The present invention relatesto a sterile composition
of a protein which retains its function as it is contained
in an aliphatic polyester.
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 sterilizationusingachemicalsterilantcomprising
a glutaraldehyde formulation or a filter. However, the
by sterilizationwithheat or radiation. Sterilizationwith
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 c o n t a i n i n g o r i m r n o b i l i z i n g a p r o t e i n , 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
containing l a r g e p a r t i c l e s or a s o l i d o r semisolid
composition.
EP0437095 teaches t h a t a n e u t r a l i z e d oxidized
celluloseproductcombinedwithheparinora heparin fragment
5 (nORC) canbe sterilizedbygarnma-ray irradiation. However,
th's document f a i l s t o t e a c h t h e s t e r i l i z a t i o n o f ORCorn-ORC
t o which a p r o t e i n is bound.
EP0562864 d i s c l o s e s a composite wound care substance
containing a collagen sponge m a t r i x , a secondbioabsorbable
10 polymer such a s an oxidized regenerated c e l l u l o s e (ORC)
dispersed f i b e r and an a c t i v e agent such as peptide. This
document teaches t h a t t h e a c t i v e agent may be contained i n
the matrix, t h e bioabsorbable polymer or both of them and
t h a t t h e composite sponge substance can be s t e r i l i z e d while
15 it is packaged.
US5730933 d i s c l o s e s a method of s t e r i l i z i n g
b i o l o g i c a l l y a c t i v e peptide with gamma-ray or electron-beam
i r r a d i a t i o n without the l o s s of the b i o l o g i c a l a c t i v i t y of
the peptide. This method is a technology comprising the
20 steps of forming a mixture of b i o l o g i c a l l y active peptide
and a foreign p r o t e i n such a s g e l a t i n , freezing or
l y o p h i l i z i n g t h i s mixture, and i r r a d i a t i n g it. This
document teaches t h a t the existence of the foreign protein
s t a b i l i z e s p e p t i d e a n d p r e v e n t s t h e r e d u c t i o n o f t h e a c t i v i t y
25 of peptide.
W02000/033893 d i s c l o s e s a complex of therapeutic
peptide and a polysaccharide s e l e c t e d from the group
c o n s i s t i n g of oxidized regenerated c e l l u l o s e , neutralized
oxidized regenerated c e l l u l o s e and mixtures thereof. This
30 document teaches t h a t when peptide is formulated together
with an e f f e c t i v e amount of the polysaccharide before
s t e r i l i z a t i o n with ionizing r a d i a t i o n , the biological
a c t i v i t y of the peptide t h e r a p e u t i c agent is not l o s t and
is s t a b i l i z e d i f peptide is s t e r i l i z e d with ionizing
radiation.
However, these documents do not suggest that the
structural change such as aggregation and deactivation of
a protein which occur during sterilization with ionizing
5 radiation can be suppressed by an aliphatic polyester.
Meanwhile, JP-A 2011-47089 discloses a process for
producing an enzyme-containing nanofiber having excellent
enzyme activity. In this process, a spinning solution
containing an enzyme and a polymer dissolved in a nonaqueous
10 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 sterile composition which retains the structure and
function of a protein.
The inventors of the present invention conducted
20 intensive studies to solve the above problem and found that,
surprisingly, when a protein is contained in an aliphatic
polyester, the structural change and functional
deterioration of the protein caused by sterilization with
radiation and either one or both of the above change and the
25 above deterioration caused by storage after sterilization
with radiation can be suppressed. The present invention was
accomplkshed based on this finding.
That is, thepresentinventionisasterilecomposition
which comprises a protein and an aliphatic polyester
30 containing the protein and is sterilized with radiation.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows each of thrombin activities obtained by
measuring the thrombin-containing sheet-like fiber molded
bodies of the present invention obtained in Examples 1 and
2, the thrombin-containing film of the present invention
obtained in Example 3, the thrombin-containing particle of
Comparative Example 1 and the comparative
5 thrombin-containing sheet-like fiber molded body obtained
inComparativeExample2 as the retention rates (%) ofavalue
aftersterilizationandavalueafterlmonthofstorageafter
sterilization based on an initial value before
sterilization; and
10 Fig. 2 shows the amounts of fibrinogen aggregates
obtained by measuring the fibrinogen-containing sheet-like
fiber molded body of the present invention obtained in
Example 4 and the fibrinogen-containing particle of
Comparative Example 3 when they are not irradiated, after
15 they are sterilized (OM) and after 1 month of storage after
sterilization (1M).
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention is a sterile composition which
20 comprises a protein and an aliphatic polyester containing
the protein and is sterilized with radiation.
The protein used in the present invention is not
particularly limited. Preferred examples of the protein
include hemostat proteins typified by fibrinogen and
25 thrombin, enzymes typified by asparaginase, catalase,
superoxidedismutase andlipase, transportproteinstypified
by hemoglobin, serum albumin and low density lipoprotein,
muscle proteins typified by actin and myosin, defense
proteins typified by antibodies and complements, toxin
30 proteins typified by diphtheria toxin, botulinum toxin and
snake venom, protein hormones typified by insulin, growth
factors andcytokine, storageproteinstypifiedbyovalbumin
and ferritin, structural proteins typified by collagen and
keratin, and growth factors typified by epidermal growth
factor (EGF), insulin-likegrowthfactor (IGF), transforming
growth factor (TGF) , nerve growth factor (NGF) ,
brain-derived neurotrophic factor (BDNF), vascular
endothelial growth factor (VEGF), granulocyte-colony
5 stimulating factor (G-CSF), granulocyte-macrophage-colony
stimulating factor (GM-CSF), platelet-derivedgrowth factor
(PDGF), erythropoietin (EPO), thrombopoietin (TPO), basic
fibroblast growth factor (bFGF or FGF2) and hepatocyte growth
factor (HGF) . Out of these, enzymes, transport proteins,
10 muscle proteins, defense proteins, toxin proteins, protein
hormones, storage proteins, structural proteins and growth
factors are particularly preferred.
The protein used in the present invention may be of
animal origin or manufactured by a genetic recombination
15 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 to another amino acid
sequence if the essentialbioactivityisthe same. Proteins
may also be used.
Tothe proteinuse dinthepresent invention, additives
which are pharmaceutically acceptable may be added.
Preferred examples of the additives include blood
25 coagulation factor XIII, albumin, isoleucine, glycine,
arginine, glutamic acid, phenylalanine, histidine,
surfactants, sodium chloride, sugar alcohols (such as
glycerol, mannitol, etc.), trehalose, sodium citrate,
aprotinin and calcium chloride. At least one selected from
30 the group of these is used.
The protein used in the present invention or a mixture
ofthe protein and additivesmaybe dispersedin an aliphatic
polyester as molecules but preferably as particles formed
by the aggregation of the molecules (may be referred to as
"protein particles" including mixed particles with the
additives).
The aliphatic polyester usedinthe present invention
is preferably a bioabsorbable or biodegradable polymer.
5 Examples of the bioabsorbable polymer include polylactic
acid, polyglycolic acid, polylactic acid-polyglycolic acid
copolymer, polycaprolacto~e, polylactic
acid-polycaprolactone copolymer, polyglycerol sebacic acid,
polyhydroxy alkanoic acid, polybutylene succinate and
10 derivatives thereof.
Out of these, polyglycolic acid, polylactic acid,
polycaprolactone, copolymers thereof and mixtures thereof
are preferred, and polylactic acid and polylactic
acid-glycolic acid copolymer are most preferred. For
15 example, a stereocomplex of poly-L-lactic acid and
poly-D-lactic acid may be used.
The molecular weight of the aliphatic polyester used
in the present invention is 1 x lo3 to 5 x lo6, preferably
1 x lo4 to 1 x lo6, much more preferably 5 x lo4 to 5 x 10'.
20 The terminal structure of the polymer and a catalyst for
polymerizing the polymer may be arbitrarily selected.
In the sterile composition of the present invention,
another polymer or another compound may be used in
combination as long as the object of the present invention
25 is not impaired. Examples of these include copolymers,
polymer blends and compound mixtures.
The aliphatic polyester usedinthe present invention
preferably has high purity. Especially, the contents of
additives and plasticizer contained in the aliphatic
30 polyester and residues such as residual catalyst, residual
monomers and residual solvent used for molding and
post-processing are preferably as low as possible.
Especiallywhenthe compositionis used formedicalpurposes,
it is necessary to reduce these contents to values below
safety standards.
In the present invention, the expr.ession "containing
the protein" means that at least part of the protein enters
the inside of the aliphatic polyester. This state is
5 distinguished from the state of a lyophilized complex in
which the protein is existent on the surface ofthe
composition or in the voids of the composition.
The form of the sterile composition of the present
invention is not particularly limited, and the composition
10 may be in the form of a fiber, film, sheet, plate-like body,
tube-likebody, linearbody, rod-likebody, cushionmaterial,
foam or porous body. The molding method for producing a
molded product is not particularly limited if it is a method
in which the structural change and the reduction of the
15 activity of the protein are suppressed. For example,
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 sterile composition of the
and films, and any one of molding techniques which have been
employed for the production of plastic fibers or films may
be employed. For example, extrusionmoldingtechniques such
as inflation extrusion molding and T die extrusion molding,
25 and calendering and casting techniques may be used. The
above molding may be melt molding or solution molding, out
of which solutionmolding is preferred in order to facilitate
the dispersionofthe protein so as to prevent the functional
deterioration of the protein.
30 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 and a
mesh obtainedbyprocessingthe nonwoven fabric are included
in the f i b e r form.
The average f i b e r diameter of the s t e r i l e composition
having a f i b e r form of the present invention is, f o r example,
0.01 t o 50 pm and may be s u i t a b l y determined by a person
5 s k i l l e d i n t h e a r t according t o the intended use.
The s t e r i l e composition having a f i b e r form of the
p r e s e n t invention may be in the form of a long f i b e r . The
longfiberisafiberformedr~ithoutaddingthestepofcutting
a f i b e r i n the course o f t r a n s i t i o n from spinning to the
10 processing of a f i b e r molded body. It can be formed by
electrospinning, spanbondingandmeltblowingmethods. Out
of these, the electrospinning method is preferred.
The e l e c t r o s p i n n i n g m e t h o d i s amethodinwhicha f i b e r
molded body is obtained on an electrode by applying a high
15 voltage t o a s o l u t i o n containing a polymer. The process
comprises the s t e p s of preparing a spinning solution
containinga polymer, applyinga highvoltage t o t h e solution,
j e t t i n g t h e s o l u t i o n , forming a f i b e r molded body by
evaporating t h e solvent from the j e t t e d solution,
an o p t i o n a l s t e p , and accumulating the f i b e r molded body by
the charge l o s s .
A d e s c r i p t i o n is subsequentlygiven o f t h e process f o r
producing a s t e r i l e composition having a f i b e r form or a
25 nonwoven f a b r i c form out of the invention, taking the
electrospinning method as an example.
The step of preparing a spinning solution i n the
electrospinning method w i l l be explained. Although the
spinning solution i n the p r e s e n t invention is not
30 p a r t i c u l a r l y l i m i t e d , an emulsion containing an organic
solvent solution o f an a l i p h a t i c polyester and an aqueous
solution of a p r o t e i n , a suspension containing an organic
solvent s o l u t i o n o f an a l i p h a t i c polyester and protein
p a r t i c l e s , or an organic solvent solution containing an
aliphaticpolyester andaproteinmaybe usedas the spinning
solution. Out of these, a suspension containing an organic
solvent solution of an aliphatic polyester and protein
particles is preferred.
5 The concentration of the aliphatic polyester in the
a l i p h a t i c p o l y e s t e r s o l u t i o n i s p r e f e r a b l y 1 t o 30vit%. When
the concentration of the aliphatic polyester is lower than
1 wt%, it is difficult to form a fiber molded body
disadvantageously. When the concentration is higher than
10 30 wt%, the fiber diameter of the obtained fiber molded body
becomes large disadvantageously. The concentration of the
aliphatic polyester contained in the organic solvent
solution is more preferably 2 to 20 bit%.
The solvent for the aliphatic polyester is not
15 particularly limited if it can dissolve the aliphatic
polyester, evaporates in the spinning step and can form a
fiber. 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,
20 dichloromethane, carbon tetrachloride, cyclohexane,
cyclohexanone, trichloroethane, methyl ethyl ketone, ethyl
acetate andmixture sthereof. To forman emulsion, a solvent
such as acetone, ethanol, methanol, tetrahydrofuran,
1,4-dioxane, 1-propanol, phenol, pyridine, acetic acid,
25 formic acid, hexafluoro-2-propanol, hexafluoroacetone,
N,N-dimethylformamide, N,N-dimethylacetamide,
acetonitrile, N-methyl-2-pyrrolidinone,
N-methylmorpholine-N-oxide or 1,3-dioxolane may be
contained. Out of these, dichloromethane or ethanol is
30 preferably used from the viewpoints of handling ease and
physical properties.
The protein in the present invention may be added to
and mixed with an organic solvent solution of an aliphatic
polyester in a solid, liquid or solution form.
In the p r e s e n t i n v e n t i o n , when the emulsion containing
an organic solvent solution of an a l i p h a t i c polyester and
an aqueous solution of a p r o t e i n is used as the spinning
s o l u t i o n , the aqueous solvent f o r the protein is not
5 p a r t i c u l a r l y limited i f it can dissolve the p r o t e i n , forms
anemulsionwiththeorganicsolventsolutionofanaliphatic
polyester, evaporates i n the spinning step and can form a
f i b e r . For example, physiological s a l i n e and buffer
solutionsmaybeused. Further, a s t a b i l i z e r f o r t h e protein
10 and a d d i t i v e s may be added. Out of these, a phosphoric acid
b u f f e r solution or physiological s a l i n e is preferably used.
The concentration of t h e protein i n the aqueous
s o l u t i o n of the p r o t e i n used i n the p r e s e n t invention is not
p a r t i c u l a r l y limited and may be s u i t a b l y determined
15 according t o the c h a r a c t e r i s t i c properties of the protein.
It is, f o r example, 0.5 t o 50 w t % .
Toprepareanemulsion fromanorganicsolvent solution
ofanaliphaticpolyesterandanaqueous s o l u t i o n o f a p r o t e i n ,
t h e mixing r a t i o of these s o l u t i o n s is not p a r t i c u l a r l y
(aqueous solution of p r o t e i n ) /(organic solvent solution of
a l i p h a t i c polyester) (volume r a t i o ) is 1/100 t o 1/2. When
t h i s value is l a r g e r than 1/2, the emulsion becomes unstable
disadvantageously
2 5 Althoughthemethod o f preparing an emulsion bymixing
together an organic solvent solution of an a l i p h a t i c
polyester and an aqueous solution of a protein is not
p a r t i c u l a r l y limited, u l t r a v i o l e t waves or s t i r r i n g means
may be used. As the s t i r r i n g means, high-speed s t i r r i n g
30 means such as a homogenizer o r s t i r r i n g means such as an
a t t r i t e r or b a l l m i l l may be used. Out of these, dispersion
with u l t r a s o n i c waves is preferred.
Also, the spinning solution may be prepared by adding
an a l i p h a t i c polyester a f t e r an emulsion is formed from an
organic solvent and an aqueous solution of a protein.
In the present invention, when a suspension containing
an organic solvent solution of an aliphatic polyester and
a protein is used as the spinning solution, the sizes of
5 protein particles are not particularly limited but
preferably 0.01 to 100 pm. It is technically difficult to
manufacture protein particles havingaparticle size smaller
than 0.01 pm, and when the particle size is larger than 100
pm, dispersibility degrades and the sterile composition
10 becomes brittle disadvantageously.
Althoughthemethodofpreparingasuspensionbymixing
together an organic solvent solution of an aliphatic
polyester andprotein particles is notparticularlylimited,
ultraviolet waves or stirring means may be used. As the
15 stirring means, 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.
Further, the spinning solution may be prepared by
from an organic solvent and 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
25 employed and also may be combined in the present invention.
Dry milling may be 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.
30 Meanwhile, wet milling is carried out by stirring with
a stirrer or kneader having high shear force while protein
particles are dispersed in a suitable dispersion medium, or
by using a ball mill or bead mill while protein particles
are dispersed in a medium. Further, protein particles
produced by a spay drier may also be used.
The sterilizationmethodusedinthepresent invention
is radiation sterilization. Examples of the radiation in
use include alpha rays, beta rays, gamma rays, neutron rays,
5 electron beams and X-rays. Out of these, gamma rays and
electron beams are 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
10 condition is not particularly limited, it is -80 to 40°C,
preferably -80 to 30°C.
Theradiationsuchas alpharays, positron, gamma rays,
neutron rays, electron beams or X-rays strips an electron
off from molecules or atoms constituting a substance when
15 it is applied to the substance. A molecular bond is broken
upon this, and a highly reactive radical is produced and
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
20 considered to be due to the destruction of 'a high-order
structure" which is a source of developing a function by the
breakage of a molecular bond by exposure. Further, as shown
in Comparative Examples ofthe specification ofthe present
application, the structural destruction or deacti~at'ion of
25 aproteinalsooccursbystorage after exposure to radiation.
However, the structural destruction and the functional
deterioration of the protein contained in the aliphatic
polyester in the present invention are suppressed even when
the protein is exposed to radiation, and also the structural
30 destruction and the functional deterioration by storage
after exposure are suppressed. This means that the
high-order structure of the protein is retained in the
composition, which is a common effect regardless of the type
of the protein. It is not considered from the thickness of
the aliphatic polyester through which the radiation is
transmitted that this effect is due to screening, and the
control mechanism is not known.
The aliphatic polyester containingthe protein before
5 radiation sterilization in the present inventionmay further
contain an electron/ion scavenger, energy transfer agent,
radical scavenger, antioxidant and plasticizer. Examples
of the electrodion scavenger Include N,Nf-tetramethyl
phenylenediamine, diphenylenediamine, pyrene and quinone.
10 Examples of the energytransfer agent include acenaphthene.
Examples of the radical scavenger include mercaptans,
octahydrophenanthrene, monoalkyl diphenyl ethers,
tocopherol, citric acid, butylated hydroxyanisole,
butylated hydroxytoluene, t-butyl hydroquinone, propyl
15 gallate and ascorbic acid derivatives. Examples of the
antioxidant include BHT, phosphite triesters, phenolic
antiaging agents andorganicthio acid salts. Additivesthat
are generally accepted as safe for use in foods and
pharmaceuticals are preferred. The amount of the additive
20 which is not particularly limited is, for example, 0.01 to
10 wt% based on the aliphatic polyester in the sterile
composition
The aliphatic polyester containing the protein in the
sterilization step preferably contains no water. The water
25 content of the aliphatic polyester is preferably not more
than 10 wt%, more preferably not more than 4 bit%, much more
preferably substantially 0 wt%.
The aliphatic polyester containing the protein may be
wrapped in a packaging material to be sterilized with
.30 radiation. Asthepackagingmaterial, amaterialhavinghigh
gas barrier properties such as aluminum is preferably used.
The aliphatic polyester 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
and the desiccant, ones which do no harm to the human body
and are not deactivated upon exposure to radiation are
preferred.
5 The sterile composition of the present invention may
be used as a medical material which requires the function
and sterility of a protein.
EXAMPLES
10 The following examples are provided for the purpose
of further illustrating the present invention but are in no
way to be taken as limiting.
1. Measurement of thrombin activity
20 pL of a sample, 60 pL of a 50 mM tris-HC1 (pH 8.5)
15 + 50 mM NaCl buffer and 20 pL of 0.1 % PLURONIC F-68 were
added to the 2008 tube of FALCON Corporation to be incubated
at 37°C for 3 minutes. Human plasma-derived purified
a-thrombin (purchased fromHaematologicTechnologies, Inc.:
HCT-0020) dilutedwiththeabovebufferto 5, 2.5, 1.25, 0.625
20 and 0.3125 U/mL was used as a standard. 100 1rL of the S-2238
testteamchromogenicsubstrate (1mM: Daiichi Pure Chemicals
Co., Ltd. ) was added to and mixed with the obtained reaction
solution under agitation to carry out a reaction at 37°C for
5 minutes, and 800 pL of a 0.1 M citric acid solution was
25 added to terminate the reaction. 200 pL of the reaction
solution was transferred to 96 well plates to measure
OD405/650.
The folloriing method was used to measure the thrombin
activity in Examples and Comparative Examples except for
30 Examples 5' to 7 and Comparative Example 4. 20 pL of a sample
a n d 8 0 p L o f a d i l u t e d s o l u t i o n f o r t h e m e a s u r e m e n t o f a c t i v i t y
(0.01 % F-68, 50 mmol/L NaCl, 50 mmol/L Tris-HC1, 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 110 IU/mL)
d i l u t e d with the above b u f f e r t o 4, 2, 1, 0.5 and 0.25 U/mL
i n the case of JPU and t o 6, 3, 1.5, 0.75 and 0.375 IU/mL
i n t h e c a s e o f IUwas usedas a standard. .I00 p L o f t h e 5-2238
5 testteamchromogenicsubstrate ( I m M : Daiichi Pure Chemicals
Co., Ltd. ) was added to and mixed with the obtained reaction
solution under a g i t a t i o n t o carry out a reaction a t 37°C for
7 minutes, and then 800 pL of a 0.1 M c i t r i c a c i d s o l u t i o n
was added t o terminate the r e a c t i o n . 200 LIL of the reaction
10 solution was t r a n s f e r r e d t o 96 well p l a t e s t o measure
OD405/650.
2. Measurement of amount of fibrinogen aggregate
~ f t e r t h e s h e e t w a s c u t t o a d i a m e t e r o f 1 c m ,f ibrinogen
15 was extracted with a d i l u t i o n solution t o measure the amount
of its aggregate by high-speed l i q u i d chromatography.
Detector: u l t r a v i o l e t absorption photometer (measurement
wavelenqth: 280 m)
20 Column: Bio Sep-SEC-s4000 (7.8 x 300 mrn, Phenomenex)
Column temperature: 25°C
Sampler temperature: 6°C
Transfer phase: 0.5 mol/L Arg-HC1/50 mmol/L phosphoric acid
b u f f e r
25 Flow r a t e : 1 mL/min
Analysis time: 20 min
3. Thickness
The thicknesses of15moldedbodies weremeasuredwith
30 a measurement force of 0.01 N by means of a high-resolution
digimatic measuring unit ((LITEMATIC VL-50 of Mitutoyo
Corporation) t o c a l c u l a t e the average value as the thickness
of the molded body. This measurement was c a r r i e d out with
m i n i m u m m e a s u r e m e n t f o r c e t h a t c o u l d b e u s e d b y t h e m e a s u r i n g
u n i t .
4 . Weight
The molded body was cut t o a s i z e of 50 rnm x 100 m t o
5 measure its weight so as t o c a l c u l a t e t h e w e i g h t ofthemolded
body.
5. Bulk density
Thebulkdensityofthemoldedbodywas calculated from
10 the above measurement thickness and weight value.
6. Measurement of thrombin ELISA
5 pg/mL of an antihuman thrombin antibody (No. SAHT-AP
of A f f i n i t y Biologicals I n c . ) was immobilized t o an ELISA
15 p l a t e (NUNC 468667). After it was washedwithPBS containing
0.05 % o f Tween 20, BlockAce (UK-B80 of DS Pharma Biomedical
Co., Ltd.) was addedtoeachwelltocarryoutmasking. After
washing with PBS containing 0.05 % of Tween 20, a t e s t body
was added. Human thrombin (HCT-0020 of Haetologic
20 Technologies, I n c . ) was used as a standard t o form a
c a l i b r a t i o n curve. Afterwashing with PBS containing 0.05 %
of Tween 20, 0.1 pg/mL of an HRP-labelled antihuman thrombin
antibody (No. SAHT-HRP of A f f i n i t y Biologicals Inc.) was
added. After a r e a c t i o n , the reaction product was washed
25 with PBS containing 0.05 % of Tween 20, a TMB reagent (DaKo
51599) was added, and the r e s u l t i n g mixture was l e f t f o r 10
minutes t o develop color. 1 N H2SO4 was added t o stop color
development so as t o measure 0D450-650 nm with a microplate
reader.
30
7. Measurement of enzyme a c t i v i t i e s of l i p a s e and
p-glucosidase
(1) Measurement of e x t r a c t i o n r a t e
The molded body was cut t o a s i z e of 2 cm x 2 cm and
immersed in 1 mL of physiological saline for 3 minutes or
3 hours to elute an immobilized enzyme. This process bias
carried out on three molded bodies to measure their weight
changes so as to obtain the average value of extraction rate
5 calculated from the following equation. The theoretical
weight of the immobilized enzyme was calculated from the
weight of the composition and wt% of the charged enzyme
powder.
Extraction rate = weight loss (mg) /theoretical weight
10 (mg) of immobilized enzyme
(2) Measurement of enzyme activity
A continuous fluorometric lipase test kit
(manufacturedby PROGEN BIOTECHNIKGMBH) was usedtomeasure
the activity of lipase. The recovery rate of activity was
15 calculated from the following equation. The amount of the
active enzyme was calculated in terms of concentration from
the value of activity. The theoretical weight of the
immobilized enzyme per unit area was calculated from wt% of
the charged enzyme powder and the weight ofthe composition.
20 Recovery rate of activity (%) = (amount of active enzyme
(mg/cm2) /theoretical weight of immobilized enzyme per unit
area (mg/cmz) x extraction rate) } x 100
Fluorescent measurement using Tokyogreen (registered'
trademark, the same shall apply hereinafter)-PGlu (of
25 Sekisui Medical Co., Ltd.) was used to measure the activity
of 0-glucosidase. The recovery rate of activity was
calculated from the following equation. The theoretical
weight of the immobilized enzyme was calculated from tit% of
the charged enzyme powder and the weight ofthe composition.
30 Recovery rate of activity (%) = {amount of active enzyme
(mg) /theoretical weight of immobilized enzyme (mg) x
extraction rate)} x 100
The retention rate of activitywas calculated fromthe
following equation
Retention rate of activity ( % ) = (recovery rate of activity
after sterilization (%)/recovery rate of activity before
sterilization ( % ) ) } x 100
5 Example 1
After thrombin-containing particles (prepared by
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
10 dispersed in ethanol, dichloromethane was added to the
resulting dispersion, and a polyglycolic acid-polylactic
acid copolymer (Purasorb PDLG5010 of PURAC) tias dissolved
in the dispersion to a concentration of 10 wt% to prepare
a spinning solution having a thrombin-containing
15 particle/polyglycolic acid-polylactic acid copolymer ratio
of 100 (1.69 as thrombin) /lo0 (w/w) . Spinning was carried
outbyanelectrospinningmethodtoobtaina sheet-like fiber
moldedbody. The obtained fibermoldedbody had athickness
of 131 pm, a weight of 1.44 mg/cm2 and a bulk density of 111
20 mg/cm3. The obtained sheet was cut to a diameter of 1 cm,
and the protein was extracted with 200 pL of physiological
salinetomeasureitsactivity. As a result, themeasurement
value of activity was 26.7 u/cm2. The obtained sheet was
sterilized by exposure to a 20 kGy electron beam and kept
25 at 40°C and 75 %RH for 1 month 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 the electron beam was
79 %. The retention rate of activity after 1 month was 78 %,
30 and no reduction in the activity of thrombin was observed
during storage.
Example 2
After thrombin-containing particles (prepared by
l y o p h i l i z i n g an aqueous solution containing 1 mg/mL of
recombinant thrombin, sodium chloride, sodium c i t r a t e ,
calcium chloride and mannitol and having a pH of 7) and
Quinizarin Green SS (of Tokyo Chemical Industry Co., Ltd.)
5 were dispersed i n ethanol, dichloromethane was added t o the
r e s u l t i n g dispersion, and a polyglycolic acid-polylactic
acid copolymer (Purasorb PDLG5010 of PURAC) was dissolved
i n the dispersion t o a concentration of 10 w t % t o prepare
a spinning s o l u t i o n having a thrombin-containing
10 p a r t i c l e / p o l y g l y c o l i c acid-polylactic acid copolymer r a t i o
of 100 (1.69 as thrombin) /I00 (w/w) . Spinning was carried
out by the electrospinning method t o obtain a sheet-like
f i b e r molded body. The obtained sheet c o n t a i n i n g t h e f i b e r
molded body (average thickness: 129 pm, weight: 1.49 mg/cm2,
15 bulk density: 124 mg/cm3) was cut t o a diameter of 1 cm, and
t h e p r o t e i n was extractedwith200 p L o f p h y s i o l o g i c a l s a l i n e
t o measure the a c t i v i t y of thrombin. As a r e s u l t , the
measurement value of a c t i v i t y was 40.2 1 u / c m 2 . The obtained
sheet was s t e r i l i z e d by exposure t o a 30 kGy electron beam
20 and k e p t a t 4O0Cand75 %RH f o r 1 m o n t h t o m e a s u r e t h e a c t i v i t y
of thrombin. When the a c t i v i t y of thrombin before
s t e r i l i z a t i o n was 100 %, the r e t e n t i o n r a t e of the a c t i v i t y
ofthrombinrightafterexposuretoanelectronbeamr~as7 0 %.
The r e t e n t i o n r a t e of a c t i v i t y a f t e r 1 month was 74 %, and
25 no reduction in the a c t i v i t y of thrombin was observedduring
storage.
Example 3
After thrombin-containing p a r t i c l e s (prepared by
30 lyophilizing an aqueous solution containing 1 mg/mL of
recombinant thrombin, sodium chloride, sodium c i t r a t e ,
calcium chloride and mannitol and having a pH of 7) was
dispersed i n ethanol, dichloromethane was added t o the
r e s u l t i n g ' d i s p e r s i o n , and a polyglycolic a c i d - p o l y l a c t i c
acid copolymer (Purasorb PDLG5010 of PURAC) was dissolved
in the dispersion to a concentration of 10 wt% to prepare
a dope solution having a thrombin-containing
particle/polyglycolic acid-polylactic acid copolymer ratio
5 of 100 (1.69 as thrombin) /I00 (w/w) . The obtained dope
solution was used to form a film by a casting method. The
coating interval was 127 btm, and the coating speed was 30.1
mm/sec. Theobtainedsheethadathickness of 58 pm, aweight
of 2.9rng/cm2 anda bulkdensityof 504 mg/cm3. The obtained
10 sheet was cut to a diameter of 1 cm, and the protein was
extracted with 200 pL of physiological saline to measure the
activity of thrombin. As a result, the measurement value
of activity was 71.1 1U/cm2. The obtained sheet was
sterilized by exposure to a 30 kGy electron beam and kept
15 at 40°C and 75 %RH for 1 month 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 75.7 % .
The retention rate of activity after 1 month was 82 %, and
20 no reduction in the activity of thrombin was observed during
storage.
Example 4
After fibrinogen-containing particles (prepared by
25 lyophilizing an aqueous solution containing 10 mg/mL of
recombinant fibrinogen, arginine, sodium chloride and
mannitol and having a pH of 8.5) were dispersed in ethanol,
dichloromethane was added to the resulting dispersion, and
a polyglycolic acid-polylactic acid copolymer (Purasorb
30 PDLG5010 of PURAC) was dissolved in the dispersion to a
c o n c e n t r a t i o n o f 1 0 w t % t o p r e p a r e a spinning solutionhaving
a fibrinogen-containing particle/polyglycolic
acid-polylactic acid copolymer ratio of 100 (50.85 as
fibrinogen)/lOO w . Spinning was carried out by the
electrospinning method to obtain a sheet-like fiber molded
body. The obtained fiber molded body had a thickness of 131
btm, a weight of 1.44 mg/cm2 and a bulk density of 110 mg/cm3.
The obtained sheet was cut to a diameter of 1 cm, and
5 fibrinogen was extracted with a dilution solution to measure
the amount of its aggregate by high-speed chromatography.
As a result, the amount of the aggregate was 9.79 %. The
obtainedsheetrias sterilizedbyexposuretoa 30 kGyelectron
beam and kept at 40°C and 75 %RH for 1 month to measure the
10 amount of the aggregate. The amount of the aggregate right
after exposure to an electron beam was 18.81 %. The weight
of the aggregate after 1 month was 24.14 %.
Comparative Example 1
15 After a 30 kGy electron beam was applied to
thrombin-containing particles (prepared by lyophilizing an
aqueous so1utioncontaining1mg/mLofrecombinantthrombin,
sodium chloride, sodium citrate, calcium chloride and
mannitol and having a pH of 7) to sterilize them, the
20 thrombin-containing particles were kept at 40°C and 75 %RH
f o r 1 m o n t h t o m e a s u r e t h e a c t i v i t y o f t h r o m b i n . Theactivity
of thrombin before exposure was 404.73 U/vial. When the
activity of thrombin before sterilization was 100 %, the
retention rate of the activity of thrombin right after
25 exposure to an electron beam was 51.8 %. The retention rate
of activity after 1 month was 17.9 %, and a reduction in the
activity of thrombin was observed during storage.
Comparative Example 2
30 After thrombin-containing particles (prepared by
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
mPaes, manufactured by Nippon Soda Co., Ltd.) was dissolved
in the resulting dispersion to a concentration of 13 wt% to
prepare a dope solution having a thrombin-containing
particle/hydroxypropyl cellulose ratio of 100/100 (w/w).
5 Spinning was carried out by the electrospinning method to
obtain a sheet-like fiber molded body. The obtained fiber
molded body had a thickness of 204 pm, 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
10 200 pL of physiological saline to measure its activity. As
a result, themeasurement value of activitywas 110.3 1u/cm2.
The obtained sheet was sterilized by exposure to a 30 kGy
e l e c t r o n b e a m a n d k e p t a t 4 0 o C a n d 7 5 % ~ ~ f o r 1 m o n t h t o m e a s u r e
the activity of thrombin. When the activity of thrombin
15 before sterilization was 100 %, the retention rate of the
activityofthrombinrightafterexposuretoanelectronbeam
was 68.4 %. The retention rate of activity after 1 month
was 54.9 % and a reduction in the activity of thrombin was
observed during storage.
20
Comparative Example 3
After a 30 kGy electron beam was applied to
fibrinogen-containing particles (prepared by lyophilizing
an aqueous solution containing 10 mg/mL of recombinant
25 fibrinogen, arginine, sodium chloride and mannitol and
having a pH of 8.5) to sterilize them, the
thrombin-containing particles were kept at 40°C and 75 %RH
forlmonthtomeasurethe amount of an fibrinogenaggregate.
The amount of the aggregate before exposure was 6.97 %. The
30 amount of the aggregate right after exposure to an electron
beam was 18.51 %. The amount of the aggregate after 1 month
was 54.72 %.
The results (the retention rates of activity of
thrombin (Th) after sterilization and after storage after
sterilization based on the value before sterilization) of
Examples 1 to 3 and Comparative Examples 1 and 2 are shown
in Fig. 1.
5 It is understood that when the protein is contained
in the aliphatic polyester, the structural change and
functional deterioration ofthe protein caused by radiation
sterilization are suppressed as compared with a case where
only protein-containing particles are used (Comparative
10 Example 1) and-further that the change and the deterioration
caused by storage after radiation sterilization are
suppressed as compared with a case where not the aliphatic
polyester but a cellulose (hydroxypropyl cellulose) is used
(Comparative Example 2).
15 The results (the amount of the fibrinogen aggregate
after sterilization and after storage after sterilization)
of Example 4 and Comparative Example 3 are shown in Fig. 2.
It is understood that when the protein is contained
inthealiphaticpolyester (Example 4), the structural change
20 of the protein caused by storage after radiation
sterilization is suppressed as compared with a case where
only protein-containing particles are used (Comparative
Example 3) .
25 Example 5
After thrombin-containing particles (Bolheal,
(registered trademark, the same shall apply hereinafter),
tissue adhesive: Vial 3) were dispersed in ethanol,
dichloromethane was added to the resulting dispersion, and
30 polylactic acid (PL18 of Purac Biomaterials) was dissolved
in the dispersion to a concentration of 10 wt% to prepare
a spinning solution having a thrombin-containing
particle/polylactic acid ratio of 40 (0.45 as thrombin) /I00
( I . Spinning bras carried out by the electrospinning
method to obtain a sheet-like fiber molded body. The
obtained sheet was sterilized with a 20 kGy electron beam.
The obtained sheet was cut to a size of 2 cm x 2 cm, and the
protein was extracted with 1 mL of physiological saline to
5 measure its activity and ELISA. As a result, the activity
measurement value was 5.0 u/cm2, and the ELISA measurement
value was 3.4 pg/cm2. When activity and ELISA measurements
were made on an unsterilized sheet likewise, the activity
measurement value was 7.5 u/cm2 and the ELISA measurement
10 value was 4.35 pg/cm2. That is, the retention rate of the
activity of the sterilized sheet was 73 % of that of the
unsterilized sheet.
Example 6
15 After thrombin-containing particles (Bolheal tissue
adhesive: Vial3) were dispersed in ethanol, dichloromethane
was added to the resulting dispersion, and polylactic acid
(PL18 of Purac Biomaterials) was dissolvedinthe dispersion
to a concentration of 10 wt% to prepare a spinning solution
20 having a thrombin-containingparticle/polylactic acid ratio
of 70 (0.78 as thrombin)/100 (w/w). Spinning was carried
out by the electrospinning method to obtain a sheet-like
fiber molded body. The obtained sheet was sterilized with
a 20 kGy electron beam. The obtained sheet was cut to a size
25 of 2 cm x 2 cm, and the protein was extracted with 1 mL of
physiological saline to measure its activity and ELISA. As
a result, the activity measurement value was 9.575 u/crn2,
and the ELISAmeasurement value was 7.0 pg/cm2. When activity
and ELISA measurements were made on an unsterilized sheet
30 likewise, the activitymeasurementvalue was 11.15 u/cmZand
the ELISA measurement value was 7.2 pg/cm2'. That is, the
retention rate of the activity of the sterilized sheet was
86 % of that of the unsterilized sheet.
Example 7
After thrombin lyophilized powders (Bolheal tissue
adhesive: Vial3) were dispersedin ethanol, dichloromethane
was added to the resulting dispersion, and polylactic acid
5 (PL18 of Purac Biomaterials) was dissolvedinthe dispersion
to a concentration of 10 wt% to prepare a spinning solution
having a thrombin lyophilized powder /polylactic acid ratio
of 100 (1.1 as thrombin) /I00 I . Spinning was carried
out by the electrospinning method at a temperature of 22'C
10 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, the voltage was 15 kV, the flow rate of the spinning
solution was 3.0 mL/h, and the distance from the jet nozzle
to a flat plate was 25 cm. The obtained sheet was sterilized
15 with a 20 kGy electron beam. The obtained sheet was cut to
a size of 2 cm x 2 cm, and the protein was extracted with
1 m L o f p h y s i o l o g i c a l s a l i n e t o m e a s u r e i t s activityandELISA.
As a result, the activity measurement value was 15 u/cm2,
andtheELISAmeasurementvaluewas11pg/cm2. Whenactivity
likewise, the activity measurement value was 23 u/cm2 and
the ELISA measurement value was 16 pg/cm2. That is, the
retention rate of the activity of the sterilized sheet was
64 % of that of the unsterilized sheet.
25
Comparative Example 4
Thrombin-containing particles (Bolheal) were
sterilized with a 20 kGy electron beam. The protein was
extracted with 1 mL of physiological saline to measure its
30 activity and ELISA. As a result, the activity measurement
value was 22.5 u/crn2 and the ELISA measurement value was 11.5
Lig/cm? When activity and ELISA measurements were made on
unsterilized thrombin-containing particles likewise, the
activity measurement value was 68.5 u/cm2 and the ELISA
measurement value was 41.5 pg/cm2. That is, the retention
rate of the activity of the sterilized sheet was 32 % of that
of the unsterilized sheet
5 Example 8
After lipase powders (derived from pig pancreas,
manufacturedbywako PureChemical Industries, Ltd., thesame
shall apply hereinafter) were dispersed in ethanol,
dichloromethane was added to the resulting dispersion, and
10 a polylactic acid-glycolicacidcopolymer (PDLG5010 of Purac
Biomaterials) was dissolved in the dispersion to a
concentrationoflOr.rt%topreparea spinningsolutionhaving
a lipase powder/polylactic acid-glycolic acid copolymer
ratio of 50/100 (w/w). Spinning was carried out by the
15 electrospinning method at a temperature of 27°C and a
humidity of not more than 25 % to obtain a sheet-like fiber
molded body. The inner diameter of a jet nozzle was 0.9 mm,
the voltage was 15 kV, the flow rate of the spinning solution
was 4.0 mL/h, and the distance from the jet nozzle to a flat
sheet was 79 %. The obtained sheet was sterilized with a
20 kGy electron beam. The obtained sterilized sheet was cut
to a size of 1 cm x 1 cm, and lipase was extracted with 1
mL of a lipase buffer contained in a kit to measure its
25 activity. As a result, the recovery rate of activity was
100 % .
Example 9
Dichloromethane was added to lipase powders, and a
30 polylactic acid-glycolic acid copolymer (PDLG5010 of Purac
Biomaterials) was dissolved in the resulting mixture to a
concentrationof10 wt%topreparea spinningsolutionhaving
a lipase powder/polylactic acid ratio of 50/100 (w/w).
Spinning was carried out by the electrospinning method at
a temperature of 26°C and a humidity of not more than 25 %
t o o b t a i n a sheet-like fibermoldedbody. The innerdiameter
of a j e t nozzle was 0.8 mm, t h e voltage was 15 kV, the flow
r a t e of t h e spinning solution was 4.0 mL/h, and the distance
5 from t h e j e t nozzle t o a f l a t p l a t e was 25 cm. The l i p a s e
e x t r a c t i o n r a t e o f t h e obtainedsheetwas 63 %. The obtained
sheet was s t e r i l i z e d with a 20 kGy electron beam. The
obtained s t e r i l i z e d sheet was cut t o a s i z e of 1 cm x 1 cm,
a n d l i p a s e r i a s extractedwith1mLofalipasebuffercontained
10 in a k i t t o measure its a c t i v i t y . As a r e s u l t , the recovery
r a t e of a c t i v i t y was 92 %.
Example 10
After P-glucosidase powders (derived from almond,
15 manufacturedbyOrienta1 Yeast Co., Ltd, the same s h a l l apply
h e r e i n a f t e r ) were dispersed i n ethanol, dichloromethane was
added t o t h e r e s u l t i n g dispersion, and a p o l y l a c t i c
acid-glycolic acid copolymer (PDLG5010 of Purac
Biomaterials) was dissolved i n the dispersion t o a
20 c o n c e n t r a t i o n o f 1 0 w t % t o p r e p a r e a spinning solutionhaving
a P-glucosidase powder/polylactic acid-glycolic acid
copolymer r a t i o of 38/62 r . Spinning was c a r r i e d out
by t h e electrospinning method a t a temperature of 27'C and
a humidity of not more than 25 % t o obtain a sheet-like f i b e r
25 molded body. The inner diameter of a j e t nozzle was 0.9 mm,
the voltage was 15 kV, the flow r a t e of the spinning solution
was 4.0 mL/h, and the distance from the j e t nozzle to a f l a t
p l a t e was 25 cm. After the obtained sheet was cut t o a s i z e
of 2 cm x 2 cm, it was s t e r i l i z e d with a 20 kGy electron beam.
30 ~ - g l u c o s i d a s e w a s e x t r a c t e d w i t h 1 m L o f p h y s i o l o g i c a l s a l i n e
t o measure its a c t i v i t y with Tokyogreen-PGlu. A s a r e s u l t ,
the recovery r a t e of a c t i v i t y was 92 %. When a c t i v i t y
measurement was made on an u n s t e r i l i z e d sheet likewise, the
recovery r a t e of a c t i v i t y was 94 %. It was understood from
above that the retention rate of the activity of the
sterilized fiber molded body was 98 % of that of the
unsterilized fiber molded body and that P-glucosidase was
not deactivated by electron beam sterilization.
5
Example 11
After P-glucosidasepowders weredispersedinethanol,
dichloromethane was added to the resulting dispersion, and
a polylactic acid-caprolactone copolymer (PLCA8812 of Taki
10 Chemical Co., Ltd.) was dissolved in the dispersion to a
concentrationof 10 wt%toprepare a spinning solutionhaving
a !3-glucosidase powder/polylactic acid-caprolactone
copolymer ratio of 38/62 (w/w). Spinning was carried out
by the electrospinning method at a temperature of 27'C and
15 a humidity of not more than 25 % to obtain a sheet-like fiber
molded body. The inner diameter of a jet nozzle was 0.9 mm,
the voltage was 15 kV, the flow rate of the spinning solution
was 3.0 mL/h, and the distance from the jet nozzle to a flat
plate tias 25 cm. After the obtained sheet was cut to a size
20 of 2 cm x 2 cm, it was sterilized with a 20 kGy electron beam.
P-glucosidasewas extractedwith 1 m L o f p h y s i o l o g i c a l s a l i n e
to measure its activity with Tokyogreen-PGlu. As a result,
the recovery rate of activity was 81 %. When activity
measurement was made on an unsterilized sheet likewise, the
25 recovery rate of activity was 80 %. It was understood from
above that the retention rate of the activity of the
sterilized fiber molded body was 101 % of that of the
unsterilized fiber molded body and that P-glucosidase was
not deactivated by electron beam sterilization.
30
Example 12
After P-glucosidasepor~dersw ere dispersedinethanol,
dichloromethane was added to the resulting dispersion, and
polylactic acid (PL18 of Purac Biomaterials) was dissolved
in the dispersion to a concentration of 11 wt% to prepare
aspinning solutionhavinga P-glucosidasepo\~ider/polylactic
acid ratio of 38/62 (ri/w). Spinning was carried out by the
electrospinning method at a temperature of 27°C and a
5 humidity of not more than 25 % to obtain a sheet-like fiber
molded body. The inner diameter of a jet nozzle was 0.9 mm,
the voltage was 15 kV, the flow rate of the spinning solution
was 3.0 mL/h, and the distance from the jet nozzle to a flat
plate was 25 cm. After the obtained sheet was cut to a size
10 of 2 cm x 2 cm, it was sterilized with a 20 kGy electron beam.
~ - g l u c o s i d a s e ~ . r a s e x t r a c t e d w i t h 1 m L o f p h y s i o l o g i c a l s a l i n e
to measure its activity with Tokyogreen-PGlu. As a result,
the recovery rate of activity was 62 %. When activity
measurement taras made on an unsterilized sheet likewise, the
15 recovery rate of activity was 71 %. It was understood from
above that the retention rate of the activity of the
sterilized fiber molded body was 87 % of that of the
unsterilized fiber molded body and that P-glucosidase was
not deactivated by electron beam sterilization.
20
Comparative Example 5
Lipase powders were sterilized with a 20 kGy electron
beam. 1mL of a lipase buffer was addedtolmgofthe powders
tomeasure theactivity oflipase. As a result, the recovery
25 rate of activity was 74 %.
Comparative example 6
P-glucosidase powders were sterilized with a 20 kGy
electron beam. 2 mg of the powders was dissolved in 1 mL
30 of physiological saline to measure the activity of
P-glucosidase with Tokyogreen-PGlu. As a result, the
retention rate of activity was 81 %.
Effect of the Invention
The sterile composition of the present invention
retains the structure and function of a protein though it
is sterilized.
5 Industrial Feasibility
The sterile composition of the present invention is
usedinthe manufacturingindustry ofmedical products which
require the function and sterility of a protein.

CLAIMS
1. A sterile composition which comprises a protein and
an aliphatic polyester containing the protein and is
5 sterilized with radiation.
2. The sterile composition according to claim 1, wherein
the protein is dispersed as particles in the aliphatic
polyester directly or as a mixture with additives which are
10 pharmaceutically acceptable.
3. The sterile composition according to claim 1 or 2,
wherein the protein is selected from the group consisting
of hemostat proteins, enzymes, transport proteins, muscle
15 proteins, defense proteins, toxin proteins, protein hormones,
storage proteins, structural proteins, growth factors and
mixtures thereof.
4. The sterile composition according to claim 1 or 2,
20 wherein the protein is a hemostat protein.
5. The sterile composition according to anyone of claims
1 to 4, wherein the aliphatic polyester is selected from the
group consisting of polyglycolic acid, polylactic acid,
25 polycaprolactone, copolymers thereof and mixtures thereof.
6. The sterile composition according to any one of claims
1 to 5 which is in a fiber form.
30 7 . The sterile composition according to claim 6 which is
produced by an electrospinning method.
8. The sterile composition according to anyone of claims
1 to 5 which is in a film form.
9. The sterile composition according to claim 8 which is
produced by a casting method.