which codes for a vaccine antigen,
therapeutic protein, or immunotherapeutic protein,
which may be operatively linked to a promoter and
inserted into a replication competent plasmid. The DNA
is free from association with transfection facilitating
proteins, viral particles, liposomal formulations,
lipids and calcium phosphate precipitating agents.
Naked DNA vaccine -- crystals of DNA coding
for a vaccine antigen or a vaccine antigen and an
immunotherapeutic. The vaccine is injected into non-
proliferating tissue where the DNA is taken up into the
cells and expressed for a period of one to six months.
The nucleic acid crystals may be covalently linked to
gold particles to aid in delivery to the site of
administration.
Organic solvents -- any solvent of non-
aqueous origin, including liquid polymers and mixtures
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thereof. Organic solvents suitable for the present
invention include: acetone, methyl alcohol, methyl
isobutyl ketone, chloroform, 1-propanol, isopropanol,
2-propanol, acetonitrile, 1-butanol, 2-butanol, ethyl
alcohol, cyclohexane, dioxane, ethyl acetate,
dimethylformamide, dichloroethane, hexane, isooctane,
methylene chloride, tert-butyl alchohol, toluene,
carbon tetrachloride, or combinations thereof
Peptide -- a polypeptide of small to
intermediate molecular weight, usually 3 to 35 amino
acid residues and frequently but not necessarily
representing a fragment of a larger protein.
Pharmaceutically effective amount -- an
amount o:f a protein or nucleic acid crystal or crystal
formulation or composition which is effective to treat
a condition in an living organism to whom it is
administered over some period of time.
Ingredients -- any excipient or excipients,
including pharmaceutical ingredients or excipients.
Excipients include, for example, the following:
Acidifying agents
acetic acid, glacial acetic acid, citric
acid, fumaric acid, hydrochloric acid, diluted
hydrochloric acid, malic acid, nitric acid, phosphoric
acid, diluted phosphoric acid, sulfuric acid, tartaric
acid
Aerosol propellants
butane, dichlorodifluoromethane,
dichlorotetrafluoroethane, isobutane, propane,
trichloromonofluoromethane
Air displacements
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carbon dioxide, nitrogen
Alcohol denaturants
denatonium benzoate, methyl isobutyl ketone,
sucrose octacetate
Alkalizing agents
strong ammonia solution, ammonium carbonate,
diethanolamine, diisopropanolamine, potassium
hydroxide, sodium bicarbonate, sodium borate, sodium
carbonate, sodium hydroxide, trolamine
Anticaking agents (see glidant)
Antifoaming agents
dimethicone, simethicone
Antimicrobial preservatives
benzalkonium chloride, benzalkonium chloride
solution, benzelthonium chloride, benzoic acid, benzyl
alcohol, butylparaben, cetylpyridinium chloride,
chlorobut:anol, chlorocresol, cresol, dehydroacetic
acid, ethylparaben, methylparaben, methylparaben
sodium, phenol, phenylethyl alcohol, phenylmercuric
acetate, phenylmercuric nitrate, potassium benzoate,
potassium sorbate, propylparaben, propylparaben sodium,
sodium benzoate, sodium dehydroacetate, sodium
propionate, sorbic acid, thimerosal, thymol
Antioxidants
ascorbic acid, acorbyl palmitate, butylated
hydroxyanisole, butylated hydroxytoluene,
hypophosphorous acid, monothioglycerol, propyl gallate,
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sodium formaldehyde sulfoxylate, sodium metabisulfite,
sodium thiosulfate, sufur dioxide, tocopherol,
tocopherols excipient
Buffering agents
acetic acid, ammonium carbonate, ammonium
phosphate, boric acid, citric acid, lactic acid,
phosphoric acid, potassium citrate, potassium
metaphosphate, potassium phosphate monobasic, sodium
acetate, sodium citrate, sodium lactate solution,
dibasic ~>odium phosphate, monobasic sodium phosphate
Capsule lubricants (see tablet and capsule lubricant)
Chelating agents
edetate disodium, ethylenediaminetetraacetic
acid and salts, edetic acid
Coating agents
sodium carboxymethylcellulose, cellulose
acetate, cellulose acetate phthalate, ethylcellulose,
gelatin, pharmaceutical glaze, hydroxypropyl cellulose,
hydroxypropyl methylcellulose, hydroxypropyl
methylcellulose phthalate, methacrylic acid copolymer,
methylcellulose, polyethylene glycol, polyvinyl acetate
phthalate, shellac, sucrose, titanium dioxide, carnauba
wax, microcystalline wax, zero
Colors
caramel, red, yellow, black or blends, ferric
oxide
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Complexing agents
ethylenediaminetetraacetic acid and salts
(EDTA), edetic acid, gentisic acid ethanolmaide,
oxyquinoline sulfate
Desiccants
calcium chloride, calcium sulfate, silicon
dioxide
Emulsifying and/or soiubilizing agents
acacia, cholesterol, diethanolamine
(adjunct), glyceryl monostearate, lanolin alcohols,
lecithin, mono- and di-glycerides, monoethanolamine
(adjunct), oleic acid (adjunct), oleyl alcohol
(stabilizer), poloxamer, polyoxyethylene 50 stearate,
polyoxyl 35 caster oil, polyoxyi 40 hydrogenated castor
oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl
ether, polyoxyl 40 stearate, polysorbate 20,
polysorbate 40, polysorbate 60, polysorbate 80,
propylene glycol diacetate, propylene glycol
monostearate, sodium lauryl sulfate, sodium stearate,
sorbitan monolaurate, soritan monooleate, sorbitan
monopalmitate, sorbitan monostearate, stearic acid,
trolamine, emulsifying wax
Filtering aids
powdered cellulose, purified siliceous earth
Flavors and perfumes
anethole, benzaldehyde, ethyl vanillin,
menthol, methyl salicylate, monosodium glutamate,
orange flower oil, peppermint, peppermint oil,
peppermint spirit, rose oil, stronger rose water,
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thymol, tofu balsam tincture, vanilla, vanilla
tincture, vanillin
Glidant and/or anticaking agents
calcium silicate, magnesium silicate,
colloidal silicon dioxide, talc
Humectants
glycerin, hexylene glycol, propylene glycol,
sorbitol
Ointment bases
lanolin, anhydrous lanolin, hydrophilic
ointment, white ointment, yellow ointment, polyethylene
glycol ointment, petrolatum, hydrophilic petrolatum,
white petrolatum, rose water ointment, squalane
Plasticizers
castor oil, diacetylated monoglycerides,
diethyl phthalate, glycerin, mono- and di-acetylated
monoglycerides, polyethylene glycol, propylene glycol,
triacetin, triethyl citrate
Polymer membranes
cellulose acetate
Solvents
acetone, alcohol, diluted alcohol, amylene
hydrate, benzyl benzoate, butyl alcohol, carbon
tetrachloride, chloroform, corn oil, cottonseed oil,
ethyl acetate, glycerin, hexylene glycol, isopropyl
alcohol, methyl alcohol, methylene chloride, methyl
isobutyl ketone, mineral oil, peanut oil, polyethylene
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glycol, propylene carbonate, propylene glycol, sesame
oil, water for injection, sterile water for injection,
sterile water for irrigation, purified water
Sorbents
powdered cellulose, charcoal, purified
siliceous earth
Carbon dioxide sorbents
barium hydroxide lime, soda lime
Stiffening agents
hydrogenated castor oil, cetostearyl alcohol,
cetyl alcohol, cetyl esters wax, hard fat, paraffin,
polyethylene excipient, stearyl alcohol, emulsifying
wax, white wax, yellow wax
Suppository bases
cocoa butter, hard fat, polyethylene glycol
Suspending and/or viscosity-increasing agents
acacia, agar, alginic acid, aluminum
monostearate, bentonite, purified bentonite, magma
bentonite, carbomer 934p, carboxymethylcellulose
calcium, carboxymethylcellulose sodium,
carboxymethycellulose sodium 12, carrageenan,
microcrystalline and carboxymethylcellulose sodium
cellulose, dextrin, gelatin, guar gum, hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, magnesium aluminum silicate,
methylcellulose, pectin, polyethylene oxide, polyvinyl
alcohol, povidone, propylene glycol alginate, silicon
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dioxide, colloidal silicon dioxide, sodium alginate,
tragacanth, xanthan gum
Sweetening agents
aspartame, dextrates, dextrose, excipient
dextrose, fructose, mannitol, saccharin, calcium
saccharin, sodium saccharin, sorbitol, solution
sorbitol, sucrose, compressible sugar, confectioner's
sugar, syrup
Tablet binders
acacia, alginic acid, sodium
carboxymethylcellulose, microcrystalline cellulose,
dextrin, ethylcellulose, gelatin, liquid glucose, guar
gum, hydroxypropyl methylcellulose, methycellulose,
polyethylene oxide, povidone, pregelatinized starch,
syrup
Tablet and/or capsule diluents
calcium carbonate, dibasic calcium phosphate,
tribasic calcium phosphate, calcium sulfate,
microcrystalline cellulose, powdered cellulose,
dextrates, dextrin, dextrose excipient, fructose,
kaolin, lactose, mannitol, sorbitol, starch,
pregelatinized starch, sucrose, compressible sugar,
confectioner's sugar
Table disintegrants
alginic acid, microcrystalline cellulose,
croscarmellose sodium, corspovidone, polacrilin
potassium, sodium starch glycolate, starch,
pregelatinized starch
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Tablet and/or capsule lubricants
calcium stearate, glyceryl behenate,
magnesium stearate, light mineral oil, polyethylene
glycol, ,odium stearyl fumarate, stearic acid, purified
stearic acid, talc, hydrogenated vegetable oil, zinc
stearate
Tonicity agent
dextrose, glycerin, mannitol, potassium
chloride, sodium chloride
Vehicle: flavored and/or sweetened
aromatic elixir, compound benzaldehyde
elixir, :iso-alcoholic elixir, peppermint water,
sorbitol solution, syrup, tolu balsam syrup
Vehicle: oleaginous
almond oil, corn oil, cottonseed oil, ethyl
oleate, :isopropyl myristate, isopropyl palmitate,
mineral oil, light mineral oil, myristyl alcohol,
octyldodecanol, olive oil, peanut oil, persic oil,
seame oi:l, soybean oil, squalane
Vehicle: solid carrier
sugar spheres
Vehicle: sterile
Bacteriostatic water for injection,
bacteriostatic sodium chloride injection
Viscosity-increasing (see suspending agent)
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Water repelling agent
cyclomethicone, dimethicone, simethicone
Wetting and/or solubilizing agent
benzalkonium chloride, benzethonium chloride,
cetylpyridinium chloride, docusate sodium, nonoxynol 9,
nonoxynol 10, octoxynol 9, poloxamer, polyoxyl 35
castor oil, polyoxyl 90, hydrogenated castor oil,
polyoxyl 50 stearate, polyoxyl 10 oleyl ether, polyoxyl
20, cetostearyl ether, polyoxyl 40 stearate,
polysorbate 20, polysorbate 40, polysorbate 60,
polysorbate. 80, sodium lauryl sulfate, sorbitan
monolaureate, sorbitan monooleate, sorbitan
monopalmitate, sorbitan monostearate, tyloxapol
Preferred ingredients or excipients include:
Salts of 1) amino acids such as glycine, arginine,
aspartic acid, glutamic acid, lysine, asparagine,
glutamine, proline, 2) carbohydrates, e.g.
monosaccharides such as glucose, fructose, galactose,
Mannose, arabinose, xylose, ribose and 3)
disaccharides, such as lactose, trehalose, maltose,
sucrose and 4) polysaccharides, such as maltodextrins,
dextrans, starch, glycogen and 5) alditols, such as
mannitol, xylitol, lactitol, sorbitol 6) glucuronic
acid, galacturonic acid, 7) cyclodextrins, such as
methyl cyclodextrin, hydroxypropyl-~i-cyclodextrin and
alike 8) inorganic salts, such as sodium chloride,
potassium chloride, magnesium chloride, phosphates of
sodium and potassium, boric acid ammonium carbonate and
ammonium phosphate, and 9) organic salts, such as
acetates, citrate, ascorbate, lactate 10) emulsifying
or solubilizing agents like acacia, diethanolamine,
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glyceryl monostearate, lecithin, monoethanolamine,
oleic acid , oleyl alcohol, poloxamer, polysorbates,
sodium lauryl sulfate, stearic acid, sorbitan
monolaurate, sorbitan monostearate, and other sorbitan
5 derivatives, polyoxyl derivatives, wax, polyoxyethylene
derivatives, sorbitan derivatives 11) viscosity
increasing reagents like, agar, alginic acid and its
salts, guar gum, pectin, polyvinyl alcohol,
polyethylene oxide, cellulose and its derivatives
10 propylene carbonate, polyethylene glycol, hexylene
glycol, tyloxapol. A further preferred group of
excipients or ingredients includes sucrose, trehalose,
lactose, sorbitol, lactitol, inositol, salts of sodium
and potssium such as acetate, phosphates, citrates,
15 borate, glycine, arginine, polyethylene oxide,
polyvinyl alcohol, polyethylene glycol, hexylene
glycol, methoxy polyethylene glycol, gelatin,
hydroxypropyl-~i-cyclodextrin.
Polymer -- a large molecule built up by the
20 repetition of small, simple chemical units. The
repeating units may be linear or branched to form
interconnected networks. The repeat unit is usually
equivalent or nearly equivalent to the monomer.
Polymeric carriers -- polymers used for
25 encapsulation of protein crystals for delivery of
proteins, including biological delivery. Such polymers
include biocompatible and biodegradable polymers. The
polymeric carrier may be a single polymer type or it
may be composed of a mixture of polymer types.
30 Polymers useful as the polymeric carrier, include for
example, poly (acrylic acid), poly (cyanoacrylates),
poly (amino acids), poly (anhydrides), poly
(depsipeptide), poly (esters) such as poly (lactic
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acid) or PLA, poly (lactic-co-glycolic acid) or PLGA,
poly (B-hydroxybutryate)~ poly (caprolactone) and poly
(dioxanone); poly (ethylene glycol), poly
((hydroxypropyl)methacrylamide, poly
[(organo)phosphazene], poly (ortho esters), poly (vinyl
alcohol), poly (vinylpyrrolidone), malefic anhydride-
alkyl vinyl ether copolymers, pluronic polyols,
albumin, natural and synthetic polypeptides, alginate,
cellulose and cellulose derivatives, collagen, fibrin,
gelatin, hyaluronic acid, oligosaccharides,
glycaminoglycans, sulfated polysaccharides, or any
conventional material that will encapsulate protein
crystals.
Protein -- a complex high polymer containing
carbon, hydrogen, oxygen, nitrogen and usually sulfur
and composed of chains of amino acids connected by
peptide linkages. Proteins in this application refer
to glycoproteins, antibodies, non-enzyme proteins,
enzymes, hormones and peptides. The molecular weight
range for proteins includes peptides of 1000 Daltons to
glycoproteins of 600 to 1000 kiloDaltons. Small
proteins, less than 10,000 Daltons, may be too small to
be characterized by a highly organized tertiary
structure, wherein said tertiary structure is organized
around a hydrophobic core.
In one embodiment of this invention, such
proteins have a molecular weight of greater than or
equal to 10,000 Daltons. According to an alternate
embodiment, that molecular weight is greater than or
equal to 20,000 Daltons. According to another
alternate embodiment, that molecular weight is greater
than or equal to 30,000 Daltons. According to a
further alternate embodiment, that molecular weight is
greater than or equal to 40,000 Daltons. According to
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another alternate embodiment, that molecular weight is
greater than or equal to 50,000 Daltons.
Protein activity -- an activity selected from
the group consisting of binding, catalysis, signaling,
transport, or other activities which induce a
functional response within the environment in which the
protein is used, such as induction of immune response,
enzymatic: activity, or combinations thereof.
Protein activity release rate -- the quantity
of protein dissolved per unit time.
Protein crystal -- protein molecules arranged
in a crystal lattice. Protein crystals contain a
pattern of specific protein-protein interactions that
are repeated periodically in three dimensions. The
protein crystals of this invention do not include
amorphous solid forms or precipitates of proteins, such
as those obtained by lyophilizing a protein solution.
Protein crystal formulation -- a combination
of protein crystals encapsulated within a polymeric
carrier t:o form coated particles. The coated particles
of the protein crystal formulation may have a spherical
morphology and be microspheres of up to 500 micro
meters in diameter or they may have some other
morphology and be microparticulates. For the purposes
of this application, "protein crystal formulations" are
included in the term "compositions".
Protein delivery system -- one or more of a
protein crystal formulation or composition, a process
for making the formulation or a method of administering
the formulation to biological entities or means
therefor.
Protein loading -- the protein content of
microspheres, as calculated as a percentage by weight
of protein relative to the weight of the dry
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formulation. A typical range of protein loading is
from 1-80g.
Protein release -- the release of active
protein from a polymeric carrier, as controlled by one
or more of the following factors: (1) degradation of
the polymer matrix; (2) rate of crystal dissolution
within the polymer matrix; (3) diffusion of dissolved
protein through the polymer matrix; (4) protein
loading; and (5) diffusion of biological medium into
the protein crystal/polymer matrix.
Prophylactically effective amount -- an
amount of: a protein or nucleic acid crystal or crystal
formulation or composition which is effective to
prevent a condition in a living organism to whom it is
administered over some period of time.
Reconstitution - dissolution of protein or
nucleic acid crystals or crystal formulations or
compositions in an appropriate buffer. or pharmaceutical
formulation.
Shelf stabilitv -- the loss of specific
activity and/or changes in secondary structure from the
native protein over time incubated under specified
conditions.
Stability -- the loss of specific activity
and/or changes in secondary structure from the native
protein over time while in solution under specified
conditions.
Stabilization -- the process of preventing
the loss of specific activity and/or changes in
secondary structure from the native proteins, by
preparing formulations of protein crystals or DNA
crystals or RNA crystals with excipients or
ingredients.
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Therapeutic protein -- a protein as described
above, which is administered to a living organism in a
formulation or composition or a pharmaceutical
formulation or composition. Therapeutic proteins
include all of the protein types described herein.
Vaccine antigen -- a protein derived from a.
pathogenic agent such as a virus, parasite, bacteria or
tumor cell. The protein activity of such vaccine
antigens is the induction of protective immune
responses specific for a pathogenic agent or tumor.
Crystallinity
Crystallinity of macromolecules is of great
value for their storage and delivery in vivo. However,
few techniques exist for the preparation of large
quantities of such crystalline macromolecules which are
stable outside of the mother liquor. Crystals of
proteins and nucleic acids must be handled with
considerable care, since they are extremely fragile and
contain a high proportion of solvent. It is well known
in x-ray crystallography that the diffraction patterns
from macromolecular crystals quickly degenerate upon
dehydration in air. Normally, a crystal is carefully
separated from its mother liquor and inserted into a
capillary tube. The tube is sealed from the air using
dental wax or silicone grease, along with a small
amount of mother liquor inside to maintain hydration
[McPhersan, A., Preparation and Analysis of Protein
Crystals, Robert E. Krieger Publishing, Malabar, p. 214
(1989)]. Another technique is to collect data from
macromolecular crystals at cryogenic temperatures. The
crystals are prepared and then rapidly cooled to
prevent i.ce lattice formation in the aqueous medium.
Instead of ice, a rigid glass forms, encasing the
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crystal with little damage. Crystals are then
maintained at 100 °K to prevent crystal disintegrations
[Rodgers, D.W., in Methods in Enzymoloay (Eds., Carter,
C.W. and Sweet, R.M.) Academic Press, v.276, p. 183
(1997)]. While this technique allows one to maintain
crystals outside of their mother liquor, it cannot be
used at temperatures higher than 100 °K.
In principle, dried crystals can be prepared
by lyophi_lization. However, this technique involves
rapid cooling of the material and can be applied only
to freeze stable products. The aqueous solution is
first frozen to between -40 and -50°C. Then, the ice
is removed under vacuum. Ice formation is usually
destructive to the protein crystal lattice, yielding a
mixture of crystals and amorphous precipitate.
It is desirable to produce macromolecules, in
the crystalline state, that are pure and stable under
storage conditions at ambient temperatures. Such
crystals constitute a particularly advantageous form of
proteins or nucleic acids for dosage preparations of
therapeutics and vaccines. The present invention
provides formulations and compositions for storage of
crystalline macromolecules as either solid particles or
dispersed in a non-aqueous solvent. Furthermore, the
invention may be applied to the storage of a single
biological macromolecule or a mixture of macromolecules
that do not interact with each other.
In another embodiment, this invention
provides a method for rendering biological
macromolecules suitable for storage in suspensions
comprising replacing the mother liquor with a non-
aqueous solvent. In yet another embodiment, the
crystalline slurry can be rendered solid by spinning
out the first solvent and washing the remaining
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crystalline solid using a second organic solvent to
remove water, followed by evaporation of the non-
aqueous solvent.
Non-aqueous slurries of crystalline
therapeutic proteins are especially useful for
subcutaneous delivery, while solid formulations are
ideally suited for pulmonary administration. Pulmonary
delivery is particularly useful for biological
macromolecules which are difficult to deliver by other
routes of administration. (See, for example, PCT
patent applications WO 96/32152, WO 95/24183 and WO
97/41833.
The proteins referred to below include
protein crystals themselves, or nucleic acid crystals
comprising DNA or RNA which encode those proteins upon
cellular uptake.
This invention advantageously provides
compositions and formulations of crystals of proteins
or nucleic acids.
Stability of Encapsulated Crvstals
Those of skill in the art will appreciate
that protein stability is one of the most important
obstacles to successful formulation of polymer
microparticulate delivery systems that control the
release of proteins. The stability of proteins
encapsulated in polymeric carriers may be challenged at
three separate stages: manufacture of the protein
crystal composition, protein release from the resulting
composition and in vivo stability after the protein
release. During preparation of microparticles or
microspheres containing soluble or amorphous proteins,
the use of organic solvents and lyophilization are
especially detrimental to protein stability.
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Subsequently, released proteins are susceptible to
moisture-induced aggregation, thus resulting in
permanent. inactivation.
In order to achieve high protein stability
during preparation of protein formulations and
compositions according to the present invention, it is
necessary to restrict the mobility of individual
protein molecules -- a result best achieved in the
crystalline solid state. For the purpose of this
application, solid state may be divided into two
categories: amorphous and crystalline. The three-
dimensional long-range order that normally exists in a
crystalline material does not exist in the amorphous
state. E'urthermore, the position of molecules relative
to one another is more random in the amorphous or
liquid states, relative to the highly ordered
crystalline state. Thus, amorphous proteins may be
less stable than their crystalline counterparts.
Figure 1 depicts the relative stability of
the following molecular states of Candida rvgosa lipase
("CRL"): crosslinked amorphous, liquid, crystalline in
200 organic solvent, crosslinked crystalline in 200
organic solvent, crosslinked crystalline without
organic solvent. Figure 1 shows that crystalline CRL
retains 80% activity for more than 175 hours in 200
organic solvent. In contrast, both amorphous and
soluble forms of the enzyme are completely inactivated
within hours. The present invention advantageously
utilizes the crystalline forms of proteins because of
their superior stability characteristics.
Maintaininct Crystallinity
In order to use protein crystals as the
protein source for preparing protein formulations and
compositions according to the present invention, the
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problem of protein crystal dissolution outside the
crystallization solution ("mother liquor") had to be
overcome. In order to maintain protein crystallinity
and hence stability, in the production of the protein
crystal formulations and compositions of this
invention, several approaches may be used:
1. Crystals remain in the mother liquor in the course
of producing protein crystals encapsulated with
polymeric carriers. Many compounds used in
protein crystallization, such as salts, PEG and
organic solvents, are compatible with polymer
processing conditions.
2. Kinetics of dissolution. The rate of crystal
dissolution outside the mother liquor depends on
conditions, such as pH, temperature, presence of
metal ions, such as Zn, Cu and Ca and
concentration of precipitants. By varying these
conditions, one can slow down the dissolution of
crystals for several hours. At the same time, the
process of microparticulate formation is very fast
and normally takes seconds to minutes to complete.
3. Dried protein crystals. The mother liquor can be
removed by filtration and the remaining
crystalline paste can be dried by air, under
vacuum, by washing with water miscible organic
solvents and/or by lyophilization.
4. Protein crystals can be chemically crosslinked to
form non-dissolvable or slowly dissolvable
crystals.
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5. The crystal size and shape can be manipulated and
controlled in the course of crystallization.
Thus, a range of crystal morphologies, each having
different dissolution kinetics and subsequently
dif:Eerent sustained release profiles compared to
amorphous proteins, is available.
Protein Constituents
The protein constituents of the formulations
and compositions of this invention may be those which
are naturally or synthetically modified. They may be
glycoproteins, phosphoproteins, sulphoproteins,
iodoproteins, methylated proteins, unmodified proteins
or contain other modifications. Such protein
constituents 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.
Tn one embodiment of this invention, such
proteins have a molecular weight of greater than or
equal to 10,000 Daltons. According to an alternate
embodiment, that molecular weight is greater than or
equal to 20,000 Daltons. According to another
alternate embodiment, that molecular weight is greater
than or equal to 30,000 Daltons. According to a
further alternate embodiment, that molecular weight is
greater than or equal to 40,000 Daltons. According to
another alternate embodiment, that molecular weight is
greater than or equal to 50,000 Daltons.
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Included among such proteins are enzymes,
such as, for example, hydrolases, isomerases, lyases,
ligases, adenylate cyclases, transferases and
oxidoreductases. Examples of hydrolases include
elastase, esterase, lipase, nitrilase, amylase,
pectinase, hydantoinase, asparaginase, urease,
subtilisin, thermolysin 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
include cellulases and oxidases.
Examples of therapeutic or prophylactic
proteins include hormones such as insulin, glucogon-
like peptide 1 and parathyroid hormone, antibodies,
inhibitors, growth factors, postridical hormones, nerve
growth hormones, blood clotting factors, adhesion
molecules, bone morphogenic proteins and lectins
trophic factors, cytokines such as TGF-~3, IL-2, IL-4,
a-IFN, (3--IFN, y-IFN, TNF, IL-6, IL-8, lymphotoxin, IL-5,
Migration inhibition factor, GMCSF, IL-7, IL-3,
monocyte-macrophage colony stimulating factors,
granulocyte colony stimulating factors, multidrug
resistance proteins, other lymphokines, toxoids,
erythropoietin, Factor VIII, amylin, TPA, dornase-a, a-
1-antitr:ipsin, human growth hormones, nerve growth
hormones, bone morphogenic proteins, urease, toxoids,
fertility hormones, FSH and LSH.
Therapeutic proteins, such as the following,
are also included:
leukocyte markers, such as CD2, CD3, CD4,
CD5, CD6, CD7, CD8, CDlla, CDllb, CDllc, CD13, CD14,
CD18, CD19, CE20, CD22, CD23, CD27 and its ligand, CD28
and its ligands B7.1, B7.2, 87.3, CD29 and its ligands,
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CD30 and its ligand, CD40 and its ligand gp39, CD44,
CD45 and isoforms, Cdw52 (Campath antigen), CD56, CD58,
CD69, CD72, CTLA-4, LFA-1 and TCR
histocompatibility antigens, such as MHC
class I or II antigens, the Lewis Y antigens, SLex,
SLey, SLea and SLeb;
integrins, such as VLA-1, VLA-2, VLA-3, VLA-
4, VLA-5, VLA-6 and LFA-1;
adhesion molecules, such as Mac-1 and
p150,95;
selectins, such as L-selectin, P-selectin and
E-selectin and their counterreceptors VCAM-1, ICAM-1,
ICAM-2 and LFA-3;
interleukins, such as IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, IL
14 and IL-15;
interleukin receptors, such as IL-1R, IL-2R,
IL-4R, IL-5R, TL-6R, IL-7R, IL-8R, IL-lOR, IL-11R, IL-
12R, IL-13R, IL-14R and IL-15R;
chemokines, such as PF4, RANTES, MIPla, MCP1,
NAP-2, Groa, Gro(3 and IL-8;
growth factors, such as TNFalpha, TGFbeta,
TSH, VEGF/VPF, PTHrP, EGF family, EGF, PDGF family,
endothel_i.n and gastrin releasing peptide (GRP);
growth factor receptors, such as TNFalphaR,
RGFbetaR, TSHR, VEGFR/VPFR, FGFR, EGFR, PTHrPR, PDGFR
family, EPO-R; GCSE-R and other hematopoietic
receptors;
interferon receptors, such as IFNaR, TFN(3R
and IFNYR;
Igs and their receptors, such as IgE, FceRI
and FceRII;
blood factors, such as complement C3b,
complement C5a, complement C5b-9, Rh factor,
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fibrinogen, fibrin and myelin associated growth
inhibitor.
The protein. constituent of the formulations
and compositions of this invention may be any natural,
synthetic or recombinant protein antigen including, for
example, tetanus toxoid, diptheria toxoid, viral
surface proteins, such as CMV glycoproteins B, H and
gCIII, HIV-1 envelope glycoproteins, RSV envelope
glycoprot:eins, HSV envelope glycoproteins, EBV envelope
glycoproteins, VZV envelope glycoproteins, HPV envelope
glycoprot:eins, Influenza virus glycoproteins, Hepatitis
family surface antigens; viral structural proteins,
viral enzymes, parasite proteins, parasite
glycoproteins, parasite enzymes and bacterial proteins.
Also included are tumor antigens, such as
her2-neu, mucin, CEA and endosialin. Allergens, such
as house dust mite antigen, lol p1 (grass) antigens and
urushiol are included.
Toxins, such as pseudomonas endotoxin and
osteopontin/uropontin, snake venom and bee venom are
included.
Also included are glycoprotein tumor-
associated antigens, for example, carcinoembryonic
antigen (CEA), human mucins, her-2/neu and prostate-
specific antigen (PSA) [R. A. Henderson and O.J. Finn,
Advances in Immunoloav, 62, pp. 217-56 (1996)].
Administration and Biological Delivery
To date, therapeutic proteins have generally
been administered by frequent injection, due to their
characteristic negligible oral bioavailability and
short plasma life. The protein crystal formulations
and compositions of the present invention, which
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include microparticulate-based sustained release
systems for protein drugs, advantageously permit
improved patient compliance and convenience, more
stable blood levels and potential dose reduction. The
slow and constant release capabilities of the present
invention advantageously permit reduced dosages, due to
more efficient delivery of active protein. Significant
cost savings may be achieved by using the protein
formulations and compositions described herein.
Formulations and compositions comprising
protein crystals in polymeric delivery carriers
according to this invention may also comprise any
conventional carrier or adjuvant used in vaccines,
pharmaceuticals, personal care formulations and
compositions, veterinary formulations, or oral enzyme
supplementation. 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,
colloida_L 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.
According to one embodiment of this
invention, protein crystals may be combined with any
conventional materials used for controlled release
administration, including pharmaceutical controlled
release administration. Such materials include, for
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example, coatings, shells and films, such as enteric
coatings and polymer coatings and films.
Protein formulations in polymeric delivery
carriers and compositions (compositions) according to
this invention, which may be devices, such as
implantable devices and may be microparticulate protein
delivery systems.
In one embodiment of this invention, the
macromolecule crystals have a longest dimension between
about 0.01 ,um and about 500 Vim, alternatively between.
about 0.1 ,um and about 100 ~cm. The most preferred
embodiment is that the protein crystal of protein
crystal formulation components are between about 50 ~m
and about 100 ~m in their longest dimension. Such
15 crystals may have a shape selected from the group
consisting of: spheres, needles, rods, plates, such as
hexagons and squares, rhomboids, cubes, bipyramids and
prisms.
According to the present invention,
encapsulation of protein crystals or protein crystal
formulations in polymeric carriers to make compositions
may be carried out on protein crystals which are
crosslinked or uncrosslinked. Such protein crystals
may be obtained commercially or produced as illustrated
herein.
Protein or nucleic acid crystals or crystal
formulations and compositions according to this
invention may be used as ingredients in personal care
compositions, including cosmetics, such as creams,
30 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,
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bleaching, cleaning, deproteinization, lipid removal,
moisturizing, decoloration, coloration or
detoxification. They may also be used as anti-oxidants
in cosmetics.
According to this invention, any individual,
including humans, animals and plants, may be treated in
a pharmaceutically acceptable manner with a
pharmaceutically effective amount of protein or nucleic
acid crystals or a crystal formulation or composition
for a period of time sufficient to treat a condition in
the individual to whom they are administered over same
period of time. Alternatively, individuals may receive
a prophyl.actically effective amount of protein or
nucleic acid crystals or crystal formulation or
composition of this invention which is effective to
prevent a condition in the individual to whom they are
administered over some period of time.
Protein or nucleic acid crystals or crystal
formulations or compositions 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
oral routes. 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, protein or nucleic
acid crystal or crystal formulations or compositions
may be topically administered to any epithelial
surface. Such epithelial surfaces include oral,
ocular, aural; anal and nasal surfaces, which may be
treated, protected, repaired or detoxified by
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application of protein or nucleic acid crystals or
crystal formulations or compositions.
Pharmaceutical, personal care, veterinary or
prophylactic formulations and compositions comprising
protein or nucleic acid crystal or crystal formulations
or compositions according to this invention may also be
selected from the group consisting of tablets,
liposomes, granules, spheres, microparticles,
microspheres and capsules.
For such uses, as well as other uses
according to this invention, protein or nucleic acid
crystals or crystal formulations and compositions may
be formu:Lated into tablets. Such tablets constitute a
liquid-free, dust-free form for storage of protein or
nucleic acid crystal or crystal formulations or
compositions which are easily handled and retain
acceptab:Le levels of activity or potency.
Alternatively, protein or nucleic acid
crystals or crystal formulations or compositions 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,
slurries, gels, creams, balms, emulsions, lotions,
powders, sprays, foams, pastes, ointments, salves,
balms and drops.
Protein or nucleic acid crystals on
formulations or compositions according to 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,
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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,
5 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,
polyacry.lates, polyoxyethylene-polyoxypropylene-block
10 polymers, polyethylene glycol and wood wax alcohols.
The most effective mode of administration and
dosage regimen of the protein or nucleic acid crystals
or crystal formulations or compositions of this
invention will depend on the effect desired, previous
15 therapy, if any, the individual's health status or
status of the condition itself and response to the
protein or nucleic acid crystals or crystal
formulations or compositions and the judgment of the
treating physician or clinician. The protein or
20 nucleic <~cid crystals or crystal formulations or
compositions may be administered in any dosage form
acceptable for pharmaceuticals, vaccinations, gene
therapy, immunotherapy, personal care compositions or
veterinary formulations, at one time or over a series
25 of treatments.
The amount of the protein or nucleic acid
crystals or crystal formulations or compositions which
provides a single dosage will vary depending upon the
particular mode of administration, formulation, dose
30 level or dose frequency. A typical preparation will
contain between about O.Olo and about 990, preferably
between about 1o and about 500, protein or nucleic acid
crystals (w/w). Alternatively, a preparation will
contain between about 0.01% and about 80o protein
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crystals, preferably between about 1% and about 50~,
protein crystals (w/w). Alternatively, a preparation
will contain between about 0.01% and about 80o protein
crystal formulation, preferably between about 1o and
about 509>, protein crystal formulation(w/w).
Upon improvement of the individual's
condition, a maintenance dose of protein or nucleic
acid crystals or crystal formulations or compositions
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.
Production of crvstals, crystal formulations
and compositions:
According to the one embodiment of this
invention, crystals, crystal farmulations and
compositions are prepared by the following process:
First, the protein or nucleic acid is crystallized.
Next, excipients or ingredients selected from sugars,
sugar alcohols, viscosity increasing agents, wetting or
solubilizing agents, buffer salts, emulsifying agents,
antimicrobial agents, antioxidants, and coating agents
are added directly to the mother liquor.
Alternatively, the crystals are suspended in an
excipient solution, after the mother liquor is removed,
for a minimum of 1 hour to a maximum of 24 hours. The
excipient concentration is typically between about 0.01
to 30~ W/W. Most preferably between about 0.1 to 100.
The ingredient concentration is between about 0.01 to
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900. The crystal concentration is between about 0.01
to 950. The mother liquor is then removed from the
crystal slurry either by filtration or by
centrifugation. Subsequently, the crystals are washed
optionally with solutions of 50 to 1000 one or more
organic solvents such as, for example, ethanol,
methanol, isopropanol or ethyl acetate, either at room
temperature or at temperatures between -20 °C to 25 °C.
The crystals are the dried either by passing a stream
of nitrogen, air, or inert gas over the crystals.
Alternatively, the crystals are dried by air drying or
by lyophilization or by vacuum drying. The drying is
carried out for a minimum 1 hour to a maximum of 72
hours after washing, until the moisture content of the
final product is below 10% by weight, most preferably
below 5~. Finally, micronizing of the crystals can be
performed if necessary.
According to one embodiment of this
invention, when preparing protein crystals, protein
crystal formulations or compositions, enhancers, such
as surfactants are not added during crystallization.
Excipients or ingredients are added to the mother
liquor after crystallization, at a concentration of
between about 1-loo W/W, alternatively at a
concentration of between about 0.1-25o W/W,
alternatively at a concentration of between about 0.1-
50° W/W. The excipient or ingredient is incubated with
the crystals in the mother liquor for about 0.1-3 hrs,
alternatively the incubation is carried out for 0.1-12
hrs, alternatively the incubation is carried out for
0.1-24 hrs.
In another embodiment of this invention, the
ingredient or excipient is dissolved in a solution
other than the mother liquor, and the protein crystals
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are removed from the mother liquor and suspended in the
excipient or ingredient solution. The ingredient or
excipient concentrations and the incubation times are
the same as those described above.
Slow Release Forms and Vaccines
In another embodiment of this invention,
encapsulation of lipases in polymeric carriers provide
compositions useful to treat patients suffering from
intestinal lipase deficiency. Such patients include
those with pancreatic steatorrhea, due to advanced
pancreatic insufficiency require oral lipase
supplementation. Unfortunately, current therapeutic
methods may not be flexible enough to protect the
active lipase during transit through the gastro-
intestinal tract and to release. the enzyme activity
where it is critically needed in the small bowel (See
L. Guarner et al., "Fate of oral enzymes in pancreatic
insuffic:iency," Gut, vol. 34, pp. 708-712, (1993)).
The flexibility of the present invention in preparing
slowly available active lipase solves the present
problems often associated with lipase supplementation.
According to one embodiment of this invention, the
combination of encapsulated lipase crystals
(compositions) and unencapsulated crosslinked lipase
crystals or formulations provides a drug therapy regime
in which enzyme activity is available early on from the
unencapsulated crosslinked lipase. As this material
undergoes proteolytic degradation, the encapsulated
enzyme (composition) begins to release enzyme activity
into the more distal bowel. A similar strategy may be
used to solve other enzyme or therapeutic protein
supplementation problems.
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The present invention may also utilize other
slow release methodologies, such as silicon based rings
or rods which have been preloaded with encapsulated
protein crystals containing hormones, antibodies or
enzymes or compositions containing them. The purpose
of this technique is to provide a constant level of
protein to the bloodstream over a period of weeks or
months. Such implants can be inserted intradermally
and can be safely replaced and removed when needed.
Other formulations and compositions according
to this :invention include vaccine formulations and
compositions comprising protein (antigen) crystals,
adjuvant and encapsulating polymer(s). The protein
antigen may be a viral glycoprotein, viral structural
protein, viral enzyme, bacterial protein, or some
engineered homolog of a viral or bacterial protein, or
any immunopotentiating protein, such as a cytokine.
One embodiment of such formulations or compositions
involves a single vaccine injection containing
microspheres having three or more different release
profiles. In this way, antigen formulations or
composition may be released over a sustained period
sufficient to generate lasting immunity. By virtue of
this formulation or composition, multiple antigen
boosts may be in single unit form. The faster
degrading preparation (composition) may contain an
immunogenic adjuvant to enhance the immune response.
One advantage of such a system is that by using protein
crystals, the native three-dimensional structures of
the epitopes are maintained and presented to the immune
system in their native form.
Once the immune system is primed, there may
be less need for an adjuvant effect. Therefore, in the
slower degrading inoculations, a less immunogenic
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adjuvant may be included and possibly no adjuvant may
be required in the slowest degrading microspheres of
the formulations and compositions . In this way,
patient populations in remote areas will not have to be
treated multiple times in order to provide protection
against infectious diseases. One of skill in the art
of biological delivery of proteins will appreciate that
many variations on this theme are feasible.
Accordingly, the examples provided here are not
intended to limit the invention.
In another embodiment of this invention, a
combination vaccine could be produced, whereby immunity
to multiple diseases is induced in a single injection.
As discussed above, microspheres having different
release profiles may be combined alone or in
formulations and compositions and may include
microspheres containing antigens from multiple
infectious agents to produce a combination vaccine
(formulations and compositions). For example,
microspheres having multiple release profiles and
containing antigen crystals of measles, mumps, rubella,
polio and hepatitis B agents could be combined and
administered to children. Alternatively, microspheres
having multiple release profiles and containing
crystals of different isolates of HIV gp120 could be
combined to produce a vaccine for HIV-1 or HIV-2.
Another advantage of the present invention is
that the protein crystals encapsulated within polymeric
carriers and forming a composition comprising
microspheres can be dried by lyophilization.
Lyophilization, or freeze-drying allows water to be
separated from the composition. The protein crystal
composition is first frozen and then placed in a high
vacuum. In a vacuum, the crystalline H~0 sublimes,
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leaving the protein crystal composition behind
containing only the tightly bound water. Such
processing further stabilizes the composition and
allows for easier storage and transportation at
typically encountered ambient temperatures.
This feature is especially desirable for
therapeutic proteins and vaccines which can be
dispensed into single dose sterile containers
("ampules") or alternatively, any desired increment of
a single dose as a slurry, in a formulation or a
composition. The ampules containing the dispensed
slurries, formulations or compositions can then be
capped, batch frozen and lyophilized under sterile
conditions. Such sterile containers can be transported
throughout the world and stored at ambient
temperatures. Such a system would be useful for
providing sterile vaccines and therapeutic proteins to
remote and undeveloped parts of the world. At the
point of use, the ampule is rehydrated with the sterile
solvent or buffer of choice and dispensed. Under this
scenario, minimal or no refrigeration is required.
Protein Crystallization
Protein crystals are grown by controlled
crystallization of.protein from aqueous solutions or
aqueous solutions containing organic solvents.
Solution conditions that may be controlled include, for
example, the rate of evaporation of solvent, organic
solvents, 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).
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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
appropriate protein crystals 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)].
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 may be 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
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and their- concentrations are determined experimentally
to facilitate crystallization.
It is critical to differentiate between
amorphous precipitates and crystalline material.
Crystalline material is a form of the solid state of
matter, which is distinct from the amorphous solid
state. Crystals display characteristic features
including a lattice structure, characteristic shapes
and optical properties such as refractive index and
birefringence. A crystal consists of atoms arranged in
a pattern that repeats periodically in three
dimensions. In contrast, amorphous material is a non-
crystalli.ne solid form of matter, sometimes referred to
as an amorphous precipitate. Such precipitates have no
molecular lattice structure characteristic of the
crystalline solid state and do not display
birefringence or other spectroscopic characteristics
typical of the crystalline forms of matter.
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. As another option,
proteins may be crystallized by using protein
precipitates as the starting material. In this case,
protein precipitates are added to a crystallization
solution and incubated until crystals form.
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, in cases in which the
crystallized protein is to be crosslinked,
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incompatibility between an intended crosslinking agent
and the crystallization medium might require exchanging
the crystals into a more suitable solvent system.
Many of the proteins for which
crystallization conditions have already been described,
may be used to prepare 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 protein crystals the present invention may be
necessary.
Crosslinkina of Protein Crystals
According to one embodiment of this
invention, for example, the release rate of the protein
from the polymeric carrier (composition) may be slowed
and controlled by using protein crystals that have been
chemical:Ly crosslinked using a crosslinker, such as for
example, a biocompatible crosslinker. Thus, once
protein crystals have been grown in a suitable medium
they may be crosslinked.
Crosslinking may be carried out using
reversib:Le crosslinkers, in parallel or in sequence.
The resu:Lting 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
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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 --> AA1-X-Y-Z-AA2
change in environment --> AA1-X+Y-Z-AA2
-- where X and Z are groups with reactive
functionality
-- where Y is a trigger
-- where AA1 and AA2 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 i.n breaking of the trigger bond and dissolution
of the protein. Thus, when the crosslinks within
protein crystals crosslinked with such reversible
crosslink:ing agents break, dissolution of protein
crystal begins and therefore the release of activity.
Alternatively, the reactive functionality of
the crosslinker and the trigger may be the same, as in:
X-Z + 2AA residues --> AA1-X-Z-AA2
change in environment --> AAA. + X-Z-AA2.
The crosslinker may be homofunctional (X=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):
I. Reactive acyl donors are exemplified by:
carboxylate esters RCOOR', amides RCONHR', Acyl azides
RCON3, carbodiimides R-N=C=N-R', N-hydroxyimide esters,
RCO-O-NR', imidoesters R-C=NH2+(OR'), anhydrides RCO-O
COR', carbonates RO-CO-0-R', urethanes RNHCONHR', acid
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halides RCOHaI (where Hal=a halogen), acyl hydrazides
RCONNR'R", 0-acylisoureas RCO-0-C=NR'(-NR"R "'),
II. Reactive carbonyl groups are exemplified
by: aldehydes RCHO and ketones RCOR', acetals
RCO(Hz)R", ketals RR'CO2R'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, Illinois (1999); S.S. Wong, Chemistrv of
Protein Con~uaation and Cross-Linking, CRC Press, Boca
Baton, Florida (1991)].
III. Alkyl or aryl donors are exemplified by:
alkyl or aryl halides R-Hal, azides R-N3, sulfate
esters RS03R', phosphate esters RPO(OR'3), alkyloxonium
salts R30+, sulfonium R3S+, nitrate esters RON02,
Michael acceptors RCR'=CR "'COB", aryl fluorides ArF,
isonitri:les RN+=C-, haloamines RZN-Hal, alkenes and
alkynes.
IV. Sulfur containing groups are exemplified
by disulfides RSSR', sulfhydryls RSH, epoxides R2C~CR'2.
V. Salts are exemplified by alkyl or aryl
ammonium salts R4N+, carboxylate RC00-, sulfate ROS03-,
phosphate ROP03" and amines R3N.
Table 1 below includes examples of triggers,
organized by release mechanism. In Table 1, R= 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.
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Table 1
Trigger Examples Release
Conditions
1. Acid Labile R-O-R H+ or Lewis
Linkers e.g. Thp, MOM, Acidic catalysts
Acetal, ketal
Aldol, Michael
adducts, esters
2. Base Labile R'OC02-R' Variety of basic
Linkers Carbonates media
R'O-CONRZ
Carbamates
RZ'NCONR2
Urethanes
Aldol, Michael
adducts, esters
3. Fluoride R-OSiR3 Aqueous F-
Labile Linkers Various Si
containing
linkers
4. Enzyme RCOOR, RCONR2' Free lipases,
Labile Linkers amidases,
esterases
5. Reduction Disulfide H2 catalyst;
Labile Linkers linkers that Hydrides
cleave via
Hydrogenolysis
Reductive
Elimination
R'-S-S-R
6. Oxidation R-OSiR3 Oxidizing
Labile Linkers Glycols R- agents: e.g.
CH (OH) -CH (OH) H202, Na0Cl, IOq
-R'
Metal based
oxidizers, other
hypervalent
oxidents
7. Thio-labile R'-S-S-R Thiols, e.g.,
linkers Cys, DTT,
mercaptoethanol
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Trigger Examples Release
Conditions
8. Heavy Metal Various Allyl Transition metal
Labile Linkers Ethers based reagents
ROCH2CH=CHR (Pd, Ir, Hg, Ag,
Alkyl, Acyl Cu, T1, Rh)
Allyl ester Pd(0)
catalysts
9. Photolabile 0-nitrobenzyl light (hv)
Linkers (ONB)
DESYL groups
in linker
10. Free Thiohydroxa- Free radical
Radical Labile mate 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-00-R temperature
13. "Safety Methylthio- Base; amines,
Catch" Labile ethyl (Mte) others
Linkers Dithianes
Additional examples of reversible crosslinkers
are described in T.W. Green, Protective Groups in
Organic Svnthesis, 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 AnQewante Chemie
Inl. Ed. Enql., 35, p. 2056 (1996) .
Other types of reversible crosslinkers are
disulfide bond-containing crosslinkers. The trigger
breaking crosslinks formed by such crosslinkers is the
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addition of reducing agent, such as cysteine, to the
environment of the crosslinked protein crystals.
Disulfide crosslinkers are described in the
Pierce Catalog and Handbook (1994-1995) and more
recently in "Bioconjugate Techniques" , By G.T.
Hermanson, (1996), Academic Press, Division of Harcourt
Brace & Company, 525 B Street, Suite 1900, San Diego,
CA 92101-4495.
Examples of such crosslinkers include:
Homobifunctional (Symmetric)
DSS - Dithiobis(succinimidylpropionate), also know as
Lomant's Reagent
DTSSP - :3-3'-Dithiobis(sulfosuccinimidylpropionate),
water soluble version of DSP
DTBP - Dimethyl 3,3'-dithiobispropionimidate~HC1
BASED - Bis- ([i- [4-azidosalicylamido] ethyl) disulfide
DPDPB - :L,4-Di-(3'-[2'-pyridyldithio]-
propionamido)butane.
Heterobifunctional (Asymmetric)
SPDP - N-Succinimidyl-3-(2-pyridyldithio)propionate
LC-SPDP -- Succinimidyl-6-(3-[2-pyridyldithio]
propionate)hexanoate
Sulfo-LC-SPDP - Sulfosuccinimidyi-6-(3-[2-
pyridyldlthio] propionate)hexanoate, water soluble
version of LC-SPDP
APDP - N-(4-[p-azidosalicylamido]butyl)-3'-(2'-
pyridyldithio) propionamide
SADP - N-Succinimidyl(4--azidophenyl)1,3'-
dithiopropionate
Sulfo-SADP - Sulfosuccinimidyl(4-azidophenyl) 1,3'
dithiopropionate, water soluble version of SADP
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5AED - Sulfosuccinimidyl-2-(7-azido-4-methycoumarin-3-
acetamidE=)ethyl-1,3'dithiopropionate
SAND - Sulfosuccinimidyl-2-(m-azido-o-
nitrobenzamido)ethyl-1,3'-dithiopropionate
SASD - Sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-
1,3'-dithiopropionate
SMPB - Succinimidyl-4-(p-maleimidophenyl)butyrate
Sulfo-SMPB - Sulfosuccinimidyl-4-(p-
maleimidophenyl)butyrate
SMPT - 4--5uccinimidyloxycarbonyl-methyl-a-(2-
pyridylthio) toluene
Sulfo-LC-SMPT - Sulfosuccinimidyl-6-(a-methyl-a-(2-
pyridylthio)toluamido)hexanoate.
In particular, see Part II, Chapters 3-5 on
Zero-length Cross-linkers, Homobifunctional Cross-
linkers and Heterobifunctional Cross-linkers in
"Bioconjugate Techniques", By G.T. Hermanson, (1996),
Academic Press, Division of Harcourt Brace & Company,
525 B Street, Suite 1900, San Diego, CA 92101-4495.
Crosslinked protein crystals useful in the
protein f=ormulations of the present invention may also
be prepared according to the methods set forth in PCT
patent application PCT/US91/05415.
Encapsulation of Protein Crystals in
Polymeric Carriers
According to one embodiment of this
invention, compositions are produced when protein
crystals are encapsulated in at least one polymeric
carrier t:o form microspheres by virtue of encapsulation
within the matrix of the polymeric carrier to preserve
their native and biologically active tertiary
structure. The crystals can be encapsulated using
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various biocompatible and/or biodegradable polymers
having unique properties which are suitable for
delivery to different biological environments or for
effecting specific functions. The rate of dissolution
and, therefore, delivery of active protein is
determined by the particular encapsulation technique,
polymer composition, polymer crosslinking, polymer
thickness, polymer solubility, protein crystal geometry
and degree and, if any, of protein crystal crosslinking
Protein crystals or formulations to be
encapsulated are suspended in a polymeric carrier which
is dissolved in an organic solvent. The polymer
solution must be concentrated enough to completely coat
the protein crystals or formulations after they are
added to the solution. Such an amount is one which
provides a weight ratio of protein crystals to polymer
between about 0.02 and about 20, preferably between
about O.:L and about 2. The protein crystals are
contacted with polymer in solution for a period of time
between about 0.5 minutes and about 30 minutes,
preferably between about 1 minutes and about 3 minutes.
The crystals should be kept suspended and not allowed
to aggregate as they are coated by contact with the
polymer.
Following that contact, the crystals become
coated and are referred to as nascent microspheres.
The nascent microspheres increase in size while coating
occurs. In a preferred embodiment of the invention,
the suspended coated crystals or nascent microspheres
along with the polymeric carrier and organic solvent
are transferred to a larger volume of an aqueous
solution containing a surface active agent, known as an
emulsifier. In the aqueous solution, the suspended
nascent microspheres are immersed in the aqueous phase,
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where the organic solvent evaporates or diffuses away
from the polymer. Eventually, a point is reached where
the polymer is no longer soluble and forms a
precipitated phase encapsulating the protein crystals
or formulations to form a composition. This aspect of
the process is referred to as hardening of the
polymerir_ carrier or polymer. The emulsifier helps to
reduce the interfacial surface tension between the
various phases of matter in the system during the
hardening phase of the process. Alternatively, if the
coating polymer has some inherent surface activity,
there may be no need for addition of a separate surface
active agent.
Emulsifiers useful to prepare encapsulated
protein crystals according to this invention include
polyvinyl alcohol) as exemplified herein, surfactants
and other surface active agents which can reduce the
surface tension between the polymer coated protein
crystals or polymer coated crystal formulations and the
solution.
Organic solvents useful to prepare the
microspheres of the present invention include methylene
chloride, ethyl acetate, chloroform and other non-toxic
solvents which will depend on the properties of the
polymer. Solvents should be chosen that solubilize the
polymer and are ultimately non-toxic.
A preferred embodiment of this invention is
that the crystallinity of the protein crystals is
maintained during the encapsulation process. The
crystallinity is maintained during the coating process
by using an organic solvent in which the crystals are
not soluble. Subsequently, once the coated crystals
are transferred to the aqueous solvent, rapid hardening
of the polymeric carrier and sufficient coating of the
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crystals in the previous step shields the crystalline
material from dissolution. In another embodiment, the
use of crosslinked protein crystals facilitates
maintenance of crystallinity in both the aqueous and
organic solvents.
The polymers used as polymeric carriers to
coat the protein crystals can be either homo-polymers
or co-polymers. The rate of hydrolysis of the
microspheres is largely determined by the hydrolysis
rate of the individual polymer species. In general,
the rate of hydrolysis decreases as follows:
polycarbonates > polyesters > polyurethanes >
polyorthoesters > polyamides. For a review of
biodegradable and biocompatible polymers, see W.R.
Gombotz and D.K. Pettit, "Biodegradable polymers for
protein and peptide drug delivery", Bioconjugate
Chemistry, vol. 6, pp. 332-351 (1995).
In a preferred embodiment, the polymeric
carrier is composed of a single polymer type such as
PLGA. In a next preferred embodiment, the polymeric
carrier can be a mixture of polymers such as 50o PLGA
and 50o albumin.
Other polymers useful as polymeric carriers
to prepare encapsulated protein crystals according to
this invention include biocompatible/biodegradable
polymers selected from the group consisting of poly
(acrylic acid), poly (cyanoacrylates), poly (amino
acids), poly (anhydrides), poly (depsipeptide), poly
(esters), such as poly (lactic acid) or PLA, poly (b-
hydroxybutryate), poly (caprolactone) and poly
(dioxanone); poly (ethylene glycol), poly
(hydroxypropyl)methacrylamide, poly
[(organo)phosphazene], poly (ortho esters), poly (vinyl
alcohol), poly (vinylpyrrolidone), malefic anhydride-
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alkyl vinyl ether copolymers, pluronic polyols,
albumin, alginate, cellulose and cellulose derivatives,
collagen, fibrin, gelatin, hyaluronic acid,
oligosaccharides, glycaminoglycans, sulfated
polysaccharides, blends and copolymers thereof.
Other useful polymers are described in J. Heller and
R.W. Balar, "Theory and Practice of Controlled Drug
Delivery from Biodegradable Polymers," Academic Press,
New York, NY, (1980); K.O.R. Lehman and D.K. Dreher,
Pharmaceutical Technology, vol. 3, pp. 5- , (1979);
E.M. Ramadan, A. E1-Helw and Y. E1-Said, Journal of
Microencapsulation, vol. 5, p. 125 (1988). The
preferred polymer will depend upon the particular
protein component of the protein crystals used and the
intended use of the encapsulated crystals (formulations
and compositions) . Alternatively, the solvent
evaporation technique may be used for encapsulating
protein crystals (see D. Babay, A. Hoffmann and S.
Benita, Biomaterials vol. 9, pp. 482-488 (1988).
In a preferred embodiment of this invention,
protein crystals are encapsulated in at least one
polymeric carrier using a double emulsion method, as
illustrated herein, using a polymer, such as
polylactic-co-glycolyic acid. In a most preferred
embodiment of this invention, the polymer is
Polylactic-co-glycolyic acid ("PLGA"). PLGA is a co-
polymer prepared by polycondensation reactions with
lactic acid ("L") and glycolic acid ("G"). Various
ratios of L and G can be used to modulate the
crystallinity and hydrophobicity of the PLGA polymer.
Higher crystallinity of the polymer results in slower
dissolution. PLGA polymers with 20-70a G content tend
to be amorphous solids, while high level of either G or
L result in good polymer crystallinity. For more
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information on preparing PLGA, see D.K. Gilding and
A.M. Reed, "Biodegradable polymers for use in surgery-
poly(glycolic)/poly(lactic acid) homo and copolymers:
l., Polymer vol. 20, pp. 1459-1464 (1981). PLGA
degrades after exposure to water by hydrolysis of the
ester bond linkage to yield non-toxic monomers of
lactic acid and glycolic acid.
In another embodiment, double-walled polymer
coated microspheres may be advantageous. Double-walled
polymer coated microspheres may be produced by
preparing two separate polymer solutions in methylene
chloride or other solvent which can dissolve the
polymers. The protein crystals are added to one of the
solutions and dispersed. Here, the protein crystals
become coated with the first polymer. Then, the
solution containing the first polymer coated protein
crystals is combined with the second polymer solution.
[See Pekarek, K.J.; Jacob, J.S. and Mathiowitz, E.
Double-walled polymer microspheres for controlled drug
release, Nature, 367, 258-260]. Now, the second
polymer encapsulates the first polymer which is
encapsulating the protein crystal. Ideally, this
solution is then dripped into a larger volume of an
aqueous solution containing a surface active agent or
emulsifier. In the aqueous solution, the solvent
evaporates from the two polymer solutions and the
polymers are precipitated.
The above process can be performed using
either protein crystals, DNA or RNA crystals, of
formulations of any of these to produce compositions.
Formulations according to this invention
comprise a protein crystal, and, at least one
ingredient. Such formulations are characterized by at
least a 60 fold greater shelf life when stored at 50 °C
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than the soluble form of said protein in solution at 50
°C, as measured by T1,2. Alternatively, they are
characterized by at least a 59 fold greater shelf life
when stored at 40 °C and 75o humidity than the
nonformulated form of said protein crystal when stored
at 40 °C and 75°s humidity, as measured by T"2.
Alternatively, they are characterized by at least a 600
greater shelf life when stored at 50 °C than the
nonformul.ated form of said protein crystal when stored
at 50 °C, as measured by T1,2. Alternatively, they are
characterized by the loss of less than 20o a-helical
structural content of the protein after storage for 4
days at 50 °C, wherein the soluble form of said protein
loses more than 500 of its a-helical structural
content after storage for 6 hours at 50 °C, as measured
by FTIR. Alternatively, they are characterized by the
loss of less than 20~ a-helical structural content of
the protein after storage for 4 days at 50 °C, wherein
the soluble form of said protein loses more than 500 of
its a-helical structural content after storage for 6
hours at 50 °C, as measured by FTIR, and wherein said
formulation is characterized by at least a 60 fold
greater shelf life when stored at 50 °C than the soluble
form of said protein in solution at 50 °C, as measured
2 5 by T1,2 .
Compositions according to this invention
comprise one of the above described protein crystal
formulations, and, at least one polymeric carrier,
wherein said formulation is encapsulated within a matix
of said polymeric carrier.
Alternatively, compositions according to this
invention comprise formulations of a protein crystal
and at least one ingredient. Such compositions may be
characterized by at least a 60 fold greater shelf life
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when stored at 50 °C than the soluble form of said
protein in solution at 50 °C, as measured by T1,2.
. Alternatively, they are characterized by at least a 59
fold greater shelf life when stored at 40 °C and 75%
humidity than the nonformulated form of said protein
crystal when stored at 40 °C and 75% humidity, as
measured by T1,2. Alternatively, they are characterized
by at least a 60o greater shelf life when stored at 50
°C than the nonformulated form of said protein crystal
when stored at 50 °C, as measured by T1,2. Alternatively
they are characterized by the loss of less than 20o a-
helical structural content of the protein after storage
for 4 days at 50 °C, wherein the soluble form of said
protein loses more than 500 of its a-helical
structural content after storage for 6 hours at 50 °C,
as measured by FTTR. Alternatively, they are
characterized by the loss of less than 20o a-helical
structural content of the protein after storage for 4
days at 50 °C, wherein the soluble form of said protein
loses more than 500 of its a-helical structural content
after storage for 6 hours at 50 °C, as measured by FTIR,
and wherein said formulation is characterized by at
least a 60 fold greater shelf life when stored at 50 °C
than the soluble form of said protein in solution at 50
°C, as measured by Tl,z .
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.
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EXAMPLES
Example 1 LIPASE
Candida ruqosa lipase crystallization:
Materials:
A - Candida rugosa lipase powder
B - Celite powder (diatomite earth)
C - MPD (2-Methyl-2,4-Pentanediol)
D - 5mM Ca acetate buffer pH 4.6
E - Deionized water
Procedure:
A 1 kg aliquot of lipase powder was mixed
well with 1 kg of celite and then 22 L of distilled
water was added. The mixture was stirred to dissolve
the lipase powder. After dissolution was complete, the
pH was adjusted to 9.8 using acetic acid. Next, the
solution was filtered to remove celite and undissolved
materials. Then, the filtrate was pumped through a 30k
cut-off hollow fiber to remove all the proteins that
were less than 30kD molecular weight. Distilled water
was added and the lipase filtrate was pumped through
the hollow fiber until the retentate conductivity was
equal to the conductivity of the distilled water. At
this point, the addition of distilled water was stopped
and 5mm C:a-acetate buffer was added. Next, Ca-acetate
buffer was delivered by pumping through the hollow
fiber until the conductivity of the retentate was equal
to the conductivity of the Ca-acetate buffer. At that
point, addition of the buffer was stopped. The lipase
solution was concentrated to 30 mg/ml solution. The
crystallization was initiated by pumping MPD slowly
into the lipase solution while stirring. Addition of
MPD was continued until a 20o vol/vol of MPD was
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r: reached. The mixture was stirred for 24 hr or until
. 900 of the protein had crystallized. The resulting
> crystals were washed with crystallization buffer to
remove all the soluble material from the crystals.
Then, the crystals were suspended in fresh
crystallization buffer to achieve a protein
concentration of 42 mg/ml.
Example 2
Formulation of lipase crystals usinq sucrose
as excibient:
In order to enhance the stability of lipase
crystals during drying and storage the crystals were
formulated with excipients. In this example, lipase
crystals were formulated in the slurry form in the
presence of mother liquor before drying. Sucrose
(Sigma Chemical Co., St. Louis, MO) was added to lipase
crystals in mother liquor as an excipient. Sufficient
sucrose was added to lipase crystals at a protein
concentration of 20 mgs/ml in mother liquor (10 mM
sodium acetate buffer, pH 4.8 containing 10 mM Calcium
chloride and 20o MPD) to reach a final concentration of
100. The resulting suspension was tumbled at room
temperature for 3 hr. After treatment with sucrose,
the crystals were separated from the liquid by
25, centrifugation as described in Example 6, method 4 or
5.
Example 3
Formulation of lipase crystals using
trehalose as excipient:
The lipase crystals were formulated as in
Example 2, by adding trehalose, instead of sucrose,
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(Sigma Chemical Co., St. Louis, MO), to a final
concentration of 10o in mother liquor. The resulting
suspension was tumbled at room temperature for 3 hr and
the crystals were separated from the liquid by
centrifugation as described in Example 6, method 4 or
5.
Example ~1
Formulation of lipase crystals using
polyethylene oxide (PEO) as excipient:
Lipase crystals were formulated using O.lo
polyethylene oxide in water as follows. The crystals,
in the mother liquor at 20 mg/ml were separated from
the mother liquor by centrifugation at 1000 rpm in a
Beckman GS-6R bench top centrifuge equipped with
swinging bucket rotor. Next, the crystals were
suspended in 0.1% polyethylene oxide for 3 hrs (Sigma
Chemical Co., St. Louis, MO) and then separated by
centrifugation, as described in Example 6, method 4 or
5.
Example 5
Formulation of lipase crystals using
methoxypalyethylene glycol (MOPEG) as excipient:
Lipase crystals were formulated as in Example
2, by adding 10o methoxypoly ethylene glycol, instead
of sucrose, (final concentration) (Sigma Chemical Co.,
St. Louis, MO) in mother liquor and separating after 3
hrs by centrifugation, as in Example 6, method 4 or 5.
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Example 6
Methods of dryina crystal formulations:
Method 1. Nz Gas Drvina at Room Temperature
Crystals as prepared in Examples 1, 10, 14 and 21
were separated from the mother liquor containing
excipient by centrifugation at 1000 rpm in a
Beckman GS-6R bench top centrifuge equipped with
swinging bucket rotor in a 50 ml Fisher brand
Disposable centrifuge tube (Polypropylene). The
crystals were then dried by passing a stream of
nitrogen at approximately 10 psi pressure into the
tube overnight.
Method 2. Vacuum Oven Dryinq
Crystals as prepared in Examples 1, 10, 14 and 21
were first separated from the mother
liquor/excipient solution using centrifugation at
1000 rpm in a Beckman GS-6R bench top centrifuge
equipped with swinging bucket rotor in a 50 ml
Fisher brand Disposable polypropylene centrifuge
tube. The wet crystals were then placed in a
vacuum oven at 25 in Hg (VWR Scientific Products)
at room temperature and dried for at least 12
hours.
Method 3. Lyophilization
Crystals as prepared in Examples 1, 10, 14 and 21
were first separated from the mother
liquor/excipient solution using centrifugation at
1000 rpm in a Beckman GS-6R bench top centrifuge
equipped with swinging bucket rotor in a 50 ml
Fisher brand Disposable polypropylene centrifuge
tube. The wet crystals were then freeze dried
using a Virtis Lyophilizer Model 24 in
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semistoppered vials. The shelf temperature was
slowly reduced to -40 °C during the freezing step.
This temperature was held for 16 hrs. Secondary
drying was then carried out for another 8 hrs.
Method 4. Organic Solvent and Air Drying
Crystals as prepared in Examples l, 10, 14 and 21
were first separated from the mother
liquor/excipient solution using centrifugation at
1000 rpm in a Beckman GS-6R bench top centrifuge
equipped with swinging bucket rotor in a 50 ml
Fisher brand Disposable polypropylene centrifuge
tube. The crystals were then suspended in an
organic solvent like ethanol or isopropanol or
ethyl acetate or other suitable solvents,
centrifuged, the supernatant was decanted and air
dried at room temperature in the fume hood for two
days.
Method 5. Air Dryina at Room Temperature
Crystals as prepared in Examples 1, 10, 14 and 21
were separated from the mother liquor containing
excipient by centrifugation at 1000 rpm in a
Beckman GS-6R bench top centrifuge equipped with
swinging bucket rotor in a 50 ml Fisher brand
Disposable centrifuge tube (Polypropylene).
Subsequently, the crystals were allowed to air dry
in t:he fume hood for two days.
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Example '7
Soluble lipase sample preparation:
For comparison, a sample of soluble lipase
was prepared by dissolving lipase crystals to 20 mg/ml
in phosphate buffered saline, pH 7.4.
Figure 2 shows the stability for soluble
lipase. Specific activity decreases extremely rapidly
with time. Within 2-3 hours, the specific activity
decreases from about 660 umoles/min/mg protein to about
100 umoles/min/mg protein or an approximate 85~
decrease. The T1,2 for soluble lipase was calculated to
be 1.12 hours.
Example 8
Olive oil assay for measurin~pase
activity:
Lipase crystals from Examples 1-7 were
assessed for activity against olive oil in pH 7.7
buffer. The assay was carried out titrimetrically
using slight modifications to the procedure described
in Pharmaceutical Enzymes - Properties and Assay
Methods, R. Ruyssen and A. Lauwers, (Eds.), Scientific
Publishing Company, Ghent, Belgium (1978).
Reagents:
1. Olive oil emulsion:
16.5 gm of gum arabic (Sigma) was dissolved in 180
ml of water, 20 ml of olive oil (Sigma) and
emulsified using a Quick Prep mixer for 3 minutes.
2. Titrant . 0.05 M NaOH
3. Solution A: 3.0 M NaCl
4. Solution B: 75 mM CaCl~.2H20
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5. Mix: 40 ml of Solution A was combined with 20
ml of Solution B and 100 ml of H20.
6. 0.5s Albumin:
7. Lipase Substrate Solution (solution 7) was
prepared by adding 50 ml of olive oil emulsion
(solution 1) to 40 ml of Mix (solution 5) and 10
ml of 0.5o albumin (solution 6).
Assay Procedure
The lipase substrate solution (solution 7)
was warmed to 37°C in a water bath. First, 20 ml of
substrate was added to a reaction vessel and the pH was
adjusted to 7.7 using 0.05 M NaOH (solution 2) and
equilibrated to 37°C with stirring. The reaction was
initiated by adding enzyme. The reaction progress was
monitored by titrating the mixture of enzyme and
substrate' with 0.05 M NaOH to maintain the pH at 7.7.
The specific activity (umoles/min/mg protein)
was equal to the initial rate x 1000 x concentration of
the titrant/the amount of enzyme. The zero point was
determined by running the reaction without enzyme,
i.e., usi.ng buffer in the place of enzyme in the
reaction mixture.
Example ~
Activity:
The shelf activity of the dried crystals from
Examples 1-5 was measured using the olive oil assay as
described in Example 8. Dried crystals (5 mg) were
dissolved in 1 ml of phosphate buffered saline ("PBS"),
pH 7.4 and the activity was measured using olive oil as
substrate.
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Shelf stability:
The shelf stability of dried crystalline
lipase formulations from Examples 2-5 was carried out
in a humidity chamber controlled at 75o relative
humidity and 40°C temperature (HOTPACK). The activity
of the crystals was measured by dissolving 5 mg of the
dried samples in PBS buffer, pH 7.4, measuring the
activity in the olive oil assay and then comparing with
the initial results.
Crystal Formulations Dried by Method 5
Figure 3 shows the shelf stability profile of
lipase crystals formulated with sucrose, trehalose and
PEO. When dried by method 5, PEO was the most
protective excipient, followed by sucrose and then
trehalose.
Crystal Formulations Dried by Method 4
Figure 4 shows the shelf stability profile of
lipase crystals formulated with sucrose, trehalose, PEO
and MOPED. When dried by method 4, the excipients PEO,
sucrose and MOPED were similar in their ability to
preserve enzyme activity as measured by their effect on
the T1,2. Trehalose was less protective of lipase
activity than the other excipients.
Shelf Stability
The time required for the specific activity
of the enzyme to decrease by 50o is known as the T1,2.
Table 2 shows the effect on the T1,; of the specific
activity for formulations of dried lipase crystals.
For lipase, sucrose was the most protective excipient,
followed by polyethylene oxide (PEO), methoxypoly
ethylene glycol (MOPED) and finally trehalose. Sucrose
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was more than 10-fold more protective, as measured by
its effect on T1,2 of specific activity.
fable 2. Lipase at 40°C and 75o humidity
Dried Lipase Crystals
Excipients Tl,z ( days )
none 1.52
Sucrose 1092
Trehalose 90
PEO 835
MOPED 434
Soluble lipase 0.0468 (1.124 hrs)
The Tl,z was calculated from the shelf life
data by non-linear regression analysis using the Sigma
Plot program. Table 2 shows that formulations of
lipase were 1,923 fold more stable than soluble when
PEO was used as the excipient. In addition,
formulations of lipase were 23,300 fold more stable
than soluble lipase when sucrose was used as the
excipient (Table 2). Formulations using MOPED and PEO
as excipients with lipase crystals were 9,270 and
17,800 fold more stable than soluble lipase (Table 2).
The stability of the formulated crystals
relative to the non-formulated crystals was greatly
enhanced, as shown in Table 2. For example, crystals
formulated with trehalose were 59 fold more stable than
non-formulated lipase crystals made without an
excipient at 40 °C, as shown in Table 2. Similarly,
crystals formulated with MOPED were 286 fold more
stable than non-formulated crystals and crystals
formulated with PEO were 549 fold more stable than non-
formulated lipase crystals made without an excipient at
°C, as shown in Table 2. Finally, crystals
formulated with sucrose were 718 fold more stable than
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non-formulated lipase crystals made without an
excipient at 40 °C, as shown in Table 2.
Moisture content:
Moisture content was determined by the Karl
Fischer method according to manufacturer's instructions
using a Mitsubishi CA-06 Moisture Meter equipped with a
VA-06 Vaporizer (Mitsubishi Chemical Corporation,
Tokyo, Japan).
Table 3. Moisture content lipase crystal formulations
TIME, DAYS% Moisture
_ TREHALOSEPEO MOPEG
SUCROSE
0 7.23 5.58 8.56 7.03
129 11.11 10.45 10.43 10.01
Crvstallinity:
The crystal integrity of the formulations
were measured by quantitative microscopic observations.
In order to visualize whether the crystals were
maintained their shape after drying, the dried crystals
were examined under an Olympus BX60 microscope equipped
with DXC-970MD 3CCD Color Video Camera with Camera
Adapter (CMA D2) with Image ProPlus software. Samples
of dried crystals were covered with a glass coverslip,
mounted and examined under lOX magnification, using an
Olympus microscope with an Olympus UPLAN F1 objective
lens 10X/0.30 PHl (phase contrast).
In this analysis, the crystals originally
formulated with sucrose, trehalose, PEO and MOPEG were
readily visualized after 129 days at 40 °C and 75%
humidity. Figure 5 shows that crystals formulated with
PEO were present at the initial time. A similar
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microscopic observation taken 129 days later and shown
in Figure 6 demonstrates that crystallinity was
maintained for the entire time period. Similar data
was obtained for crystals formulated with sucrose,
MOPEG and trehalose (data not shown).
Secondary structure characterization by FTIR:
The fourier transform infrared ("FTIR")
spectra were collected on a Nicolet model 550 Magna
series spectrometer as described by Dong et al. [bong,
A., Caughey, B., Caughey, W.S., Bhat, K.S. and Coe,
J.E. Biochemistry, 1992; 31:9364-9370; Dong, A.
Prestrelski, S.J., Allison, S.D. and Carpenter, J.F.
J.Pharm. Sci., 1995; 84: 415-424.] For the solid
samples, 1 to 2 mg of the protein was lightly ground
with 350 mg of KBr powder and filled into small cups
used for diffuse reflectance accessory. The spectra
were collected and then processed using Grams 32 from
Galactic software for the determination of relative
areas of the individual components of secondary
structure using second derivative and curve-fitting
program under amide I region (1600-1700 crri~).
For comparison, a soluble lipase sample was
prepared by dissolving lipase crystals in phosphate
buffered saline and analyzed for stability by FTIR.
Secondary structure was determined as
follows: FTIR spectra were collected on a Nicolet model
550 Magna series spectrometer. A 1 ml sample of
soluble lipase was placed on a Zinc selenide crystal of
ARK ESP. The spectra were collected at initial (0)
time and after the loss of most of the activity or,
near-zero activity. The acquired data was then
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processed using Grams 32 software from Galactic
Software for the determination of relative areas of the
individual components of secondary structure using
second derivative and curve-fitting program under amide
I region ( 1600-1700 cm' ) .
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Table 4. Lipase at 40°C and 75~ Humidity
Extended
Sample a- Helix~i-Sheets (3-Turn coil Random
Soluble 25.08 59.89 4.57 3.21 7.25
Lipase
initial
time
After 3 13.14 16.11 13.59 6.74 50.42
days at
4 0C
Lipase 19.46 49.94 0.00 30.60 0.00
-
Sucrose
initial
time
After 66 17.70 60.70 0.00 21.60 0.00
days at
40C and
75$
Humidity
Lipase 23.30 52.33 0.00 23.17 1.20
-
2 Trehalose
0
initial
time
After 66 18.38 55.51 0.00 26.11 - 0.00
days at
40C and
75$
Humidity
Lipase-PEO23.22 48.33 0.00 24.53 3.92
initial
3 time
0
After 66 21.79 54.90 1.65 21.66 0.00
days at
40C and
75$
Humidity
Lipase 24.66 49.37 0.00 19.20 6.77
-
MOPEG
initial
time
4 After 66 26.59 51.09 0.00 22.32 0.00
0
days at
4 0C and
75$
Humidity
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CONCLUSION:
Table 4 shows that for soluble lipase,
approximately 50% and 75 ~ of the a-helix and ~i-sheet
structure content was lost over three days. There was
a corresponding increase in the content of random
structure.
In contrast, Table 4 shows that crystals
which were formulated and dried were much more stable
than the soluble enzyme. Such crystals showed a loss
of a-helical structure which ranged from 6.1s to 21.1%
after 66 days at 40 °C and 75% humidity. At the same
time, random coil in solution increased from 7-500 over
3 days, while crystallinity showed minimum random coil
content even after 66 days.
The data in Table 4, obtained using FTIR to
monitor changes in secondary structure, correlated with
the activity data shown in Figure 4. In particular,
lipase formulated with sucrose, PEO and MOPEG showed
significantly less loss of a-helical structure and
maintained a higher specific activity over the 66 day
time period at elevated temperature and humidity than
crystals formulated with trehalose, which showed a 21g
loss of a-helical structure and had the lowest activity
profile.
Example 10 HUMAN SERUM ALBUMIN
Crystallization of Human Serum Albumin
Ten grams of powdered human serum albumin was
added to a 75 ml stirred solution of 100 mM phosphate
buffer (pI-I 5.5) at 4°C. Final protein concentration was
120 mg/ml, estimated from the OD28~ value of the
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solution. First, a saturated ammonium sulfate solution
(767 g/1), prepared in deionized water, was added to
the protein solution to a final concentration of
350 g/1 or 50o saturation. Next, the crystallization
solution was "seeded" with 1 ml of 50 mg/ml albumin
crystals in 50o ammonium sulfate at pH 5.5. Seed
crystals were prepared by washing a sample of crystals
free of precipitate with a solution of 50o saturated
ammonium sulfate in 100 mM phosphate buffer at pH 5.5.
The seeded crystallization solution was incubated
overnight at 4°C on a vigorously rotating platform.
Crystals in the shape of rods (20 um) appeared in the
solution overnight after approximately 16 hr.
Example 11
Formulation of HSA crystals using gelatin as
excipient:
In order to enhance the stability of human
serum albumin (HSA) crystals during drying and storage
the crystals were formulated with excipients. In this
example, HSA crystals were formulated in the slurry
form in the presence of mother liquor before drying.
Gelatin (Sigma Chemical Co., St. Louis, MO) was added
to lipase crystals in mother liquor as an excipient.
Sufficient gelatin was added to lipase crystals at a
protein concentration 20 mgs/ml in mother liquor (2.5M
ammonium sulfate in 100mM phosphate buffer, pH 5.5) to
reach a final concentration of 10%. The resulting
suspension was tumbled at room temperature for 3 hr.
After treatment with gelatin, the crystals were
separated from the liquid by centrifugation as
described in Example 6, method 1.
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Drying of HSA crystals
The HSA crystals were then dried by the four
methods described in Example 6. The crystals were
suspended in cold (4°C) ethanol as the organic solvent
of method 4.
Example 12
Soluble human serum albumin preparation:
For comparison, the soluble HSA sample was
prepared by dissolving HSA crystals at 20mg/ml in
water.
Example 1.3
Shelf stability:
Shelf stability of HSA dried crystal
formulations were carried out in a waterbath at 50°C
temperature. The stability of the crystals was
monitored by following structural degradation by FTIR
analysis.
Moisture content:
Moisture content was determined by the Karl
Fischer method according to manufacturer's instructions
using a Mitsubishi CA-06 Moisture Meter equipped with a
VA-06 Vaporizer (Mitsubishi Chemical Corporation,
Tokyo, Japan) .
Table 5. Moisturecontent HSA crystals
%Moisture
TIME, DAYS Gelatin
0 5.0464
12 6.3841
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Crystallinity:
The crystal integrity of the formulations was
measured by quantitative microscopic observations, as
described in Example 9. Figure ? shows that HSA
crystals were readily visualized immediately after
preparing the formulation with gelatin. Figure 8 shows
that crystallinity was maintained after four days at 50
°C .
Secondary structure characterization by FTIR:
The FTIR spectra were collected on a Nicolet
model 550 Magna series spectrometer as described by
Dong et al.[Dong, A., Caughey, B., Caughey, W.S., Bhat,
K.S. and Coe, J.E. Biochemistry, 1992; 31:9364-93?0;
Dong, A. Prestrelski, S.J., Allison, S.D. and
Carpenter, J.F. J.Pharm. Sci., 1995; 84: 415-424]. For
the solid samples, 1 to 2 mg of the protein was lightly
ground with 350 mg of KBr powder and filled into small
cups used for diffuse reflectance accessory. The
spectra were collected and processed using Grams 32
from Galactic software for the determination of
relative areas of the individual components of
secondary structure using a second derivative and
curve-fitting program under amide I region (1600 -1?00
cn1 1 ) .
For comparison, the soluble HSA sample was
prepared by dissolving HSA crystals in water and tested
for stability by FTTR. The secondary structure was
determined as in Example 9.
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Table 6. Secondary Structure content of HSA
a- Vii- Extended
Sample Helix Sheets (3-Turn coil Random
HSA- 45.47 6.83 6.79 40.91 0.00
Soluble
HSA- 28.71 30.39 8.59 28.07 4.24
Sol. 15
min
HSA- 19.15 34.69 8.16 27.91 10.09
Sol. 30
min
HSA- 10.04 15.82 0.00 42.83 31.31
Sol. 60
min
HSA crystals
HSA 49.08 24.77 0.00 26.15 0.00
-Gelatin
HSA 40.93 7.65 7.48 43.94 0.00
-Gelatin
After 12
days at
50C
CONCLUSION:
Soluble HSA showed a rapid 78a decrease in a-
helical content after 1 hour in solution and a
corresponding increase in random coil structure.
Dry formulated crystals showed an
approximately 16o decrease in a-helical content, a
large decrease in ~i-sheet content and no increase in
the content of random structure.
Example 14 PENICILLIN ACYLASE
Crystallization of Penicillin Acylase
An ammonium sulfate suspension of Penicillin
acylase from Boehringer Mannheim was the raw material.
The suspension of Penicillin acylase was diluted 1:3
with deionized water. This solution was concentrated
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by diafilration using a 30K membrane to a final
concentration of 200 OD at AZeonm /ml. The enzyme
solution was then diluted with 4 M NaH2PO4.Hz0 to 150 OD
A280nm /ml.
A biphasic solution of 4 M and 1 M sufficient
NaHzPOq.H20 to yield a final solution concentration of
1.9 M NaP04, was prepared. In this case, 280 ml of 1 M
NaH2PO~.H20 was carefully overlaid on top of to the top
of 549 ml of 4 M NaH2PO4.H20. The enzyme solution was
poured gently into the side of the container to form a
layer above the 1 M layer. An overhead stirrer was set
up with a marine impeller. The agitator was placed
into the container with the blades just below the 4M/1M
interface. The speed was adjusted to a setting of 8.0,
or 600 rpm. The agitator was switched on and stopped
after 10 minutes. The impeller was removed from the
container. The volume of seed crystals was measured
with a graduated pipette using between 0.5 and 0.1o by
volume and added to the 20 L sterile polycarbonate
container. The seed was not allowed to be static for
more than 10 seconds before addition. The solution was
mixed by hand using a flat-blade impeller for about 1
minute. The crystallization mixture was allowed to
stand for 24 hours.
After 24 hours, the container was opened and
the solution was mixed by hand for 30 seconds. The
solution was allowed to sit for an additional 24 hours.
After a total of 48 hours, a 10 ml sample was taken of
the supernatant. The sample was filtered with a 0.2
micron Acrodisc. The AZSOnm of the supernatant was
measured. When supernatant Az~onm was greater than 1.0
mg/ml, the crystallization was allowed to continue for
24 more hours. When the supernatant concentration was
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less than. 1.0 mg/ml, the AZ~o~m of crystal slurry and
supernatant directly were determined.
Example 15
Penicillin G Assay for PA Crystals
The basis of the activity assay for
Penicillin acylase involves a titrimetric assay which
measures the enzymatic hydrolysis of benzyl penicillin
by the enzyme. The enzyme catalyzes cleavage of a
phenyl acetyl group from penicillin G thus causing a
decrease in pH. This activity was followed by
measuring the volume of 50 mM NaOH needed (to maintain
a pH of 8 at 28°C) per minute of reaction. The assay
uses a substrate buffer made with potassium chloride
and Tris and was reported in U/mg.
Penicillin acylase assay:
Chemicals and solutions used in the assay:
1. Penicillin-G, Sigma, Potassium salt
2. 0.05 N Sodium hydroxide
3. 1.0 M KCl, 20 mM Tris buffer, pH 8.0
4. 10 mM Tris, 10 mM CaCl2 buffer, pH 8.0
5. 0.01 M PBS buffer, pH 7.5
Preparation of substrate solution
1 g of penicillin-G was added to 10 ml of
1.0 M KC1, 20 mM Tris buffer, pH 8.0 solution and about
70 ml of DI water, 1 ml of 10 mM Tris, 10 mM CaCl2.Then
the pH of the solution was adjusted to 8.0 using 0.05 N
NaOH. Next, final solution volume was adjusted to 100
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ml. The solution was prepared fresh before each use
and used within five hours of preparation.
Preparation of PA sample solution
A sample of Penicillin acylase was dissolved
in PBS buffer, pH 7.4. Typically, 2 to 4 mg (dry
weight) of protein were used for each assay.
Assay for hydrolysis of penicillin G
20 ml of penicillin acylase substrate
solution was added to a titration vessel and
equilibrated to 28 °C. After adding the enzyme solution
to the reaction vessel, the hydrolysis of penicillin-G
was monitored by titrating the reaction mixture with
0.05 N NaOH to maintain the pH at 8Ø
Example 16
Formulation of PA crystals using
hydroxypropyl-Q-cyclodextrin (HPCD)
as excipient:
In order to enhance the stability of PA
crystals during drying and storage, the crystals were
formulated with excipients. In this example, PA
crystals were formulated in the slurry form in the
presence of mother liquor before drying.
Hydroxypropyl-~i-cyclodextrin (HPCD) (Sigma Chemical
Co., St. Louis, MO) was added to PA crystals in mother
liquor as an excipient. Sufficient HPCD was added to
lipase crystals at a protein concentration 20 mgs/m in
mother liquor (1.9M NaHzP04, pH 6.6), to reach a final
concentration of 100. The resulting suspension was
tumbled at room temperature for 3 hr. After treatment
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with HPCD, the crystals were separated from the liquid
by centrifugation as described in Example 6, method 1.
Example 17
Formulation of PA crystals using mother
lictuor itself as excipient:
The PA crystals were formulated using mother
liquor (1.9M NaHzPOq, pH 6.6). The crystals were
separated by centrifugation as in Example 16.
Example 18
Dryina PA
The PA crystals from Examples 15-17 were then
dried according to the methods 1, 3 or 4 of Example 6.
The organic solvent used for method 4 of Example 6 was
ethyl acetate.
Example 1.9
Soluble PA preparation:
As a standard of comparison, a sample of
soluble PA was prepared by dissolving PA crystals in
water at 20 mg/ml.
The stability of soluble PA was measured over
time and a plot specific activity at 55 °C versus time
is shown in Figure 9. PA enzyme activity in solution
decayed rapidly and was undetectable after
approximately 20 hours.
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Example 20
Activity of the dried crystals:
The activity of the dried crystals from
Example .L8 was tested in the Pen G assay as described
in Example 15. The dried crystals (5 mg) were
dissolved in 1 ml of water and the activity was
measured using Pen G as substrate
Shelf stability:
The shelf stability of PA dried crystal
formulations was determined using a Reactive-Therm III
- Heating /Stirring module by Pierce at 55°C
temperature. Activities were measured by dissolving 5
mg of the dried sample in water and measuring the
enzyme activity in the Pen G assay described in Example
15 and compared with the initial results. Figure 10
depicts the shelf stability profiles of PA crystals
with and without excipient. Figure 10 also depicts
shelf stability profiles of PA crystal formulations
dried with nitrogen (method 1) or air dried (method 4).
Table 7. Penicillin acylase activity at 55°C
Dry Crystals-Penicillin acylase T1/2 days
Penicillin acylase -crystals dried by 123.25
lyophilization
Penicillin acylase - HPCD dried by 99.24
lyophilization
Penicillin acylase - dried using 24.85
nitrogen
Penicillin acylase -HPCD dried by 87.91
nitrogen
Soluble Penicillin acylase 0.21 (4.92
hrs)
The T1,2 was calculated from the shelf life
data by non-linear regression analysis using the Sigma
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Plot program. The stability of the formulated crystals
relative to the non-formulated crystals was enhanced,
as shown in Table 7. For example, crystals formulated
with HPCD were 418 fold more stable soluble PA at 55 °C
(Table 7). Crystals formulated with HPCD were 3.5 fold
more stable at 55 °C than non-formulated PA crystals
made without an excipient, as shown in Table 7.
Moisture content:
Moisture content was determined by the Karl
Fischer method according to manufacturer's instructions
using a Mitsubishi CA-06 Moisture Meter equipped with a
VA-06 Vaporizer (Mitsubishi Chemical Corporation,
Tokyo, Japan).
Table 8. Moisture content of PA crystals
Moisture
DAYS _PA HPCD
0 2.6198 1.5722
43 2.1792 3.1963
Crystallinity:
The crystal integrity of the formulations was
measured by quantitative microscopic observations as
described in Example 9. Crystallinity was maintained
through out the process as indicated by the crystals,
which were readily visible (data not shown).
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Secondary structure characterization by FTIR:
Stability was assessed by quantifying the
secondary stucture content of the dried and formulated
PA crystals by FTIR as described in Example 9. Soluble
PA was u:>ed for comparative purposes.
Table 9. Secondary Structure of PA at 55°C
Extended
Sample a- Helix(3-Sheets(3-Turn coil Random
Soluble 37.74 33.53 11.57 17.16 0.00
PA
Init. time
After 2 25.27 35.32 7.29 21.93 10.19
hrs at 55"C
After 9.29 50.66 0.00 26.77 13.28
24hrs at
55C
1 PA -dried 33.83 27.99 5.32 13.66 0.00
5
by lyoph.
init. time
After 22.53 18.9 17.03 18.42 23.12
43days at
55C
PA - HPCD 35.9 36.96 0.00 27.14 0.00
dried by
lyoph.
init. time
2 After 19.05 16.77 23.11 16.65 24.42
5
43days at
55C
PA -dried 25.73 34.93 6.68 28.73 3.93
by N2
init. time
no
excipient
After 11.88 34.73 10.22 30.07 13.10
43days at
55C
PA -HPCD 31.26 35.78 12.24 20.72 0.00
dried by
nitrogen
init. time
4 After 22.17 25.31 0.00 42.23 10.29
0
43days at
55C
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CONCLUSION:
The loss of a-helical content was 76Q after 1
day for soluble Penicillin acylase. In contrast, when
HPCD was used as an excipient and the formulation was
dried by method 1 of Example 6, only 310 of the a
helical content was lost after 43 days at 55 °C
temperature.
Example 21 GLUCOSE OXIDASE
Preparation of Glucose Oxidase Crystals
We prepared crystals of glucose oxidase as
follows. First, the glycoprotein was purified by anion
exchange chromatography and then the crystallization
parameters were optimized (data not shown).
As a result of these studies, we found that
the conditions for crystallization of glucose oxidase
fall generally in the range of 7 to 17°s PEG 4000 or
6000, 8 to 200 2-propanol or ethanol and buffer to
adjust th.e pH to between 3 and 6. It should be
understood, however, that many sets of experimental
conditions within and near this range can produce
satisfactory results for the crystallization of glucose
oxidase and other glycoproteins. Those of skill in the
art will appreciate that the precise conditions which
efficiently produce crystals of the desired size and
quality, will vary due to differences in experimental
conditions, such as protein and reagent purity, rates
of stirring, shear force effects and carbohydrate
content.
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Larne Scale Crystallization of Glucose
Oxidise
We determined preferred conditions for
preparative scale crystallization of glucose oxidise.
Preparative scale crystallization generally involves
100 to 900 mls of glycoprotein.
A. Crystallization at Constant pH Without Seed
Glucose oxidise was diafiltered in water and
concentrated to an AZeo of between 5 and 15. The glucose
oxidise concentrate was mixed (1:1) with one volume of
the crystallizing reagent containing 18o PEG 6000, 320
2-propanol in 0.2 M Na-Acetate at pH 5Ø After
mixing, the solution was cooled to 6°C. The glucose
oxidise crystallization solution was stirred for 24
hours at 100 rpm with a propeller stirrer. During this
time, the crystals formed gradually.
Examtale 22
Formulation of glucose oxidise crystals using
trehalose as excipient:
In order to enhance the stability of glucose
oxidise (GOD) crystals during drying and storage, the
crystals were formulated with excipients. In this
example, GOD crystals were formulated in the slurry
form in i~he presence of mother liquor before drying.
Trehalose (Sigma Chemical Co., St. Louis, MO) was added
to GOD crystals in mother liquor as an excipient.
Sufficient trehalose was added to GOD crystals at a
protein concentration 20 mgs/ml in mother liquor (100
mM sodium acetate buffer, pH 5.5 containing 320
isopropanol and 9o PEG 6000) to reach a final
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concentration of 10%. The resulting suspension was
tumbled at room temperature for 3 hr. After treatment
with trehalose, the crystals were separated from the
liquid by centrifugation as described in Example 6,
method 1.
Example 23
Formulation of crlucose oxidase crystals using
lactitol as excipient:
Glucose oxidase crystals were formulated as
in Example 22 by adding lactitol (Sigma Chemical Co.
St. Louis, MO), (instead of trehalose) to a final
concentration of 10o to the mother liquor. The
crystals were separated from the mother liquor/lactitol
solution after three hours by centrifugation.
Example 24
Formulation of glucose oxidase crystals using
hydroxypropyl-f3-cyclodextrin (HPCD)
as excipient:
Glucose oxidase crystals were formulated
using hydroxypropyl-~-cyclodextrin (HPCD) as in Example
22 (instead of trehalose) by adding HPCD to a final
concentration of 10o in mother liquor and incubated for
3 hrs (Sigma Chemical Co. St. Louis, MO). The crystals
were then separated from the mother liquor/HPCD
solution after 3 hr. by centrifugation as described in
Example F, method 1.
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Example 25
Formulation of glucose oxidase crystals using
gelatin as excipient:
Glucose oxidase crystals were formulated as
in Example 22 by adding gelatin to a final
concentration of 100 (Sigma Chemical Co. St. Louis, MO)
in mother liquor (instead of trehalose). The crystals
were separated from the mother liquor/gelatin solution
after three hours by centrifugation.
Example 26
Formulation of Glucose oxidase crystals using
Methoxypolyethylene Glycol as excipient:
The glucose oxidase crystals were formulated
as in Example 22 by adding methoxypoly ethylene glycol
to a final concentration of loo (Sigma Chemical Co. St.
Louis, M0) in mother liquor. The crystals were
separated from the mother liquor/methoxypoly ethylene
glycol solution after 3 hrs by centrifugation as
described in Example 6, method 1.
Example 27
Formulation of glucose oxidase crystals using
sucrose as excipient:
Glucose oxidase crystals were formulated as
in Example 22 by adding sucrose to a final
concentration of 10~ (Sigma Chemical Co., St. Louis,
MO) in the mother liquor. The crystals were separated
from the mother liquor/sucrose solution after three
hours by centrifugation.
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Example 28
Drying Glucose Oxidase Formulations
The glucose oxidase crystal formulations
described above were dried according to the methods
described in Example 6. Method 4 utilized cold (4°C)
isopropanol as the organic solvent.
Example 29
Soluble glucose oxidase preparation:
For comparison, the soluble glucose oxidase
sample was prepared by dissolving glucose oxidase
crystals at 20 mg/ml in 50 mM citrate buffer, pH 6Ø
Figure 11 shows the stability of the soluble
glucose oxidase over time at 50 °C. The specific
activity declines rapidly with time. After 24 hours,
the specific activity decreases by more than 900. The
T1,2 for soluble glucose oxidase was calculated to be
0.91 hours.
Example 30
Glucose Oxidase Activity Assay:
The following protocol was used to determine
the activity of dried glucose oxidase crystals and
crystal formulations.
Chemicals and solutions:
1. Phosphate buffer (20 mM, pH 7.3), NaCl
(0.1 M) solution,
2. 21 mM O-Dianisidine dihydrochloride
stock solution, diluted to 0.21 mM as
working solution before use,
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3. 2 M glucose solution,
4. Peroxide solution (2 mg/ml),
5. 50 mM citrate buffer, pH 6.0
Sample preparation:
1. Add 2 mg of glucose oxidase to 1 ml of
50 mM citrate buffer, pH 6.0 and vortexed well for
about 2 min. The solution was mixed by tumbling
at room temperature for 1 hour to reconstitute.
2. Prepare a dilute enzyme solution by
mixing 0.1 ml of the above enzyme solution with
4.9 ml of the same citrate buffer.
Assay procedure for enzyme activity measurement:
1. The assay was monitored by a UV-Vis
spectrophotometer. Use the kinetic mode and set
wavelength at 460 nm and temperature at 25 °C.
2. Warm up the O-dianisidine/phosphate
working solution in the 25 °C water bath and bubble
the solution with oxygen for at least 20 min.
before use.
3. Measure the blank using the reagent
solution without the enzyme solution added.
4. Pipette 2.4 ml of oxygenated O-
dianisidine/phosphate working solution, 0.4 ml of
2 M glucose solution and 0.1 ml of peroxidase into
a disposable cuvette.
5. Add 10 ml of the enzyme sample on the
cuvette wall (tilt the cuvette to prevent the
sample from mixing with the reagent at this step)
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and cover with a piece of parafilm. Mix quickly
by inverting the cuvette twice, insert the cuvette
into the spectrophotometer's cell compartment and
start to collect data.
Calculate the enzyme specific activity using the
following formula
Specific activity = A*B*C/D*E*F
Where:
A = The changed in units of absorbance at
460 nm per minute
B = Reaction mixture volume (ml)
C = Dilution factor
D = 11.3 (a constant)
E = Weight (mg) of the enzyme used
F = Sample volume (ml)
Example 31
Activity of the dried crystals:
The activity of the dried crystal
formulations of glucose oxidase was measured as
described in Example 30.
Shelf stability:
A study of the shelf stability of
formulations of glucose oxidase crystals was performed.
In this case, the formulations were dried by method 4
of Example 6 and were stored in a 2 ml screw cap
Eppendorf tube in a waterbath at 50 °C temperature for
13 days. Activities at specific time points were
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obtained by dissolving 2 mg of the dried sample in 50
mM citrate buffer, pH 6.0 and then measuring the
activity according to Example 30.
The shelf stabilities of the various glucose
oxidase formulations stored at 50 °C were determined.
The data are presented in Figure 12. Lactitol was the
most effective excipient at preserving glucose oxidase
specific activity over time at elevated temperature.
Table 10. Glucose Oxidase at 50°C
Dried glucose oxidase
crystals
Excipi.ent T1,2 days
none 1.52
Trehal.ose 3.8
Lactit:ol 4. 85
HPCD 2.4
MOPEG 13.1
Gelatin 3.95
Sucrose 3.27
Soluble GOD 0.04 (0.91 hrs)
The Tl,z was calculated from the shelf life
data by non-linear regression analysis using the Sigma
Plot program. Table 10 shows that formulations of
glucose oxidase were 95 fold more stable than soluble
when trehalose was used as the excipient. In addition,
formulations of glucose oxidase were 121 fold more
stable than soluble when lactitol was used as the
excipient (Table 10). Formulations using HPCD or MOPEG
as excip:ients with glucose oxidase crystals were 60 and
325 fold more stable than soluble glucose oxidase,
respectively(Table 10). Finally, formulations using
gelatin or sucrose as excipients with glucose oxidase
crystals were 99 and 82 told more stable than soluble,
respectively (Table 10).
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Formulations made with either trehalose,
lactitol, HPCD, MOPEG, gelatin or sucrose, as
excipients with glucose oxidase crystals were 2.5 fold,
3.2 fold, 1.6 fold, 8.6 fold, 2.6 fold, or 2.2 fold
more stable than glucose oxidase crystals made without
an excipient at 50 °C, as measured by T1,2, as shown in
Table 10.
Moisture content:
Moisture content was determined by the Karl
Fischer method according to manufacturer's instructions
using a Mitsubishi CA-06 Moisture Meter equipped with a
VA-06 Vaporizer (Mitsubishi Chemical Corporation,
Tokyo, Japan).
Table 11. Moisture content of GOD crystal formulations
1 5 TIME Moisture
DAYS Trehalose Lactitol HPCD MOPEG Gelatin Sucrose
0 4.3819 4.5274 8.3817 4.2008 4.7090 4.2083
13 E3.7292 11.4808 8.7582 8.0763 13.7541 9.8284
Cr~stallinity:
The crystal integrity of the GOD formulations
was measured by quantitative microscopic observations
as described in Example 9. In this example, the
crystals were readily visualized, indicating that
crystallinity was maintained throughout the process.
Figure 13 show that glucose oxidase crystals
were readily visualized immediately after preparing the
lactitol formulation. Figure 14 demonstrates that
crystallinity was maintained after 13 days at 50 °C.
Crystalline material was also readily visualized after
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formulating glucose oxidase with trehalose, as depicted
in Figure 15. Likewise, crystalline material remained
readily visualized after after 13 days at 50 °C, as
shown in Figure 16.
Secondary structure characterization by FTIR:
Stability was assessed by quantifying the
secondary stucture content of the dried and formulated
GOD crystals by FTIR as described in Example 9. For
comparison, a soluble glucose oxidase sample was
prepared by dissolving glucose oxidase crystals in 50
mM citrate buffer at pH 6.0 and placing about 1 ml on a
Zinc selenide crystal of ARK ESP, which then analyzed
for stability by FTIR.
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Table 12. Soluble Glucose Oxidase at 50°C
a- ~3- Extended
sample Helix Sheets ~i-Turn coil Random
soluble GoD 35.65 29.14 14.02 8.32 12.87
init. time
After lhr 23.05 34.78 6.17 18.56 17.44
at 50C
After 6 hr 9.92 25.45 8.44 32.04 24.15
at 50C
GOD- 30.34 29.10 8.28 23.13 9.15
1 Trehalose
0
init. time
After 4 24.63 33.32 5.78 27.17 9.10
days at 50C
coD 33.82 25.88 8.82 20.41 11.07
-Lactitol
init. time
After 4 27.24 27.53 8.56 19.82 16.85
days at 50C
con -HPCD 29. 25 . 75 8 . 46 23 . 10 12 .
B1 88
2 init. time
0
After 4 17.15 15.75 6.12 31.67 29.31
days at 50C
con -MOPED 25.45 28.23 9.62 22.52 14.18
init. time
After 4 15.87 30.91 8.91 28.68 15.63
days at SOC
MoD-gelatin 31.05 34.05 7.05 23.13 4.72
init. time
After 4 23.49 26.2 8.58 16.24 25.49
days at 50C
coD - 30.12 29.68 11.03 19.75 9.92
Sucrose
init. time
After 4 24 ; 23 . 75 9. 58 31 . 53 10. 46
68
-
da s at 50C
CONCLUSION:
Soluble glucose oxidase lost 75~ of its a-
helical content within only 6 hours at 50 °C.
The sugars lactitol, sucrose and trehalose
were the most effective excipients in preventing loss
of a-helical content upon storage at an elevated
temperature. Glucose oxidase crystals formulated in
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lactitol, sucrose and trehalose and dried by method 4
showed only an 18.1-19.4 0 loss of a-helical structural
content after 9 days at 50 °C.
Example 32
Dryina of Candida ruaosa lit~ase crystals:
Materials:
A - Candida rugosa lipase (Example 1)
B - Poly(ethylene glycol), 100 PEG
200, 300, 400, or 600
C - acetone
Procedure:
A 4 ml aliquot of crystal suspension (140 mg)
is added to four 15 ml tubes. Next, the suspension is
centrifuged at between 1000 to 3000 RPM for between 1
to 5 minutes or until the crystallization buffer is
removed. Then, 4 ml of liquid polymer (any PEG between
200 to 600 is suitable) is added to each tube and the
contents are mixed until homogeneous. The suspension
is centrifuged at between 1000 to 3000 RPM for between
1 to 5 minutes or until the liquid polymer is removed.
Next, 4 ml of acetone (isopropanol, butanol and other
solvents are also suitable) is added to each tube and
mixed well. The crystal/organic solvent suspensions
are transferred to 0.8 cm X 4 cm BIO-RAD poly-prep
chromatography columns (spin columns). The columns are
centrifuged at 1000 RPM for 1 to 5 minutes to remove
the organic solvent. Finally, nitrogen gas is passed
through the column to dry the crystals until a free
flowing powder results.
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Example 33
Purafect (protease) 4000L crystallization:
Materials:
A - crude purafect 4000L
B - 15o NaSOq solution
Procedure:
One volume of crude purafect enzyme solution
is mixed with two volumes of 15o Na2S04 solution. The
mixture .is stirred for 24 hr at room temperature or
until the crystallization is completed. The crystals
are washed with 15~ Na2S04 solution to eliminate the
soluble enzyme. The crystals are suspended in fresh
15o NazS04 solution to yield a protein concentration of
27 mg/ml.
Example 34
Drying of Purafect crystals:
Materials:
A - purafect crystals suspension
B - Poly(ethylene glycol), 1000 PEG
200, 300, 400, or 600
C - Organic solution
Procedure:
A 4 ml aliquot of crystal suspension (140 mg)
is added to four 15 ml tubes. Next, the suspension is
centrifuged at between 1000 to 3000 RPM for between 1
to 5 minutes or until the crystallization buffer is
removed. Then, 4 ml of liquid polymer (any PEG between
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200 to 600 is suitable) is added to each tube and the
contents are mixed until homogeneous. The suspension
is centrifuged at between 1000 to 3000 RPM for between
1 to 5 minutes or until the liquid polymer is removed.
Next, 4 ml of acetone (isopropanol, butanol and other
solvents are also suitable) is added to each tube and
mixed well. The crystal/organic solvent suspensions
are transferred to 0.8 cm X 4 cm BIO-RAD poly-prep
chromatography columns (spin columns). The columns are
centrifuged at 1000 RPM for 1 to 5 minutes to remove
the organic solvent. Finally, nitrogen gas is passed
through 'the column to dry the crystals until a free
flowing powder results.
Example 35
Producing DNA for Crystallization
Plasmids derived from pUC plasmids, such as
pSP64, may be used to produce either DNA or mRNA for
crystallization. In this example, plasmid pSP64
(availab=Le from Promega Biological Research Products)
is used to generate DNA for crystallization. The cDNA
coding for the protein of interest is inserted into any
of a number restriction sites available in the multiple
cloning site. The recombinant plasmid is then used to
transform E. coli bacteria. Next, large amounts of
plasmid are obtained by growth of the bacteria in
ampicillin containing medium. The techniques for
producing the recombinant plasmid, transforming E. coli
cells and for bacterial growth and plasmid DNA
preparation are described in detail in "Molecular
Cloning, 2nd Edition" (1989) Sambrook, J., Fritsch, E.
F. and T. Maniatis.
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Plasmid DNA is subsequently purified from the
bacterial cultures by first lysing the cells and then
separating the plasmid DNA from the genomic DNA, RNA
and other cellular materials using CsCl gradients.
These techniques are well known in the art and are
discussed in detail in Sambrook et al. The gradient
purified DNA is extracted with Tris-EDTA buffer
saturated N-butanol and finally ethanol. Next, plasmid
DNA is subjected to either linearization of the
plasmid, for use in the generation of mRNA for RNA
crystallization (Example 36) or excision of the gene of
interest for DNA crystallization (Example 37).
Example 36
Producing mRNA for Crystallization
The SP64 plasmid in combination with SP6 RNA
Polymerise (available from Promega Biological Research
Products) are used for the generation of milligram
quantities of 5' capped RNA transcripts. The plasmid
prepared in Example 35, prior to excision of the gene,
is linearized with a restriction enzyme downstream of
the poly A tail. The linear plasmid is purified by 2
phenol/chloroform and 2 chloroform extractions. DNA is
next precipitated with NaOAc (0.3M) and 2 volumes of
EtOH. Next, the pellet is resuspended at approximately
1 mg/ml in DEPC-treated distilled and deionized water.
Transcription is carried out in a buffer
composed of 400 mM Tris HC1 (pH 8.0), 80 mM MgCl2, 50 mM
DTT and 40 mM spermidine. The subsequent reagents are
added in order to one volume of DEPC-treated water at
room temperature: 1 volume SP6 RNA polymerise
transcription buffers rATP, rCTP and rUTP to 1 mM
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concentration; rGTP to 0.5 mM concentration;
7meG(5')ppp(5')G cap analog (New England Biolabs,
Beverly, Mass., 01951) to 0.5 mM concentration; the
linearized DNA template prepared above to 0.5 mg/ml
5 concentration; RNAs in (Promega, Madison, Wis.) to 2000
U/ml concentration; and SP6 RNA polymerase (Promega,
Madison, Wis.) to 3000 U/ml concentration. The
transcription mixture is incubated for 1 hour at 37°C.
The DNA template is then digested by adding 2
U RQ1 DNAse (Promega) per microgram of DNA template
used. The digestion reaction is carried out for 15
minutes. The transcribed RNA is extracted twice with
chloroform/phenol and twice with chloroform. The
supernatant solution is precipitated with 0.3M NaOAc in
15 2 volumes of EtOH and the pellet is resuspended in 100
ml DEPC-treated deionized water per 500 ml
transcription product. Finally, the supernatant
solution is passed over an RNAse-free Sephadex G50
column (Boehringer Mannheim # 100 411). The resultant
mRNA is sufficiently pure to be used crystallization.
Example 37
DNA crystallization
Materials:
A - Purified plasmid DNA (Example 35)
B - spermine
C - MPD (2-Methyl-2,4-Pentanediol)
D - 5mM Ca Acetate buffer pH 7.0
E - Deionized water
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Procedure:
The amplified DNA (Example 35) is removed
from the plasmid by restriction digestion. The
inserted gene is purified from the plasmid vehicle by
agarose gel electrophoresis and extraction of the gene
of interest from the gel band of the appropriate
molecular weight.
Using the hanging drop technique, DNA at 5
mg/ml in 5 mM Ca Acetate/20o MPD/1 mM spermine buffer
at pH 7.0 is incubated at room temperature until 90o of
the DNA has crystallized. The resulting crystals are
washed with crystallization buffer to remove all the
soluble material from the crystals. Then, the crystals
are resuspended in fresh crystallization buffer to
achieve a DNA concentration of 5 mg/ml.
Example 38
RNA crystallization
Materials:
A - Purified mRNA (Example 36)
B - spermine
C - MPD (2-Methyl-2,4-Pentanediol)
D - 5mM Ca Acetate buffer pH 7.0
E - Deionized water
Procedure:
Using the hanging drop technique, RNA
(Example 36) at 5 mg/ml in 5 mM Ca Acetate/20o MPD/1 mM
spermine buffer at pH 7.0 is incubated at room
temperature until 90% of the RNA has crystallized. The
resulting crystals are washed with crystallization
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buffer to remove all the soluble material from the
crystals. Then the crystals are resuspended in fresh
crystallization buffer to achieve RNA concentration of
mg/ml.
5 Example 39
Induction of an Immune Response to HIV ap120
Usinct DNA Crystals
DNA, coding for HIV gp160, is prepared
according to the methods of Examples 36 and 37.
Crystals of HIV gp160 are then used for immunization of
mice. Many genetic clones of both primary and
laboratory isolates of HIV are available from the Aids
Research and Reagent Program, National Institutes of
Allergy and Infectious Diseases, Rockville MD 20852,
for designing vaccines which induce broad neutralizing
immunity .
The DNA crystals are maintained in the
crystallization buffer and 200 ~1/mouse is injected
into the rear hind leg. The development of an immune
response to gp120 is determined by measuring serum
antibodies to the corresponding V3 loop peptide in
ELISA on a monthly basis.
Example 40
Induction of an Immune Response to HIV ap120
Usina mRNA Crystals
RNA coding HIV gp160 is prepared according to
the methods of Examples 35, 36 and 38. Crystals of HIV
gp160 mRNA are used for the immunization of mice.
Various primary and laboratory isolates of HIV are
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available from the Aids Research and Reagent Program,
National Institutes of Allergy and Infectious Diseases,
Rockville MD 20852, for designing vaccines which induce
broad neutralizing immunity.
The RNA crystals are maintained in the
crystallization buffer and 200 ,ul/mouse is injected
into the rear hind leg. The development of an immune
response to gp120 is determined by measuring serum
antibodies to the corresponding V3 loop peptide in
ELISA on a monthly basis.
Example 41
Olicro DNA crystallization
Materials:
A - Synthetic Oligo DNA
B - spermine
C - MPD (2-Methyl-2,4-Pentanediol)
D - 5mM Ca Acetate buffer pH 7.0
E - Deionized water
Procedure:
Using the hanging drop technique, synthetic
oligo DNA at 5 mg/ml in 5 mM Mg Acetate/30o MPD/1 mM
spermine buffer at pH 7.0 is incubated at room
temperature until. 900 of the DNA has crystallized. The
resulting crystals are washed with crystallization
buffer to remove all the soluble material from the
crystals. Then, the crystals are resuspended in fresh
crystallization buffer to achieve a DNA concentration
of 5 mg/ml.
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ExamQle 42
Antisense DNA Administration For Inhibition
of Gene Expression
Oligo DNA crystals coding for DNA sequences
which are complementary to the sense strand of an mRNA
species which is to be suppressed are generated as in
Example 41. Next, the crystals or a formulation
containing the crystals is administered to the site
where gene expression is intended to be inhibited.
Subsequently, cells will take up the DNA crystals or
dissolved DNA and the oligo DNA and host mRNA will form
complementary base pairs and gene expression will be
inhibited for a time.
ENCAPSULATED PROTEIN CRYSTALS
Example 43
Large Scale Crystallization of
Pseudomonas cepacia Lipase
A slurry of 15 kg crude Pseudomonas cepacia
lipase (PS 30 lipase - Amano) ("LPS") was dissolved in
100 L distilled deionized water and the volume brought
to 200 L with additional distilled 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 ~m filter to remove celite. The mixture
was then ultrafiltered and concentrated to 10 L (121.4
g) using a 3K hollow fiber filter membrane cartridge.
Solid calcium acetate was added to a concentration of
20 mM Ca(CH3C00)2. The pH was adjusted to 5.5 with
concentrated acetic acid, as necessary. The mixture
was heated to and maintained at a temperature of 30°C.
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Magnesium sulfate was added to a 0.2 M concentration,
followed by glucopon to a to concentration.
Isopropanol was then added to a final concentration of
23%. The resulting solution was mixed for 30 minutes
at 30°C, then cooled from 30°C to 12°C over a 2 hour
period. Crystallization was then allowed to proceed
for 16 hours .
The crystals were allowed to settle and
soluble protein was removed using a peristaltic pump
with tygon tubing having a 10 ml pipette at its end.
Fresh crystallization solution (23o isopropyl alcohol,
0.2 M Mg509, 1% glucopon, 20 mM Ca (CH3C00) 2, pH 5. 5) was
added to bring the concentration of protein to 30 mg/ml
(O.D. 280 of a 1 mg/ml solution = 1.0, measured using a
spectrophotometer at wavelength 280). The crystal
yield was about 120 grams.
Crosslinked LP5 Crystals
Crosslinked Pseudomonas cepacia lipase
crystals, sold under the name ChiroCLEC-PC''~'", are
available from Altus Biologics, Inc. (Cambridge,
Massachusetts) were used to produce formulations
according to Example 48. Alternatively, lipase
crystals as prepared above may be crosslinked using any
conventional method.
Example 44
Crosslinkina of Glucose Oxidase Crystals
We then crosslinked the glucose oxidase
crystals prepared in Example 21 as follows. The
crosslinking procedure involved glutaraldehyde, or
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glutaraldehyde pretreated with either Tris buffer (2-
amino-2-hydroxymethyl-1,3-propanediol), lysine or
diaminoor_tane .
The crosslinking was performed using 60 mg of
protein. Tris pretreated glutaraldehyde (48 mg Tris-
base/g glutaraldehyde) was added to concentrations of
0.2 and c).6 g/g GO crystals suspended in 0.2 M sodium
phosphate at pH 7Ø Reactions were allowed to proceed
for two hours at room temperature. After two hours,
the crystals were filtered and washed over glass fiber
paper.
The crosslinked crystals were encapsulated as
described in Example 48. No differences were
encountered in the encapsulation process between the
variously crosslinked crystals. A representative
sample is shown in Figure 21.
Example 45
Crosslinked Candida Ruaosa Lipase Crystals
Crosslinked Candida ruaosa lipase crystals,
sold under the name ChiroCLEC-CR't", are available from
Altus Biologics, Inc. (Cambridge, Massachusetts) and
were used to produce formulations according to Example
48. Alternatively, lipase crystals as prepared above,
may be crosslinked using any conventional method.
Example 46
Crosslinkina of Human Serum Albumin Crystals
Crosslinking was performed on the human serum
albumin crystals prepared in Example 10. The
crosslinking reaction was performed at 4°C in a stirred
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solution of crystals in the mother liquor containing
50o saturated ammonium sulfate. The crystals were not
washed prior to crosslinking with borate-pretreated
glutaraldehyde.
Pretreated glutaraldehyde was prepared by
adding one volume of 50o glutaraldehyde ("GA") to an
equal volume of 300 mM sodium borate at pH 9. The
glutaraldehyde solution was then incubated at 60°C for 1
hour. The solution was cooled to room temperature and
the pH was adjusted to 5.5 with concentrated HC1.
Next, the solution was rapidly cooled on ice to 4°C.
The pretreated glutaraldehyde (25°) was added
to the crystallization solution in a stepwise fashion,
using 0.050 increments (total concentration) at 15
minute intervals to a concentration of 20. Aliquots of
the crystallization solution used ranged between 1 ml
and 500 ml volume. The crystals were then brought to
5% GA and incubated at 4°C for 4 hours to allow
crosslinking. Finally, albumin crosslinked crystals
were collected by low speed centrifugation and washed
repeatedly with pH 7.5, 100 mM Tris HC1. Washing was
stopped when the crosslinked crystals could be
centrifuged at high speed without aggregation.
Example 47
Crosslinkina of Penicillin Acvlase
Pretreated glutaraldehyde was prepared by the
method of Example 46.
The pretreated glutaraldehyde (25%) was added
to the crystallization solution in a stepwise fashion,
using 0.05 increments (total concentration) at 15
minute intervals to a concentration of 1.5~. Aliquots
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of the crystallization solution used ranged between 1
ml and 500 ml volume. Finally, crosslinked crystals
were col:Lected by low speed centrifugation and washed
repeatedly with pH 7.5, 100 mM Tris HC1. Washing was
stopped when the crosslinked crystals could be
centrifuged at high speed without aggregation.
Example 48
Microencapsulation of Protein Crystals in
Polylactic-co-crlycolic acid (PLGA)
A. G1ycoproteins, Proteins, Enzymes
Hormones, Antibodies and Peptides
Microencapsulation was performed using
uncrosslinked crystals of lipase from Candida rugosa
and Pseudomonas cepacia, glucose oxidase from
Aspergillus niger and Penicillin acylase from
Escherichia coli. Further, microencapsulation was
performed using crosslinked enzyme crystals of lipase
from Candida rugosa, glucose oxidase from Aspergillus
niger and Penicillin acylase from Escherichia coli.
Table 13 shows the approximate average diameters of
samples of the microspheres which were produced by this
example. In addition, human serum albumin or any other
protein crystals or protein crystal formulation
produced may be encapsulated by this technique.
Table 13. Microspheres Produced
Crosslinked Crosslinked
Microspheres Crystals Crystals
Diameter /.cm Diameter /.cm
Candida rugosa lipase 90 90
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Glucose oxidase 50 50
Penicillin acylase 90 70
Lipase from Pseudomonas 60
cepacia (slurry)
B. Preparation of Dry Crystals:
Crystals or crystal formulations dried
according to Example 6 may each be used to produce the
microspheres of this invention. One process for drying
protein crystals for use in this invention involves air
drying.
Approximately 500 mg each of Candida rugosa
lipase crystals from Example 1 (uncrosslinked and
crosslinked), glucose oxidase from Examples 21 and 44
(uncrosslinked and crosslinked) and Penicillin acylase
from Examples 14 and 47 (uncrosslinked and crosslinked)
were air dried. First, the mother liquor was removed
by centrifugation at 3000 rpm for 5 minutes. Next, the
crystals were at 25°C in the fume hood for two days.
C. Polymer and Solvents
The polymer used to encapsulate the protein
crystals was PLGA. PLGA was purchased as 50/50
Poly(DL-lactide-co-glycolide) from Birmingham Polymers,
Inc. from Lot No. D97188. This lot had an inherent
viscosity of 0.44 dl/g in HFIP@ 30°C.
The methylene chloride was spectroscopic
grade and was purchased from Aldrich Chemical Co.
Milwaukee, WI. The poly vinyl alcohol was purchased
from Ald:rich Chemical Co. Milwaukee, WI.
D. Encapsulation of Crystals in PLGA:
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The crystals were encapsulated in PLGA using
a double emulsion method. The general process was as
follows, either dry protein crystals or a slurry of
protein crystals was first added to a polymer solution
in methylene chloride. The crystals were coated with
the polymer and became nascent microspheres. Next, the
polymer in organic solvent solution was transferred to
a much larger volume of an aqueous solution containing
a surface active agent. As a result, the organic
solvent began to evaporate and the polymer hardened.
In this example, two successive aqueous solutions of
decreasing concentrations of emulsifier were employed
for hardening of the polymer coat to form microspheres.
The following procedure was one exemplification of this
general process. Those of skill in the art of polymer
science will appreciate that many variations of the
procedure may be employed and the following example was
not meant to limit the invention.
1.0 Use of Dry Protein Crystals
Dry crystals of crosslinked and uncrosslinked
Candida rugosa lipase produced according to Example l,
crosslinked and uncrosslinked glucose oxidase produced
according to Examples 21 and 45, crosslinked and
uncrosslinked penicillin acylase produced according to
Example 14 and 47, were weighed into 150 mg samples.
The weighed protein crystals were then added directly
into a 15 ml polypropylene centrifuge tube (Fisher
Scientific) containing 2 ml of methylene chloride with
PLGA at 0.6 g PLGA/ml solvent. The crystals were added
directly to the surface of the solvent. Next, the tube
was throughly mixed by vortexing for 2 minutes at room
temperature to completely disperse the protein crystals
in the solvent with PLGA. The crystals were allowed to
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become completely coated with polymer. Further
vortexing or agitation may be used to keep the nascent
microspheres suspended to allow further coating. The
polymer may be hardened as described in section 3Ø
2.0 Use of a Protein Crystal Slurry
A crystal slurry of Pseudomonas cepacia
lipase was produced using approximately 50 mg of
crystals per 200 ~1 of mother liquor. The crystal
slurry was rapidly injected into a 15 ml polypropylene
centrifuge tube (Fisher Scientific) with 2 ml of a
solution of methylene chloride and poly(lactic-co-
glycolic acid) at 0.6 g PLGA/ml solvent. The needle
was inserted below the surface of the solvent and
injected into the solution. In this case, 150 mg of
total protein, or 600 ~cl of aqueous solution, was
injected. The injection was made using a plastic
syringe Leur-lok (Becton-Dickinson & Company) and
through a 22 gauge (Becton-Dickinson & Company)
stainless steel needle. Next, the protein crystal-PLGA
slurry was mixed thoroughly by vortexing for 2 minutes
at room temperature. The crystals were allowed to be
completely coated with polymer. Further vortexing or
agitation was optionally used to keep the nascent
microspheres suspended to allow further coating.
3.0 Hardening the Polymer Coating
A two step process was employed to facilitate
the removal of methylene chloride from the liquid
polymer coat and allow the polymer to harden onto the
protein crystals. The difference between the steps is
that the concentration of emulsifier was much higher in
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the first solution and the volume of the first solution
is much smaller than the second.
In the first step, the polymer coated crystal
and methylene chloride suspension was added dropwise to
a stirred flask of 180 ml of 5°s polyvinyl alcohol
(hereinafter "PVA") in water with 0.5~ methylene
chloride at room temperature. This solution was mixed
rapidly for 1 minute.
In step two, the first PVA solution
containing the nascent microspheres was rapidly poured
into 2.4 liters of cold (4°C) distilled water. This
final bath was mixed gently at 4°C for 1 hr with the
surface of the solution under~nitrogen. After 1 hr,
the microspheres were filtered using 0.22 ~m filter and
washed with 3 liters of distilled water containing O.lo
Tween 20 to reduce agglomeration.
Example 49
Production of Encapsulated Crystals
Encapsulated microspheres of Pseudomonas
cepacia lipase are prepared by phase separation
techniques. The crystalline LPS prepared in Example 43
is encapsulated in polylactic-co-glycolic acid ("PLGA")
using a double emulsion method. A 700 mg aliquot of
protein crystals is injected in methylene chloride
containing PLGA (0.6 g PLGA/ml solvents 10 ml). The
mixture is homogenized for 30 sec at 3,000 rpm, using a
homogenizer with a microfine tip. The resulting
suspension is transferred to a stirred tank (900 ml)
containing 6% poly (vinyl alcohol) ("PVA") and
methylene chloride (4.5 ml). The solution is mixed at
1,000 rpm for 1 min. The microspheres in the PVA
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solution are precipitated by immersion in distilled
water, washed and filtered. The microspheres are then
washed with distilled water containing O.lo Tween, to
reduce agglomeration and dried with nitrogen for 2 days
at 4 °C.
Example 50
A. Protein Content of Microspheres
The total protein content of the microspheres
prepared in Example 98 was measured. Triplicate
samples of 25 mg of the PLGA/PVA microspheres were
incubated in 1 N sodium hydroxide with mixing for 48
hrs. The protein content was then estimated using
Bradford's method (M. M. Bradford, Analytical
Biochemistry, vol. 72, page 248-254 (1976)) and a
commercially available kit from BioRad Laboratories
(Hercules, CA). The protein containing microspheres
were compared to PLGA microspheres without any crystals
and is shown in Table 14.
Table 14. Protein Content of Microspheres
Protein (s) Protein (o)
Microspheres Crosslinked Uncrosslinked
Crystals Crystals
Unloaded PLGA 0
Microspheres
Candida rugosa lipase 30 20
Glucose oxidase 35 28
Penicillin acylase 27 25
Lipase from Pseudomonas 39
cepacia (slurry)
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The activity per milligram or specific
activity of selected samples from Table 14 was
determined and is shown in Table 15.
Table 15. Specific Activity
Activity/mg Activity/mg
Microspheres Crosslinked Uncrosslinked
Crystals Crystals
Unloaded PLGA 0 0
Microspheres
Candida rugosa lipase 413
Penicillin acylase 9.63
Lipase from Pseudomonas 1414
cepacia (slurry)
Example 51
Protein Release from Microspheres
The release of protein from the PLGA
microspheres prepared in Example 48 was measured by
placing 50 mg of protein encapsulated PLGA microspheres
in microcentrifuge filtration tubes containing 0.22 ~m
filters. Next, 600 ul of release buffer (phosphate
buffered saline with 0.020 Tween 20 at pH 7.4) was
added to the microspheres on the retentate side of the
filter. The tubes were incubated at 37°C to allow
dissolution. To measure the amount of protein released
with time, samples were taken at different time
intervals. The tube was centrifuged at 3000 rpm for 1
minute and the filtrate was removed for protein
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activity and total protein measurements. The
microspheres were then resuspended with another 600 ul
of release buffer.
A. Total Protein Released From Micros~heres
Table 16 shows that almost 90% of the input
protein was recovered in all cases. In addition, the
percentage of total Pseudomonas cepacia lipase released
from the microencapsulated protein crystals was
relatively constant for approximately 5.7 days or until
more than 80% of the input protein had been released at
a rate of 15.8%/day. This long rapid release was
followed by eight days with a only 0.6% release per
day.
In contrast, Table 16 further shows that
Candida :rugosa lipase crystals displayed the opposite
profile, displaying first a slow release which was
followed by a rapid release phase. In the first three
days, about 10% of the protein was released with a
shallow slope of 2.4%/day. From day 4 to day 14,
another 80% of the protein was released in a linear
fashion and a slope of 7.5%/day. The release profiles
shown in Table 16 were obtained at 37°C and at pH 7.4.
These data illustrate that the encapsulated
proteins of this invention are suitable for biological
delivery of therapeutic proteins. Various rates of
delivery can be selected by manipulating the choice of
protein crystal, size of the crystals, crosslinking of
the crystals, the hydrophobic and hydrophilic
characteristics of the encapsulating polymer, the
number of encapsulations, dose of microspheres and
other easily controllable variables.
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Table 16. Protein Release From Microspheres
Time (hr) o Input Pseudomonas o Input Candida
cepacia Lipase rugosa lipase
Released Released
0 0 0
18 28 2
41 56 5
89 75 10
137 82 22
210 84 34
239 85 47
306 86 70
330 87 86
B. Protein Activity Released From
Microspheres
The biological activity of the protein
released with time was measured using the olive oil
assay for lipase microspheres. These results are shown
in Table 17.
The biological activity of the released
protein, as shown in Table 17, demonstrates that the
microspheres protect and release active protein. The
cumulative percent activity released, calculated based
on the amount of input protein, was closely correlated
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with the total protein released (compare Table 16 and
Table 17). The two different crystal lipases released
essentially 100 active protein. Even after 7 days of
immersion at 37°C, the protein that was released from
the microspheres was fully active.
Table 17. Activity of Released Protein
Time (hr) o Input Pseudomonas Input Candida
cepacia Lipase Rugosa lipase
Activity Released Activity Released
0 0 0
18 28 2
41 56 5
89 75 10
137 82 22
210 84 34
234 85 47
306 86 70
330 87 86
The activity measurements set forth above
were made using the olive oil assay described in
Example 8.
Example 52
Microscopic Examination of PLGA Microspheres
In order to visualize whether the crystals
were intact after encapsulation, PLGA microspheres
prepared according to Example 48 were examined under an
Olympus (3X60 microscope equipped with DXC-970MD 3CCD
Color Video Camera with Camera Adapter (CMA D2) with
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Image ProPlus software. Samples of dry microspheres
were covered with a glass coverslip, mounted and
examined under 10X magnification, using an Olympus
microscope with an Olympus UPLAN F1 objective lens
10X/0.30 PH1 (phase contrast), the crystals were
readily visualized and the crystal size determined.
Microsphere and crystal sizes were determined using
Image Pro Software from Olympus and 0.5-150 ~cm sizing
beads provided by the manufacturer. The size of the
outer PLGA microspheres was determined, as well as for
the crystals.
Figure 17 depicts crosslinked enzyme crystals
of lipase from Candida rugosa encapsulated by the
method of Example 48. The crystal size was
approximately 25 ~m and the microspheres were
approximately 90 /,cm. The magnification was 250X.
Figure 18 depicts uncrosslinked enzyme
crystals of lipase from Candida rugosa encapsulated by
the method of Example 48. The crystal size was
approximately 25 ~m and the microspheres were
approximately 120 ,um. The magnification was 250X.
Figure 19 depicts crosslinked enzyme crystals
of Penicillin acylase from Escherichia coli
encapsulated by the method of Example 48. The crystal
size was approximately 25 /cm and the microspheres were
approximately 70 /.cm. The magnification was 250X.
Figure 20 depicts uncrosslinked enzyme
crystals of Penicillin acylase from Escherichia coli
encapsulated by the method of Example 48. The crystal
size was approximately 50 ~m and the microspheres were
approximately 90 Vim. The magnification was 250X.
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Figure 21 depicts crosslinked enzyme crystals
of glucose oxidase from Aspergillus niger encapsulated
by the method of Example 48. The crystal size ranged
from 0.5 to 1 ~m and the microspheres were
approximately 50 ~cm. The magnification was 500X.
Figure 22 depicts uncrosslinked enzyme
crystals of glucose oxidase from Aspergillus niger
encapsulated by the method of Example 48. The crystal
size ranged from 0.5 to 1 /.cm and the microspheres were
approximately 50 Vim. The magnification was 500X.
Figure 23 depicts uncrosslinked enzyme
crystals of lipase from Pseudomonas cepacia,
encapsulated as a slurry in the mother liquor by the
method of Example 48. The crystal size was
approximately 2.5 ~m and the microspheres were
approximately 60 /.cm. The magnification was 500X.
Figure 24 depicts uncrosslinked enzyme
crystals of lipase from Pseudomonas cepacia. The
crystal size was approximately 2.5 ,um. The
magnification was 1000X.
Example 53
Protein Release
The release of proteins from the PLGA
microspheres is measured by placing 50 mg of PLGA
microspheres in micro-centrifuge filtration tubes
containing 0.22 ,um filters. A 600 ul aliquot of
release buffer (10 mM HEPES, pH 7.4, 100 mM NaCl, 0.020
Tween, 0.020 azide) is added to suspend the
microspheres on the retentate side of the filter. The
tubes are sealed with 3 cc vial stoppers and covered by
parafilm. The microspheres are then incubated at 37°C.
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Samples are taken over time by centrifugation (13,000
rpm, 1 min) of the tubes. The filtrate is removed and
the microspheres are resuspended with 600 ul of the
release buffer. The quality of the released protein is
assayed by SEC-HPLC and enzymatic activity.
The shape and size of the protein crystals
may be chosen to adjust the rate of dissolution or
other properties of the protein crystal formulations of
this invention.
Example 54
Encapsulation of Lipase Crvstals Using a
Biological Polymer
Biological polymers are also useful for
encapsulating protein crystals. The present example
demonstrates encapsulation of crosslinked and
uncrosslinked crystals of Candida rugosa lipase
crystals. The uncrosslinked and crosslinked crystals
were prepared as described in Example 1 and 45.
Antibodies and chemicals were purchased from Sigma.
1.0 Preparation of coated crystals
A solution of 1.5 ml of bovine serum albumin
("BSA") at 10 mg/ml was prepared, in 5 mM phosphate
buffer adjusted to pH 7. Next, 15 ml of a 10 mg/ml
suspension of Candida rugosa lipase crystals was
prepared in 5 mM K/Na phosphate buffer, 1 M NaCl, at pH
7 ("buffer"). The BSA solution was added to the
crystal solution and the two solutions were mixed
thoroughly. The crystals were incubated in the BSA for
min with slow mixing using an orbital shaker.
30 Following the incubation with BSA, the crystals were
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dryed overnight by vacuum filtration. The dryed
crystals were resuspended in buffer without albumin.
The crystals were washed with buffer until no protein
could be detected in the wash as measured by absorbance
at280nm or until the A28onm was < 0.01. The crystals
were recovered by low speed centrifugation.
2.0 Detection of the Albumin Coat
The coated crystals were evaluated by Western
blotting to confirm the presence of the albumin layer
Following washing, coated protein crystals were
incubated in 100 mM NaOH overnight to dissolve the
microspheres into the constituent proteins. The
samples were neutralized, filtered and analyzed by
SDS-PAGE immunoblot according to Sambrook et al.
"Molecular Cloning: A Laboratory Manual", Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York
(1989) .
The results of SDS-PAGE immunoblot of both
albumin coated crosslinked and uncrosslinked crystal
microspheres of Candida rugosa lipase revealed a single
immunoreactive species having the same molecular weight
as albumin.
Samples of the albumin coated crosslinked and
uncrosslinked crystal microspheres of Candida rugosa
lipase were incubated with a fluorescene-labeled
anti-BSA antibodies which specifically recognize and
bind to bovine serum albumin. Next, excess antibody
was removed thorough washing with phosphate buffer.
Microscopic examination of these fluorescently labeled
albumin coated crystal microspheres under a fluorscent
microscope revealed specific fluorescene-labeling of
the microspheres. Uncoated lipase crystals were used
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as control and these showed no specific binding of the
antibody.
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.
