TECHNICAL FIELD
The present invention relates to a protein composition
which comprises specific amino acids and/or a cellulose ether
derivative and has resistance to radiation sterilization.
10 BACKGROUND ART
Natural and synthetic proteins are becoming more and
more important as drugs. When they are used for medical
applications, their products must be sterilized. As means
of sterilization, there are known heat sterilization in an
15 autoclave, sterilization with ionizing radiation such as a
y ray or electron beam, gas sterilization with an ethylene
oxide gas, plasma sterilization with hydrogen peroxide, and
separate sterilization using a chemical sterilant comprising
a glutaraldehyde formulation or a filter. However, the
20 activities of proteins such as bioactive proteins are reduced
by sterilization with heat or radiation. Sterilization with
ethylene oxide has possibilities that a by-product may be
produced by a chemical reaction and that a highly toxic
residual gas may adversely affect the human body.
25 Sterilization with a chemical sterilant has a problem that
the resistance to a sterilant of a protein and changes in
pH, ion intensity and temperature must be taken into
consideration. Then, to manufacture pharmaceuticals and
medical products containing or immobilizing a protein, their
30 production processes must be entirely made in sterile
conditions and a huge amount of production cost is required.
Although a solution containing a protein is subjected
to separate sterilization with a filter, it is difficult to
apply this separate sterilization to a composition
*•
2
containing large particles or a solid or semisolid
composition.
EP0437095 teaches that a neutralized oxidized
cellulose product combined with heparin or a heparin fragment
5 (nORC) can be sterilized by gamma-ray irradiation. However,
this document fails to teach the sterilization of ORG or n-ORC
to which a protein is bound.
EP0562864 discloses a composite wound care substance
containing a collagen sponge matrix, a second bioabsorbable
10 polymer (such as an oxidized regenerated cellulose (ORC)
dispersed fiber) and an activator (such as peptide) . This
document teaches that the activator may be contained in the
matrix, the bioabsorbable polymer or both of them and that
the composite sponge substance can be sterilized while it
15 is packaged.
US5730933 discloses a method of sterilizing
biologically active peptide by gamma-ray or electron-beam
irradiation without the loss of the biological activity of
the peptide. This method is a technology comprising the
20 steps of forming a mixture of biologically active peptide
and a foreign protein such as gelatin, freezing or
lyophilizing this mixture, and irradiating it. This
document teaches that the existence of the foreign protein
stabilizes peptide and prevents the reduction of the activity
25 of peptide.
WO2000/033893 discloses a complex of therapeutic
peptide and a polysaccharide selected from the group
consisting of oxidized regenerated cellulose, neutralized
oxidized regenerated cellulose and mixtures thereof. This
30 document teaches that when peptide is formulated together
with an effective amount of the polysaccharide before
sterilization with ionizing radiation, the biological
activity of the peptide therapeutic agent is not lost and
is stabilized if peptide is sterilized with ionizing
3
radiation.
However, these documents do not suggest that the
deactivation of a protein at the time of sterilizing with
ionizing radiation can be suppressed by making a cellulose
5 ether derivative and specific amino acids coexistent with
the protein.
Meanwhile, JP-A 2011-47089 discloses a process for
producing an enzyme-containing nanofiber having excellent
enzyme activity. In this process, a spinning solution
10 containing an enzyme and a polymer dissolved in a nonaqueous
solvent is spun by an electrostatic spinning method to form
a zymogen nanofiber which is then imparted with water and
dried. However, this document is silent about the
sterilization of the enzyme-containing nanofiber.
15
DISCLOSURE OF THE INVENTION
It is an object of the present invention to provide
a protein composition having resistance to radiation
sterilization.
20 The inventors of the present invention conducted
intensive studies to solve the above problem and found that,
surprisingly, the resistance to radiation sterilization of
a protein is improved by making a mixture of glycine,
phenylalanine and histidine and/or a cellulose ether
25 derivative coexistent with the protein. The present
invention was accomplished based on this finding.
That is, the present invention is a protein composition
which comprises a mixture of glycine, phenylalanine and
histidine and/or a cellulose ether derivative as an additive.
30
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the sterilization resisting effect for
a protein of a combination of a cellulose ether derivative
and specific additives of the present invention (axis of
4
ordinate: gel intensity relative value {before
sterilization: 100) ) ; and
Fig. 2 shows the sterilization resisting effect of a
combination of a cellulose ether derivative and specific
5 additives of the present invention (axis of ordinate: an
increase in the amount of a protein aggregate (%)).
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention is a protein composition which
10 comprises a mixture of glycine, phenylalanine and histidine
and/or a cellulose ether derivative as an additive.
The protein used in the present invention is not
particularly limited. Examples of the protein include
hemostat proteins typified by fibrinogen and thrombin,
15 enzymes typified by asparaginase, catalase, superoxide
dismutase and lipase, transport proteins typified by
hemoglobin, serum albumin and low density lipoprotein,
muscle proteins typified by actin and myosin, defense
proteins typified by antibodies and complements, toxin
20 proteins typified by diphtheria toxin, botulinum toxin and
snake venom, protein hormones typified by insulin, growth
factors and cytokine, storage proteins typified by ovalbumin
and ferritin, structural proteins typified by collagen and
keratin, and growth factors typified by epidermal growth
25 factor (EGF), insulin-like growth factor (IGF), transforming
growth factor (TGF), nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF), vascular
endothelial growth factor (VEGF), granulocyte-colony
stimulating factor (G-CSF), granulocyte-macrophage-colony
30 stimulating factor (GM-CSF)-, platelet-derived growth factor
(PDGF), erythropoietin (EPO), thrombopoietin (TPO), basic
fibroblast growth factor (bFGF or FGF2) and hepatocyte growth
factor (HGF). Out of these, hemostat proteins, enzymes,
transport proteins, muscle proteins, defense proteins, toxin
5
proteins, protein hormones, storage proteins, structural
proteins and growth factors are preferred, and fibrinogen
is particularly preferred.
The protein used in the present invention may be of
5 animal origin or manufactured by a genetic recombination
technique. If it is of animal origin, it is preferably of
human origin. The protein manufactured by the genetic
recombination technique may be a variant obtained by
replacing the amino acid sequence by another amino acid
10 sequence if the essential bioactivity is the same. Proteins
obtained by modifying these proteins and mixtures thereof
may also be used.
To the protein used in the present invention, a
stabilizer and an additive which are pharmaceutically
15 acceptable (to be referred to as "stabilizer, etc."
hereinafter and distinguished from the additive which is made
coexistent with the protein to provide resistance to
radiation sterilization in the present invention) may be
added. Preferred examples of the stabilizer, etc. include
20 arginine, isoleucine, glutamic acid, citric acid, calcium
chloride, sodium chloride, protease inhibitors {such as
aprotinin), albumin, surfactants, phospholipids,
polyethylene glycol, sodium hyaluronate, glycerin,
trehalose and sugar alcohols (such as glycerol andmannitol) .
25 At least one selected from arginine, sodium chloride,
trehalose, mannitol and citric acid is preferred, and citric
acid is particularly preferred.
A mixture of the protein and the stabilizer, etc. used
in the present invention contains the protein in an amount
30 of not more than 35 parts by weight, preferably not more than
30 parts by weight based on 100 parts by weight of the mixture.
When the additive used in the present invention is a
mixture of glycine, phenylalanine and histidine, the content
of glycine is generally 5 to 90 parts by weight, preferably
6
15 to 60 parts by weight, more preferably 20 to 40 parts by
weight, the content of phenylalanine is generally 1 to 80
parts by weight, preferably 2 to 40 parts by weight, more
preferably 4 to 20 parts by weight, and the content of
5 histidine is generally 2 to 70 parts by weight, preferably
5 to 40 parts by weight, more preferably 8 to 20 parts by
weight based on 100 parts by weight of the total of the
additive and the protein.
When the additive in the present invention is a
10 cellulose ether derivative, the protein or a mixture of the
protein and the stabilizer, etc. used in the present
invention may be supported on the cellulose ether derivative
but preferably contained in the cellulose ether derivative
(the word "contained" refers to a state that at least part
15 of the protein enters the inside of the cellulose ether
derivative) . In this case, the molecules of the protein and
the stabilizer, etc. may be dispersed in the cellulose ether
derivative but preferably as particles formed by the
aggregation of the molecules of the protein and the
20 stabilizer, etc. (may be referred to as "protein particles"
including mixed particles with the stabilizer, etc.)
This preferred existence of the protein or the protein
particles in the cellulose ether derivative remains
unchanged when the additive is only the cellulose ether
25 derivative and also when the additive consists of a mixture
of glycine, phenylalanine and histidine and the cellulose
ether derivative.
Examples of the cellulose ether derivative used in the
present invention include hydroxypropyl cellulose, methyl
30 cellulose, hydroxyethyl cellulose, hydroxypropylmethyl
cellulose, sodium carboxymethyl cellulose and mixtures
thereof.
One selected from the group consisting of
hydroxypropyl cellulose, hydroxyethyl cellulose,
7
hydroxypropylmethyl cellulose and mixtures thereof is
preferred, and hydroxypropyl cellulose is most preferred.
Although the molecular weight of the cellulose ether
derivative used in the present invention is not particularly
5 limited, when viscosity measurement is carried out at a
concentration of 2 % and 20°C, a molecular weight which
exhibits a viscosity of 1 to 10,000 mPa*s, preferably 2 to
5,000 mPa's, more preferably 2 to 4,000 mPa*s is selected.
In the protein composition of the present invention,
10 another polymer or another compound may be used in
combination as long as the object of the present invention
is not impaired.
The cellulose ether derivative used in the present
invention preferably has high purity. Especially, the
15 contents of additives and plasticizer contained in the
cellulose ether derivative and residues such as residual
catalyst, residual monomers and residual solvent used for
molding and post-processing are preferably as low as possible.
Especially when the composition is used for medical purposes,
20 it is necessary to reduce these contents to values below
safety standards.
The form of the protein composition of the present
invention is not limited to a particular form including an
indeterminate form, and the composition may be in the form
25 of a film, fiber, sheet, plate-like body, tube-like body,
linear body, rod-like body, cushion material, foam or porous
body. The molding method for producing a molded product is
not particularly limited if it is a method in which the
activity of the protein is not reduced. For example,
30 suitable molding techniques such as extrusion molding,
injection molding, calender molding, compression molding,
blow molding, vacuum forming, powder molding, cast molding
and casting may be employed. The protein composition of the
present invention is suitable for the production of films
8
and fibers. The fiber form as used herein refers to a 3-D
molded body formed by the lamination, weaving, knitting or
another technique of one or a plurality of fibers. The fiber
form is, for example, a nonwoven fabric. Further, a tube
5 and a mesh obtained by processing the nonwoven fabric are
included in the fiber form.
For the production of these, any one of techniques which
have been employed for the production of films or plastic
fibers may be employed. For example, extrusion molding
10 techniques such as casting, electrospinning, inflation
extrusion molding and T die extrusion molding, and
calendering technique may be used. The above molding may
be solution molding or melt molding, out of which solution
molding is preferred in order to facilitate the dispersion
15 of the protein or the protein particles so as to prevent the
functional deterioration of the protein.
The process for producing the protein composition
having a film form out of the present invention will be
explained, taking the casting technique as an example.
20 Protein particles having an average particle diameter
(generally 0.1 to 200 urn, preferably 1 to 100 urn) suitable
for dispersion in a solvent are prepared by pounding
lyophilized protein powders in a mortar. After the protein
particles are dispersed in one or more suitable solvents
25 (such as 2-propanol and ethanol) which can dissolve the
cellulose ether derivative, can form a suspension with the
protein particles and evaporate in the film forming step to
form a film, the cellulose ether derivative and further
optionally a plasticizer such as MACROGOL are dissolved in
30 the resulting dispersion so as to prepare a dope solution
containing the protein particles dispersed in the cellulose
ether derivative solution. A film is formed by the casting
technique using the obtained dope solution.
The protein composition having a film form out of the
9
present invention comprises the protein or the protein
particles .in an amount of generally not less than 100 wt%,
preferably not less than 500 wt%, more preferably 800 to 950
wt% based on the cellulose ether derivative though this
5 depends on the type of the protein and the type of the
cellulose ether derivative. When the content of the protein
or the protein particles falls below the above range, the
function of the protein may not be obtained fully and when
the content exceeds the above range, film ^voidability may
10 become unsatisfactory.
The average thickness of a film of the protein
composition having a film form out of the present invention
which differs according to the intended use is preferably
10 to 1,000 um.
15 The average fiber diameter of the protein composition
having a fiber form out of the present invention is, for
example, 0.01 to 50 um and may be suitably determined by a
person skilled in the art according to the intended use. The
protein composition may be in the form of a long fiber. The
20 long fiber is a fiber formed without adding the step of cutting
a fiber in the course of transition from spinning to the
processing of a fiber molded body. It can be formed by
electrospinning, span bonding and melt blowing methods . Out
of these, the electrospinning method is preferred as the long
25 fiber can be molded without adding heat and the functional
deterioration of the protein can be suppressed.
The electrospinning method is a method in which a fiber
molded body is obtained on an electrode by applying a high
voltage to a solution containing a polymer. This process
30 comprises the steps of preparing a spinning solution
containing a polymer, applying a high voltage to the solution,
jetting the solution, forming a fiber molded body by
evaporating the solvent from the jetted solution,
eliminating the charge of the formed fiber molded body as
10
an optional step, and accumulating the fiber molded body by
the charge loss.
A description is subsequently given of the process for
producing the protein composition having a fiber form or a
5 nonwoven fabric form out of the present invention, taking
the electrospinning method as an example.
The step of preparing a spinning solution in the
electrospinning method will be explained. A suspension of
a cellulose ether derivative solution and protein particles
10 is preferably used as the spinning solution in the present
invention.
The concentration of the cellulose ether derivative
in the suspension is preferably 1 to 30 wt%. When the
concentration of the cellulose ether derivative is lower than
15 1 wt%, it is difficult to form a fiber molded body
disadvantageously. When the concentration is higher than
30 wt%, the fiber diameter of the obtained fiber molded body
becomes large and the viscosity of the suspension becomes
high disadvantageously. The concentration of the cellulose
20 ether derivative in the suspension is more preferably 1.5
to 20 wt%.
The solvent for the cellulose ether derivative is not
particularly limited if it can dissolve the cellulose ether
derivative, forms a suspension with the protein particles
25 and evaporates in the spinning step so that a fiber can be
formed. Only one solvent or a combination of two or more
solvents may be used. Examples of the solvent include
chloroform, 2-propanol, toluene, benzene, benzyl alcohol,
dichloromethane, carbon tetrachloride, cyclohexane,
30 cyclohexanone, trichloroethane, methyl ethyl ketone, ethyl
acetate, acetone, ethanol, methanol, tetrahydrofuran,
1,4-dioxane, 1-propanol, phenol, pyridine, acetic acid,
formic acid, hexafluoro-2-propanol, hexafluoroacetone,
N,N-dimethylformamide, N,N-dimethylacetamide,
11
acetonitrile, N-methy1-2-pyrrolidinone,
N-methylmorpholine-N^oxide, 1,3-dioxolan, water and
mixtures thereof. Out of these/ 2-propanol or ethanol is
preferably used from the viewpoints of handling ease and
5 physical properties.
Although the method of preparing a suspension by mixing
together the cellulose ether derivative solution and the
protein particles is not particularly limited, ultraviolet
waves or stirring means may be used. As the stirring means,
10 high-speed stirring means such as a homogenizer or stirring
means such as an attriter or ball mill may be used. Out of
these, dispersion with ultrasonic waves is preferred.
Also, the spinning solution may be prepared by adding
the cellulose ether derivative after a suspension is formed
15 from a solvent and the protein particles.
Before the preparation of the suspension, protein
particles may be microfabricated. For microfabrication,
there are dry milling and wet milling both of which may be
employed and also may be combined in the present invention.
20 Dry milling is carried out by milling with a ball mill,
planetary mill or oscillating mill, by pounding in a mortar
with a pestle, or by grinding with a medium stirring type
pulverizer, jet mill or stone mill.
Meanwhile, wet milling is carried out by stirring with
25 a stirrer or kneader having high shear force while the protein
particles are dispersed in a suitable dispersion medium, or
by using a ball mill or bead mill while the protein particles
are dispersed in a medium.
Further, protein particles produced by a spay drier
30 may also be used.
In the protein composition having a fiber form or a
nonwoven fabric form out of the present invention, the sizes
of the protein particles are not particularly limited but
preferably 0.01 to 100 urn. It is technically difficult to
12
manufacture protein particles having a particle size smaller
than 0.01 urn, and when the particle size is larger than 100
urn, dispersibility degrades and the fiber molded body becomes
brittle disadvantageously.
5 The sterilization method used for the protein
composition of the present invention is radiation
sterilization. Examples of the radiation include alpha rays,
beta rays, gamma rays, neutron rays, electron beams and
X-rays. Out of these, gamma rays and electron beams are
10 preferred, and electron beams are most preferred. Although
the sterilization method is not particularly limited, the
dose of the radiation is 10 to 80 kGy, preferably 20 to 30
kGy. Although the temperature condition is not particularly
limited, it is -80 to 40°C, preferably -80 to 30°C.
15 The radiation such as alpha rays, positron, gamma rays,
neutron rays, electron beams or X-rays strips an electron
off from*molecules or atoms constituting a substance when
it is applied to the substance. A molecular bond is broken
upon this, and a highly reactive radical is produced and
20 chemically reacts with a surrounding substance secondarily.
It is well known that a protein tends to lose its
function (activity) upon exposure to radiation. This is
considered to be due to the destruction of "a high-order
structure" which is a source of developing a function by the
25 breakage of a molecular bond by exposure. However, the
functional deterioration of the protein composition of the
present invention is suppressed even when it is exposed to
radiation. This means that the high-order structure of the
protein is retained in the composition of the present
30 invention, which is a common effect regardless of the type
of the protein. It is not considered from the thickness of
the cellulose ether derivative through which the radiation
is transmitted that this effect is due to screening, and the
control mechanism is not known. The mechanism of a
13
phenomenon that the effect of the cellulose ether derivative
is remarkably improved by the addition of specific amino
acids is not known as well.
The protein composition of the present invention may
5 further comprise an electron/ion scavenger, energy transfer
agent, radical scavenger, antioxidant and plasticizer.
Examples of the electron/ion scavenger include
N, N' -tetramethyl phenylenediamine, diphenylenediamine,
pyrene and quinone. Examples of the energy transfer agent
10 include acenaphthene. Examples of the radical scavenger
include mercaptans, octahydrophenanthrene, monoalkyl
diphenyl ethers, tocopherol, citric acid, butylated
hydroxyanisole, butylated hydroxytoluene, t-butyl
hydroquinone, propyl gallate and ascorbic acid derivatives.
15 Examples of the antioxidant include BHT, phosphite triesters,
phenolic antiaging agents and organic thio acid salts,
Additives that are generally accepted as safe for use in foods
and pharmaceuticals are preferred. The amount of the
additive which is not particularly limited is, for example,
20 0.01 to 10 wt% based on the cellulose ether derivative in
the protein composition.
The cellulose ether derivative containing the protein
in the sterilization step preferably contains no water. The
water content of the cellulose ether derivative is preferably
25 not more than 10 wt%, more preferably not more than 4 wt%,
much more preferably substantially 0 wt%.
The protein composition of the present invention may
be wrapped in a packaging material to be sterilized with
radiation. As the packaging material, a material having high
30 gas barrier properties such as aluminum is preferably used.
The protein composition may be hermetically sealed and
packaged together with a deoxidant or desiccant or while an
inert gas is filled into the package after degasification,
or both methods may be combined together. As the deoxidant
14
and the desiccant, ones which do no harm to the human body
and are not deactivated upon exposure to radiation are
preferred.
The protein composition of the present invention may
5 be used as a medical material which requires the function
and sterility of a protein.
The present invention includes a sterile protein
composition obtained by sterilizing the protein composition
of the present invention with radiation.
10
EXAMPLES
The following examples are provided for the purpose
of further illustrating the present invention but are in no
way to be taken as limiting.
15
Measurement methods for Examples 1 to 4 and Comparative
Examples 1 and 2
1. Average fiber diameter:
The diameters of fibers at 20 locations selected at
20 random from a photo of the surface of the obtained fiber molded
body taken by a scanning electron microscope (VE8800 of
Keyence Corporation) at 3,000-fold magnification to obtain
the average value of all the fiber diameters as average fiber
diameter. N = 20.
25
2. Average thickness:
The film thicknesses of 15 fiber molded bodies cut to
a size of 50 mm x 100 mm were measured with a measurement
force of 0.01 N by means of a high-resolution digimatic
30 measuring unit {LITEMATIC VL-50 of Mitutoyo Corporation) to
calculate the average value. This measurement was carried
out with minimum measurement force that could be used by the
measuring unit.
15
3. ELISA measurement
(1) Fibrinogen
10 ug/mL of an antihuman fibrinogen antibody (DAKO
A0080) was immobilized to an ELISA plate (NUNC 468667).
5 After it was washed with PBS containing 0.05 % of Tween 20,
Block Ace (UK-B80 of DS Pharma Biomedical Co. , Ltd.) was added
to each well to carry out masking. After washing with PBS
containing 0.05 % of Tween 20, a test body v/as added. Human
fibrinogen (No. FIB3 of Enzyme Research Laboratories) was
10 used as a standard to form a calibration curve. After washing
with PBS containing 0.05 % of Tween 20, an HRP-labelled
antihuman fibrinogen antibody (CPL5523) was added. After
a reaction, the reaction product was washed with PBS
containing 0.05 % of Tween 20, a TMB reagent (KPL 50-76-02
15 50-65-02) was added, and the resulting mixture v/as left for
6 minutes to develop color. 1 M H3PO4 was added to stop color
development so as to measure OD450-650 nm with a microplate
reader.
(2) Thrombin
20 5 ng/mL of an antihuman thrombin antibody (No. SAHT-AP
of Affinity Biologicals Inc.) was immobilized to an ELISA
plate (NUNC 468667) . After it was washed with PBS containing
0.05 % of Tween 20, Block Ace (UK-B8 0 of DS Pharma Biomedical
Co., Ltd. ) was added to each well to carry out masking. After
25 washing with PBS containing 0.05 % of Tween 20, a test body
v/as added. Human thrombin (HCT-0020 of Haematologic
Technologies, Inc.) was used as a standard to form a
calibration curve. After washing with PBS containing 0.05 %
of Tween 20, 0.1 jj.g/mL of an HRP-labelled antihuman thrombin
30 antibody (No. SAHT-HRP of Affinity Biologicals Inc.) was
added. After a reaction, the reaction product v/as v/ashed
with PBS containing 0.05 % of Tween 20, a TMB reagent (DaKo
S1599) was added, and the resulting mixture was left for 10
minutes to develop color. 0. 5M H2S04 was added to stop color
16
development so as to measure 0D450-650 nm with a microplate
reader.
4 . Measurement of thrombin activity
5 20 uL of a sample and 80 \xL of a dilution solution for
activity measurement (0.01 % F-68, 50 mmol/L NaCl, 50 mmol/L
Tris-HCl, pH 8.4) were added to the polystyrene tube of BD
to be incubated at 37 °C for 3 minutes . Recombinant thrombin
(JPU Thrombin Standard 400 U/mL or WHO/US Thrombin Standard
10 110 I U/mL: prepared by their ovm companies) diluted with the
above buffer to 4, 2, 1, 0.5 and 0.25 U/mL in the case of
JPU and to 6, 3, 1.5, 0.75 and 0.375 IU/mL in the case of
IU was used as a standard. 100 uL of the S-2238 test team
chromogenic substrate (1 mM: Daiichi Pure Chemicals Co.,
15 Ltd.) was added to and mixed with the obtained reaction
solution under agitation to carry out a reaction at 37 °C for
7 minutes, and then 800 uL of a 0.1 M citric acid solution
was added to terminate the reaction. 200 uL of the reaction
solution was transferred to 96 well plates to measure
20 OD405/650.
Example 1
After lyophilized fibrinogen powders (Bolheal,
(registered trademark, the same shall apply hereinafter),
25 tissue adhesive: Vial 1) were dispersed in 2-propanol,
hydroxypropyl cellulose (6-10 mPa-s, manufactured by Wako
Pure Chemical Industries, Ltd.) was dissolved in the
resulting dispersion to a concentration of 16 wt% so as to
prepare a spinning solution having a fibrinogen-containing
30 particle/hydroxypropyl cellulose ratio of 20 (9.2 as
fibrinogen)/100 (w/w). Spinning was carried out by an
electrospinning method at a temperature of 22°C and a
humidity of not more than 26% to obtain a sheet-like fiber
molded body. The inner diameter of a jet nozzle was 0.8 mm,
17
the voltage was 11 kV, the flow rate of the spinning solution
was 1.2 inL/h, and the distance from the jet nozzle to a flat
plate was 15 cm. The obtaxned fiber molded body had an
average fiber diameter of 0.86 ]xm and an average thickness
5 of 137 urn. The obtained sheet was sterilized with a 20 kGy
electron beam. The sterilized sheet was cut to a size of
0.5 cm x 0.5 cm, and the protein was extracted with 62.5 |j.L
of physiological saline to carry out ELISA measurement. As
a result, the amount of the immobilized protein was 0.15
10 mg/cm2. Meanwhile, when ELISA measurement was made on an
unsterilized sheet likewise, the amount of the immobilized
protein was 0.16 mg/cm2. Therefore, the recovery rate of the
protein of the sterilized sheet was 94 % of that of the
unsterilized sheet.
15
Example 2
After lyophilized fibrinogen powders (Bolheal tissue
adhesive: Vial 1) were dispersed in 2-propanol,
hydroxypropyl cellulose (6-10 mPa*s, manufactured by Wako
20 Pure Chemical Industries, Ltd.) was dissolved in the
resulting dispersion to a concentration of 16 wt% so as to
prepare a spinning solution having a lyophilized fibrinogen
powder/hydroxypropyl cellulose ratio of 4 0 (18 as
fibrinogen)/100 (w/w). Spinning was carried out by the
25 electrospinning method at a temperature of 22°C and a
humidity of not more than 26% to obtain a sheet-like fiber
molded body. The inner diameter of the jet nozzle was 0.8
mm, the voltage was 12.5 kV, the flow rate of the spinning
solution was 1.2 mL/h, and the distance from the jet nozzle
30 to the flat plate was 15 cm. The obtained fiber molded body
had an average fiber diameter of 0.43 um and an average
thickness of 152 um. The obtained sheet was sterilized with
a 20 kGy electron beam. The sterilized sheet was cut to a
size of 0.5 cm x 0.5 cm, and the protein was extracted with
18
62.5 (J.L of physiological saline to carry out ELISA
measurement. As a result, the amount of the immobilized
protein was 0.27 mg/cm2. Meanwhile, when ELISA measurement
was made on an unsterilized sheet likewise, the amount of
5 the immobilized protein v/as 0.30 mg/cm2. Therefore, the
recovery rate of the protein of the sterilized sheet was 90 %
of that of the unsterilized sheet.
Example 3
10 After lyophilized fibrinogen powders (Bolheal tissue
adhesive: Vial 1) were dispersed in 2-propanol,
hydroxypropyl cellulose (6-10 mPa-s, manufactured by Wako
Pure Chemical Industries, Ltd.) was dissolved in the
resulting dispersion to a concentration of 16 wt% so as to
15 prepare a spinning solution having a lyophilized fibrinogen
powder/hydroxypropyl cellulose ratio of 100 (46 as
fibrinogen)/100 (w/w). Spinning was carried out by the
electrospinning method at a temperature of 22°C and a
humidity of not more than 26% to obtain a sheet-like fiber
20 molded body. The inner diameter of the jet nozzle v/as 0.8
mm, the voltage was 12.5 kV, the flow rate of the spinning
solution was 1.2 mL/h, and the distance from the jet nozzle
to the flat plate was 15 cm. The obtained fiber molded body
had an average fiber diameter of 0.35 um and an average
25 thickness of 191 um. The obtained sheet v/as sterilized with
a 20 kGy electron beam. The sterilized sheet v/as cut to a
size of 0.5 cm x 0.5 cm, and the protein was extracted with
62.5 JiL of physiological saline to carry out ELISA
measurement. As a result, the amount of the immobilized
30 protein was 0.78 mg/cm2. Meanwhile, when ELISA measurement
v/as made on an unsterilized sheet likewise, the amount of
the immobilized protein was 0.76 mg/cm2. Therefore, the
recovery rate of the protein of the sterilized sheet was 102 %
of that of the unsterilized sheet.
19
Comparative Example 1
Lyophilized fibrinogen powders {Bolheal tissue
adhesive: Vial 1) were sterilized with a 20 kGy electron beam.
5 The protein was extracted with 1 mL of physiological saline
to carry out ELISA measurement. As a result, the ELISA
measurement value was 31 ug/mL. Meanwhile, when ELISA
measurement was made on unsterilized lyophilized fibrinogen
powders (Bolheal) likewise, the ELISA measurement value was
10 90 |ag/mL. Therefore, the recovery rate of the protein of the
sterilized sheet was 34 % of that of the unsterilized sheet.
Example 4
After thrombin-containing particles (prepared by
15 lyophilizing an aqueous solution containing 1 mg/mL of
recombinant thrombin, sodium chloride, sodium citrate,
calcium chloride and mannitol and having a pH of 7) were
dispersed in 2-propanol, hydroxypropyl cellulose (2.0-2.9
mPa's, manufactured by Nippon Soda Co., Ltd.) was dissolved
20 in the resulting dispersion to a concentration of 13 wt% so
as to prepare a dope solution having a thrombin-containing
particle/hydroxypropyl cellulose ratio of 100/100 (w/w).
Spinning was carried out by the electrospinning method to
obtain a sheet-like fiber molded body. The obtained fiber
25 molded body had a thickness of 204 um, a weight of 2. 08 mg/cm2
and a bulk density of 101 mg/cm3. The obtained sheet was cut
to a diameter of 1 cm, and the protein was extracted with
200 uL of physiological saline to measure its activity. As
a result, the activity measurement value was 110.3 U/cm2.
30 The obtained sheet was sterilized by exposure to a 30 kGy
electron beam to measure the activity of thrombin. When the
activity of thrombin before sterilization was 100 %, the
retention rate of the activity of thrombin right after
exposure to an electron beam was 68.4 %.
20
Comparative Example 2
After a 30 kGy electron beam was applied to
thrombin-containing particles {prepared by lyophilizing an
5 aqueous solution containing 1 mg/mL of recombinant thrombin,
sodium chloride, sodium citrate, calcium chloride and
mannitol and having a pH of 7) to sterilize them, the activity
of thrombin was measured. The activity of thrombin before
exposure was 404.73 U/vial. When the activity of thrombin
10 before sterilization was 100 %, the retention rate of the
activity of thrombin right after exposure to an electron beam
was 51.8 %.
Measurement methods for Examples 5 and 6 and Comparative
15 Examples 3 and 4
1. Average thickness:
The film thicknesses of 9 fiber molded bodies obtained
by cutting the composition to a suitable size were measured
with a measurement force of 0.01 N by means of a
20 high-resolution digimatic measuring unit (LITEMATIC VL-50
of Mitutoyo Corporation) to calculate the average value.
This measurement was carried out with minimum measurement
force that could be used by the measuring unit.
25 2. Measurement of enzyme activity
A continuous fluorometric lipase test kit
(manufactured.by PROGEN BIOTECHNIK GMBH) was used to measure
the activity of lipase. The retention rate of activity was
calculated from the following equation. The amount of the
3 0 active enzyme was calculated in terms of concentration from
the value of activity.
Retention rate of activity (%) = {amount of active enzyme
after sterilization (mg/cm2) /amount of active enzyme before
sterilization (mg/cm2) } x 100
21
Fluorescent measurement using Tokyogreen (registered
trademark, the same shall apply hereinafter)-pGlu {of
Sekisui Medical Co., Ltd.) was employed to measure the
activity of p-glucosidase . The recovery rate of activity was
5 calculated from the following equation. The theoretical
weight of the immobilized enzyme was calculated from wt% of
the charged enzyme powder and the weight of the composition.
Recovery rate of activity (%) = {amount of active enzyme
(mg)/theoretical weight of immobilized enzyme (mg)} x 100
10 The retention rate of activity was calculated from the
following equation.
Retention rate of activity {%) = {recovery rate of activity
after sterilization (%)/recovery rate of activity before
sterilization (%))} x 100
15
Example 5
After lipase powders (derived from pig pancreas,
manufactured by Wako Pure Chemical Industries, Ltd. , the same
shall apply hereinafter) were dispersed in 2-propanol,
20 hydroxypropyl cellulose (6-10 mPa*s, manufactured by Wako
Pure Chemical Industries, Ltd.) was dissolved in the
resulting dispersion to a concentration of 13 wt% so as to
prepare a spinning solution having a lipase
powder/hydroxypropyl cellulose ratio of 50/100 (w/w).
25 Spinning was carried out by the electrospinning method at
a temperature of 27°C and a humidity of not more than 27 %
to obtain a sheet-like fiber molded body. The inner diameter
of the jet nozzle was 0.8 mm, the voltage was 18 kV, the flow
rate of the spinning solution was 1.2 mL/h, and the distance
30 from the jet nozzle to the flat plate was 16.5 cm. The
obtained fiber molded body (10cm x 14 cm) had an average
thickness of 168 f.im. The obtained fiber molded body was
sterilized with a 20 kGy electron beam. After the sterilized
fiber molded body was cut to a size of 1 cm x 1 cm, lipase
22
was extracted with 1 mL of a lipase buffer contained in a
kit to measure its activity. As a result, the amount of the
active enzyme was 0.46 mg/cm2. Meanwhile, when activity
measurement was made on an unsterilized sheet likewise, the
5 amount of the active enzyme was 0.40 mg/cm2. It is understood
from above that the retention rate of the activity of the
sterilized fiber molded body was 115 % of that of the
unsterilized fiber molded body and that lipase was not
deactivated by sterilization with an electron beam.
10
Example 6
After lipase powders were dispersed in 2-propanol,
hydroxypropyl cellulose (6-10 mPa*s, manufactured by Wako
Pure Chemical Industries, Ltd.) was dissolved in the
15 resulting dispersion to a concentration of 13 wt% so as to
prepare a cast solution having a lipase powder/hydroxypropyl
cellulose ratio of 50/100 (w/w). Casting was carried out
by using a doctor blade (YBA-3 of YOSHIMITSU) at a coating
width of 15 mil to obtain a sheet. The obtained sheet (4
20 cm x 6 cm) had an average thickness of 180 um. The obtained
sheet was sterilized with a 20 kGy electron beam. After the
sterilized sheet was cut to a size of 1 cm x 1 cm, lipase
was extracted with 1 mL of a lipase buffer contained in a
kit to measure its activity. As a result, the amount of the
25 active enzyme was 0.69 mg/cm2. Meanwhile, when activity
measurement was made on an unsterilized sheet likewise, the
amount of the active enzyme was 0 . 64 mg/cm2. It is understood
from above that the retention rate of the activity of the
sterilized sheet was 108 % of that of the unsterilized sheet
30 and that lipase was not deactivated by sterilization with
an electron beam.
Example 7
After p-glucosidase powders (derived from almond,
23
manufactured by Oriental Yeast Co., Ltd, the same shall apply
hereinafter) were dispersed in 2-propanol, hydroxypropyl
cellulose (6-10 raPa's, manufactured by Wako Pure Chemical
Industries, Ltd.) was dissolved in the resulting dispersion
5 to a concentration of 13 wt% so as to prepare a spinning
solution having a p-glucosidase powder/hydroxypropyl
cellulose ratio of 38/62 (w/w). Spinning was carried out
by the electrospinning method at a temperature of 27°C and
a humidity of not more than 27 % to obtain a sheet-like fiber
10 molded body. The inner diameter of the jet nozzle v/as 0.9
mm, the voltage was 18 kV, the flow rate of the spinning
solution was 1.2 mL/h, and the distance from the jet nozzle
to the flat plate was 16.5 cm. The obtained fiber molded
body (10 cm x 10 cm) had an average thickness of 207 urn. After
15 the obtained fiber molded body was cut to a size of 2 cm x
2 cm, it was sterilized with a 20 kGy electron beam.
p-glucosidase v/as extracted from the obtained sterilized
sheet with 1 raL of physiological saline to measure its
activity with Tokyogreen-pGlu. As a result, the recovery
20 rate of activity was 42 %. Meanwhile, when activity
measurement v/as made on an unsterilized sheet likewise, the
recovery rate of activity was 46 %. It is understood from
above that the retention rate of the activity of the
sterilized fiber molded body was 91 % of that of the
25 unsterilized fiber molded body and that the deactivation of
the enzyme can be suppressed by containing it in the cellulose
ether derivative.
Comparative Example 3
30 Lipase powders were sterilized with a 20 kGy electron
beam. 1 mL of a lipase buffer was added to 1 mg of the powders
to measure its activity. As a result, the activity value
was 0 . 25 pmol/mL*min. Meanwhile, when activity measurement
was made on unsterilized lipase powders likewise, the
24
activity value was 0.34 pmol/mL*min. Therefore, the
retention rate of the activity of the sterilized powders was
7 4 % of that of the unsterilized powders.
5 Comparative Example 4
p-glucosidase powders were sterilized with a 20 kGy
electron beam. 2 mg of the powders was dissolved in 1 mL
of physiological saline to measure its activity with
Tokyogreen-pGlu. As a result, the retention rate of activity
10 was 81 %.
Measurement method for Examples 8 to 10
When an electron beam is applied to'lyophilized
fibrinogen powders (fibrinogen-containing particles), the
15 changes of fibrinogen (increase in the amount of its
aggregate, reduction in gel strength) occur. To investigate
the effect of suppressing the changes of fibrinogen by
exposure to an electron beam {sterilization resisting
effect), a bulk solution of fibrinogen was prepared and 1
20 mL of the bulk solution was charged into a 5 mL glass vial
to be lyophilized. A 30 kGy electron beam was applied to
part of the vial in which lyophilization was completed to
compare each lyophilized product before and. after
sterilization.
25 Comparison evaluation was carried out by measuring gel
strength by means of the EZTest small-sized bench-top tester
(of Shimadzu Corporation) and the content of the aggregate
by means of BioSep-SEC-s4000 (of Phenomenex) (analyzing
conditions : fractionating with a 50 mM phosphoric acid buffer
30 solution {pH of 7.0} and 0.5 M arginine hydrochloride salt
as mobile phases at a flow rate of 1.0 ml/min, detecting a
target substance with a wavelength of 280 nm; and determining
the quantity of the aggregate from a peak detected earlier
than a monomer peak).
25
As for the procedure of preparing a sample for analysis
{analytical sample), an unsterilized lyophilized product
vial and a sterilized lyophilized product vial were each
dissolved in 1 iriL of distilled water. The resulting
5 solutions were centrifuged by a centrifugal tube at 15,000
rpm for 5 minutes and let pass through a 0.45 Jim filter to
be used as analytical samples.
Example 8
10 The sterilization resisting effect for a protein of
a combination of a cellulose ether derivative and specific
additives was investigated by the following method.
(method) The function of fibrinogen was evaluated by
measuring the gel strength of each of fibrinogen bulk
15 solutions of compositions comprising "a cellulose ether
derivative + specific additives" (compositions (1) shown in
Nos. 1 to 6 in Table 1 below) and fibrinogen bulk solutions
of compositions (2) prepared by eliminating the cellulose
ether derivative from the compositions (1), and the gel
20 strengths before and after sterilization of these solutions
were compared with each other to investigate the
sterilization resisting effect. The results are shown in
Table 2.
The compositions (1) (lyophilized powders and
25 hydroxypropyl cellulose were suspended in 2-propanol to form
a sheet) and the compositions (2) (lyophilized powders) were
dissolved in water to an Fbg concentration of 1 % and diluted
with a buffer solution containing 10 mM arginine and 270 mM
sodium chloride and having a pH of 8.5 to a concentration
30 of 2 mg/mL.
After 10 uL of fibrogammin (240 units/mL) and 110 |xL
of a thrombin solution (containing 0 . 2 mg/mL of 100 mM calcium
chloride) were added to a 2 mL polypropylene tube and the
resulting solution was pipetted, 900 }.iL of a 2 mg/mL
26
fibrinogen solution was added in such a manner that air
bubbles were not contained and left to stand at 37°C for 1
hour to measure the gel strength by means of the EZTest
small-size bench -top tester (of Shimadzu Corporation).
27
Table 1 compositions (1): compositions comprising
cellulose ether derivative + specific additives
Composition
No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
composition of bulk solution
1 % of Fbg, 10 mM arginine, 110 mM sodium
chloride, 1.0 % of glycine, 0.1 % of mannitol,
0.4 % of hydroxypropyl cellulose
1 % of Fbg, 10 mM arginine, 110 mM sodium
chloride, 1.0 % of glycine, 0.2 % of mannitol,
0.4 % of hydroxypropyl cellulose
1 % of Fbg, 10 mM arginine, 110 mM sodium
chloride, 1.0 % of glycine, 0.25 % of
phenylalanine, 0.2 % of trehalose, 0.4 % of
histidine, 0.1% of trisodium citrate, 0.4% of
hydroxypropyl cellulose
1 % of Fbg, 10 mM arginine, 110 xoM sodium.
chloride, 1.0 % of glycine, 0.1 % of mannitol,
0.25 % of phenylalanine, 0.2 % of trehalose,
0.4 % of histidine, 0.1 % of trisodium citrate,
0.4 % of hydroxypropyl cellulose
1 % of Fbg, 10 mM arginine, 110 mM sodium
chloride, 1.0 % of glycine, 0.1 % of mannitol,
0.25 % of phenylalanine, 0.4 % of histidine,
0.1 % of trisodium citrate, 0.4 % of
hydroxypropyl cellulose
1 % of Fbg, 10 mM arginine, 110 mM sodium
chloride, 1.0 % of glycine, 0.2 % of mannitol,
0.25 % of phenylalanine, 0.4 % of histidine,
0.1 % of trisodium citrate, 0.4 % of
hydroxypropyl cellulose
28
10
Compositions (2): compositions comprising specific
additives
These were prepared by eliminating the cellulose ether
derivative (hydroxypropyl cellulose: HPC) from the
compositions (1).
(results)
The values of gel strength after sterilization are
shown in Table 2 and Fig. 1 when the values before
sterilization are 100.
Table 2 sterilization resisting effect for protein of
a combination of cellulose ether derivative and specific
additives
composition
No.l
No. 2
No. 3
No. 4
No. 5
No. 6
Compositions (1) (cellulose
ether derivative + specific
additives)
51.5
51.1
81.5
84.0
77.6
84.4
compositions (2)
(specific additives)
49.8
36.5
58.1
57.9
57.7
59. 6
15
20
The sterilization resistance improving effect due to
the existence of the cellulose ether derivative was not
observed in the composition No. 1 whereas the above effect
due to the existence of the cellulose ether derivative was
observed in the compositions Nos. 2 to 6. This effect was
marked especially in the composition Nos. 3 to 6.
Example 9
The sterilization resisting effect of glycine was
29
10
investigated with the compositions (two) shown in Table 3
below in the same manner as in Example 8. The results are
shown in Table 4.
Table 3 compositions for evaluating the sterilization
resisting effect of glycine
Composition
G(-)
G( + )
fibrinogen
1.0%
1.0%
Arginine
(pH 8.5)
10 mM
10 mM
sodium
chloride
110 mM
110 mM
mannitol
0.2%
0.2%
glycine
0%
1.0%
Table 4 results of evaluating the sterilization resisting
effect of glycine
Composition
G{-)
G( + )
s4000 (aggregate)
Before
sterilization
(%)
14.0
14.0
after
sterilization
(%)
28.0
21.6
increase in
amount
(%)
14.0
7.6
An increase in the content of the fibrinogen aggregate
was suppressed by the addition of glycine.
15 Example 10
The sterilization resisting effect of a combination
of a cellulose ether derivative and specific additives was
investigated by the same method as in Example 8.
Hydroxypropyl cellulose (HPC) as the cellulose ether
20 derivative and eight different fibrinogen bulk solutions
shown in Table 5 below were used. 1.0 % of fibrinogen, 110
mM sodium chloride, 1.0 % of glycine and 0.2 % of mannitol
were used in the following eight compositions. The results
30
a r e shovm in Table 6 and Fig. 2.
Table 5 sterilization resisting effect of a| combination of cellulose ether d
additives
Composition
lHPC(-)
1HPC( + )
2HPC(-)
2HPC( + )
3HPC(-)
3HPC(+)
4HPCC-)
4HPC(+)
Arginine
(pH8.5)
lOmM
lOmM
lOmM
lOmM
lOmM
lOmM
0.4mM
0.4mM
tris
! (pH8.5)
OmM
OmM
OmM
OmM
OmM
OmM
lOmM
lOmM
histidine
0%
0%
0.4%
0.4%
0.4%
0.4%
0.4%
0.4%
phenylalanine
0%
0%
0.25%
0.25%
0.25%
0.25%
0.25%
0.25%
32
Table 6 evaluation results of sterilization resisting
effect of a combination of cellulose ether derivative and
specific additives
Composition
IHPC(-)
1HPC{+)
2HPC(-)
2HPC{ + )
3HPC{-)
3HPC{+)
4HPC(-)
4HPC(+)
s4000 (aggregate)
Before
sterilization
(%)
14.0
12.8
13.8
13.9
15.1
14.8
14.9
14.2
after
sterilization
(%)
21.6
18.4
19.2
15.8
19.8
17.8
19.5
15.5
increase
in amount
(%)
7.6
5.7
5.4
1.9
4.7
2.9
4 .6
1.3
An increase in the content of the protein aggregate
was suppressed by the addition of the cellulose ether
derivative. The effect of suppressing an increase in the
above content due to the existence of the cellulose ether
10 derivative was marked in compositions 2 to 4 comprising
phenylalanine and histidine . It is understood from this that
the reason that the effect of improving sterilization
resistance due to the existence of the cellulose ether
derivative is not observed in composition No. 1 whereas the
15 effect is observed and marked in composition Nos. 3 to 6 in
Example 7 is considered to be due to the fact that the
coexistence of phenylalanine and histidine with the
cellulose ether derivative in composition Nos. 3 to 6
provides a marked sterilization resisting effect for a
20 protein.
33
Effect of the Invention
The protein composition of the present invention has
resistance to radiation sterilization. The sterile
5 composition of the present invention retains the structure
and function of a protein though it is sterilized.
Industrial Feasibility
The protein composition of the present invention is
10 used in the manufacturing industry of medical products which
requires the function and sterility of a protein.

CLAIMS
1. A protein composition which comprises a mixture of
glycine, phenylalanine and histidine and/or a cellulose
5 ether derivative as an additive.
2. The protein composition according to claim 1, wherein
the additive is a cellulose ether derivative and a protein
is contained in the cellulose ether derivative.
10
3. The protein composition according to claim 1, wherein
the additive is a mixture of glycine, phenylalanine and
histidine.
15 4. The protein composition according to claim 1, wherein
the additive consists of a mixture of glycine, phenylalanine
and histidine and a cellulose ether derivative, and a protein
is contained in the cellulose ether derivative.
20 5. The protein composition according to any one of claims
1, 2 and 4, wherein the cellulose ether derivative is selected
from the group consisting of hydroxypropyl cellulose, methyl
cellulose, hydroxyethyl cellulose, hydroxypropylmethyl
cellulose, sodium carboxymethyl cellulose and mixtures
25 thereof.
6. The protein composition according to any one of claims
1, 2 and 4, wherein the cellulose ether derivative is selected
from the group consisting of hydroxypropyl cellulose,
30 hydroxyethyl cellulose, hydroxypropylmethyl cellulose and
mixtures thereof.
7. The protein composition according to any one of claims
1, 2 and 4, wherein the cellulose ether derivative is
35
hydroxypropyl cellulose.
8 . The protein composition according to any one of claims
1 to 7, wherein the protein is selected from the group
5 consisting of enzymes, transport proteins, muscle proteins,
defense proteins, toxin proteins, protein hormones, storage
proteins, structural proteins, growth factors and mixtures
thereof. :
10 9. The protein composition according to any one of claims
1 to 7, wherein the protein is fibrinogen.
10. The protein composition according to any one of claims
1 to 9 which' is in a film form.
15
11. The protein composition according to any one of claims
1 to 9 which is in a fiber form or a nonwoven fabric form.
12. The protein composition according to any one of claims
20 1 to 11 which is sterilized with radiation.