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(19) State Intellectual Property Office of the People's Republic of China

(12)
Invention (10)Authorized Publication No. CN 109620962 B
(45) Authorization announcement date

patent 2021.11.26

(21) Application No. 201710929420 .1 a61k 47/32

(2006 .01) a61k 47/10
(22) Application date 2017 .10 .09
(2006 .01) a61k 39/39
(65) Published document (2006 .01)
number of the same
application Application (56) Comparison of documents
Publication No. CN CN 101143131 A ,2008 .03 .19
109620962 A CN 105816873 A ,2016 .08 .03
WO 0224211 A1 ,2002 .03 .28
(43) Application Publication Date
CN 106176634 A ,2016 .12 .07
2019 .04 .16
Reviewer Pang Xiuping
(73) Patentee Zhongmu Industrial Co.
Address: Building 16-19, No. 188, South
4th Ring Road West, Fengtai District,
Beijing 100070, China

(72) Inventor Pan Jingxue Yan Shi

Yang Xiaorong
(74) Patent Agency Beijing Luhao Intellectual
Property Agency Co.
Company 11002
Represented by Wenjun Wang, Lu
Wang
(51)In t.Cl .
A61K 47/40 (2006 .01)
A61K 47/36 (2006 .01) 1 page of claims 8 pages of
instructions
(54) Name of the invention
A vaccine diluent and its preparation method and application
(57) Summary
The present invention relates to the field of
immunology, and specifically discloses a vaccine
diluent whose raw materials include water for
injection and polymer, optionally with the addition of
an immune booster. Said vaccine diluent is prepared
by weighing the raw materials into the bottom of
a stainless steel high-pressure reactor,puttingina
magnet, and after sealing, the reactor is put into a water
bath with magnetic stirring, setting the water bath
temperature, turning on a single-cylinder syringe
pump to charge carbon dioxide to the pressure
required for the experiment, stopping the press, closing the
reactor valve, keeping its internal pressure constant,
and setting the stirring rate. After the reaction is placed
in the water bath at constant temperature for a certain
time, the pressure is removed and the result is obtained.
The combination of diluent provided by the present
invention after supercritical carbon dioxide
treatment is easy for industrial production of vaccines,
safe and effective. The diluent made in the present
invention can be applied to both live vaccine
antigens and inactivated vaccine antigens or
synthetic peptide vaccine antigens.
CN 109620962 B Rights Rights Requirements Request
Book 1/1 页

1 . A diluent for inactivated vaccine antigens, characterized by raw materials of 89 .98%

aqueous solution for injection, 7% poly(lactic acid) hydroxyethylic acid, 2% poly(vinyl pyrrolidone), 1%
chitosan, and 0 .02% imiquimod, an immune booster.
2. The method of preparing the diluent of claim 1, characterized in thatitcomprises:
Put the raw materials into the bottom of the high-pressure reactor, put in the magnet, seal the reactor
and put it into the water bath with magnetic stirring, set the water bath temperature from 30 to 4 5 ° C turn
,
on the single-cylinder syringe pump to charge carbon dioxide to the pressure required for
the experiment 8 to 15 M P a ,stop stamping, close the reactor valve, keep its internal pressure
constant, set the stirring rate from 200 to 2000 rpm; place it in the water bath at a constant
temperature After reacting for 10 to 120 minutes, remove the pressure, and the diluent is obtained.
3. The diluent of claim 1 for use in the preparation of animal vaccines.
4 . An animal vaccine, characterized in that it is obtained by the preparation with the participation of the
diluent of claim 1.
5 . Animal vaccine according to claim 4, characterized in that said animal vaccine is an inactivated swine fever
vaccine.

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CN 109620962 B S Mi B 3 /8
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A vaccine diluent and its preparation method and application

Technology Field
[0001] The present invention relates to the field of immunology and, specifically, to a vaccine diluent.

Background Technology
[0002] Vaccine diluent, a reagent that is used for the immunization of the organism after dilution of the vaccine.
[0003] T h e role of vaccine diluents is very important, not only to dilute the vaccine, but also to
protect the vaccine and improve the immune effect of the vaccine.
[0004] The type, source and production process of the vaccine will have a certain impact on the selection of
diluent. The differences in manufacturers, immunization effects, etc., make the requirements for dilution
multiples, dilution methods and diluents of vaccines in the actual application process also differ greatly,
and if they are chosen arbitrarily, not only will they fail to provide good dilution effects, but also may produce
adverse reactions.
[0005] Currently, animal vaccines are often diluted using deionized water or physiological saline
or PBS buffer for vaccination of animals, which often leads to unsatisfactory immunization effect
or immunization failure.

Invention content
[0006] In order to solve the problems in the prior art, the present invention aims to
provide a vaccine diluent and its preparation method and application.
[0007] In order to achieve the purpose of the present invention, the technical solution of the present invention
is as follows:
[0008] In a first aspect, the present invention provides a vaccine diluent having a raw material
comprising water for injection in combination with cyclodextrin, modified starch, polycaprolactone,
polyfumaric acid-sebacic acid, chitosan, mannan, polyoxyethylene polyoxypropylene ether block copolymer,
polymethacrylate, polyacrylate, polyvinylpyrrolidone, poloxamer series, polylactic acid one or a combination of
one or more of -hydroxyethyl acid, poly(cross-linked acrylate), peptidoglycan, polyhydroxybutyric acid,
polyvinylpyrrolidone and polyethylene glycol;
[0009] It was prepared as follows:
[0010] Put the raw material into the bottom of the high-pressure reactor, put in the magnet,
seal the reactor and put it into a water bath with magnetic stirring, set the water bath temperature from 32 to
5 0 ° C ,turn on the single-cylinder syringe pump to charge carbon dioxide to the pressure
required for the experiment 7 .3 to 15 MPa, stop stamping, close the reactor valve, keep its internal
pressure constant, set the stirring rate from 200 to 2000 rpm; keep the temperature in the water
bath at a constant temperature Set the stirring rate at 200 to 2000 rpm; keep the reaction
at a constant temperature in a water bath for 10 to 120 minutes, then remove the pressure and
obtain the diluent.
[0011] Further as a preference, the raw material of the vaccine diluent is one of (1) to (3):

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CN 109620962 B S Mi B 4 /8
[0012] ay n o
(1) Water for injection, cyclodextrin, chitosan and polyethylene glycol; pages

[0013]
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(2) Water for injection, polycaprolactone, polyfumaric acid-decanedioic acid and water-
soluble starch;
[0014] (3) Water for injection, mannan, peptidoglycan, poly(lactic acid-hydroxyethylic acid) and
poly(hydroxybutyric acid).
[0015] On the basis of the above preferred solution, the raw materials and their mass
percentages are further optimized as (1)
～One of (3):
[0016] (1) 90% water for injection, 4% cyclodextrin, 4% chitosan and 2% polyethylene glycol;
[0017] (2) 90% water for injection, 3% polycaprolactone, 3% polyfumaric acid-decanedioic
acid and 4% water-soluble starch;
[0018] (3) Water for injection 90%, mannoprotein 3%, peptidoglycan 1%, poly(lactic acid-
hydroxyethylic acid) 1% and poly(hydroxybutyric acid) 5%.

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[0019] It has been experimentally verified that
s the vaccine
g ok dilution using the vaccine diluent provided
by the present invention can effectively improve the immunization effect of the vaccine.
[0020] In a second aspect, the present invention further introduces an immune booster on the basis of the
above-mentioned vaccine diluent for better immune effect of the diluted vaccine.
[0021] Specifically, a vaccine diluent having as raw materials water for injection, an immune booster, and one or
more of cyclodextrin, modified starch, chitosan, mannan, polyoxyethylene propylene oxide block copolymer,
polymethacrylate, polyacrylate, polyvinylpyrrolidone, poly(lactic acid-hydroxyglycolic acid) (PLGA),
poly(cross-linked acrylate), and poly(ethylene glycol) one or a combination of t h e s e ; [0022]
prepared as follows:
[0023] The raw material is put into the bottom of the high-pressure reactor, put into the
magnet, and after sealing, the reactor is put into a water bath with magnetic stirring, set the water bath
temperature from 30 to 4 0 ° C ,turn on the single cylinder syringe pump to charge carbon
dioxide to the pressure required for the experiment from 8 to 15 M P a stop , stamping, close the
reactor valve, keep its internal pressure constant, and set the stirring rate from 200 to 2000 rpm. Set the
stirring rate at 200 to 2000 rpm. After the reaction is placed at constant temperature in the water
bath for 10 to 120 minutes, the pressure is removed and the diluent is obtained.
[0024] Further, said immune enhancer is preferably selected from one or more of astragalus polysaccharide,
purple daisy extract, saponin, imiquimod and polyinosinic acid-polycytidylic acid.
[0025] Further as a preferred option, the raw material of the vaccine diluent is one of (1) to
(3):
[0026] (1) Water for injection, polyvinylpyrrolidone, poloxamer 407 and saponin Quil-A;
[0027] (2) Water for injection, polycaprolactone, poloxamer 188 and astragalus polysaccharide;
[0028] (3) Water for injection, polylactic acid-hydroxyethylic acid, polyvinylpyrrolidone, chitosan and
imiquimod.
[0029] On the basis of the above preferred solution, the raw materials and their mass
percentages are further optimized as (1)
～One of (3):
[0030] (1) Water for injection 89 .9%, polyvinylpyrrolidone 9%, poloxamer 407 1 % , and
saponin Quil-A 0 .1%;
[0031] (2) water for injection 89 .9%, polycaprolactone 6%, poloxamer 188 4 % and
astragalus polysaccharide 0 .1%;
[0032] (3) Water for injection 89 .98%,PLA-hydroxyethylic acid 7%, polyvinylpyrrolidone
2%, chitosan 1% and mirex
Mott 0 .02%.
[0033] In a third aspect, the present invention provides the application of the aforementioned
vaccine diluents without immune enhancers/vaccine diluents containing immune enhancers in
the preparation of animal vaccines.
[0034] O n this basis, the present invention provides an animal vaccine obtained by the involvement
of the vaccine diluent described herein in the preparation of the vaccine. In particular, this can be shown
by preparing a diluent using the vaccine diluent described in the present invention and diluting the antigen of a
commercially available live swine fever cell vaccine in accordance with the standard ratio.
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[0035] Further, the vaccine to be diluted is any vaccine commonly used in the art, which is not otherwise
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limited by the present invention, and optionally, said vaccine may contain live or inactivated vaccine
antigens or synthetic peptide vaccine antigens, etc.
[0036] The percentages mentioned in the present invention refer to the mass percentages if not
otherwise specified; the raw materials or reagents involved are ordinary commercially available
products, and the operations involved are conventional in the art if not otherwise specified.
[0037] O n t h e basis of common knowledge in the art, each of the above preferred conditions,
can be combined with each other to obtain specific embodiments.
[0038] The beneficial effect of the present invention is that:
[0039] The present invention provides a diluent for animal vaccines with immune enhancing
effect and achieves good immunity after vaccination.
[0040] Currently in the production of vaccine diluents are mostly used in liquid or solid dissolved,
sterilized and used directly, the present invention

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To provide a diluent product prepared by
s applying
g ok supercritical technology to the diluent
product after pretreatment and then decontamination, the method is simple and the made diluent
has good immunity. A supercritical fluid is a high-density fluid in a state of supercritical temperature and
critical pressure. Supercritical fluids have many unique properties, such as small viscosity, density, diffusion
coefficient, solubilization capacity and other properties are very sensitive to changes in temperature
and pressure: viscosity and diffusion coefficient are close to gases, while density and solubilization capacity
are close to liquids. Supercritical fluids have both gas and liquid properties, their viscosity is similar to
that of gas, but their diffusion coefficient is much larger than that of liquid, and their density is similar to that of
liquid. Supercritical CO2 is one of the most widely studied fluids because it has the following
characteristics: CO2 critical temperature is 31 .26℃, critical pressure is 72 .9atm, critical conditions are easy to
achieve; CO2 is chemically inactive, colorless, tasteless and non-toxic, good safety; cheap, high purity and easy to
obtain. Since the critical temperature of supercritical CO2 is close to room temperature, the critical pressure is not
high, chemically inert, and does not pollute the environment, making it a green medium, and the
thermodynamic activity of supercritical fluids can be controlled by changing the pressure, which
provides a flexible and adjustable way to control material morphology and properties. The use of
supercritical fluids to dissolve and plasticize polymers after modification treatment, the material
volume increases, the pores also gradually expand, the polymer backbone structure is opened, and the
ability to carry and transport substances dissolved in carbon dioxide into the polymer matrix, the
polymer
Modification and tuning at the multi-scale structural level to achieve controlled "tailoring"of polymer
properties, resulting in micron or
nanometer-sized microporous networks and lead to changes in their basic physical properties, such
as surface tension, crystallization behavior and rheological behavior of the polymer. The supercritical CO2-
treated diluent combination provided by the present invention is easy to produce industrially for vaccines in a
safe and effective manner. The diluents made in the present invention can be applied to both live
vaccine antigens, inactivated vaccine antigens or synthetic peptide vaccine antigens.

Specific implementation
[0041] Preferred embodiments of the present invention will be described in detail below in
connection with embodiments. It is to be understood that the following embodiments are
given for illustrative purposes only and are not intended to limit the scope of the present
invention. Various modifications and substitutions may be made to the present invention by those
skilled in the art without departing from the purpose and spirit of the present invention.
[0042] The experimental methods used in the following embodiments are conventional if
not specifically described.
[0043] The materials, reagents, etc. used in the following embodiments are available commercially if not
otherwise specified.
[0044] Example 1
[0045] The raw materials and mass percentages are as follows:
[0046] 1、Aqueous solution for injection 90%;
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CN 109620962 B S Mi B 8 /8
[0047] 2. Polymer combinations: ay n o pages

[0048] Cyclodextrin 4 %;
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[0049] Chitosan 4 %;
[0050] Polyethylene glycol 4000 2 %.
[0051] Preparation method:
[0052] Weigh the above aqueous solution for injection and polymer combination into
the bottom of the stainless steel high-pressure reactor,putinthemagnet,dense
After closing, the reactor is put into a water bath with magnetic stirring, set the water bath temperature
from 32 to 50°C, turn on the single cylinder syringe pump to charge carbon dioxide to the
required pressure of 7 .3 to 15 MPa for the experiment, stop stamping, close the reactor valve,
keep its internal pressure unchanged, and set the stirring rate from 200 to 2000 rpm. After applying
supercritical carbon dioxide treatment in a water bath at constant temperature for 10 to 120
minutes, unpressure, open the reactor, and remove to obtain the diluent.
[0053] Example 2
[0054] This embodiment differs from embodiment 1 in that:

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[0055] The raw materials and masss g ok
percentages are as follows:
[0056] 1、Aqueous solution for injection
90%;
[0057] 2. Polymer combinations:
[0058] Polycaprolactone 3 %;
[0059] Polyfumaric acid-decanedioic
acid 3 %;
[0060] Water-soluble starch 4 %.
[0061] Example 3
[0062] The difference between this
embodiment and embodiment 1
is that
[0063] The raw materials and mass
percentages are as follows:
[0064] 1、Aqueous solution for injection
90%;
[0065] 2. Polymer combinations:

[0066]

[0067] Example 4
[0068] The raw materials and mass percentages are as follows:
[0069] 1. aqueous solution for injection 89 .9%;
[0070] 2. Polymer combinations:
[0071] Polyvinylpyrrolidone 9 %;
[0072] Peloxam 407 1 %;
[0073] 3. Immune booster:
[0074] Saponin Quil-A 0 .1 %.
[0075] Preparation method:
[0076] Weigh the above aqueous solution for injection and polymer combination into
the bottom of the stainless steel high-pressure reactor and add immune enhancing
The reactor is placed in a water bath with magnetic stirring, set the water bath temperature from 30 to 45°C,
turnonthe singlecylinder syringe pump to charge carbon dioxide to the required pressure of
8 to 15 MPa for the experiment, stop stamping, close the reactor valve, keep its internal pressure
constant, and set the stirring rate from 200 to 2000 rpm. Set the stirring rate at 200 to 2000 rpm.
After the reaction is placed at constant temperature in the water bath for 10 to 120 minutes, remove the
pressure, open the reactor, and take out the diluent.

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[0077] Example 5 ay n o pages
[0078] This embodiment differs froms g ok
embodiment 4 in that
[0079] The raw materials and mass
percentages are as follows:
[0080] 1. Aqueous solution for injection
89 .9%;
[0081] 2. Polymer combinations:
[0082] Polycaprolactone 6 %;
[0083] Poloxam 188 4 %;
[0084] 3. Immune booster:
[0085] Astragalus polysaccharide
0 .1 %.

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[0086] Example 6 s g ok
[0087] This embodiment differs from embodiment 4 in that
[0088] The raw materials and mass percentages are as follows:
[0089] 1. Aqueous solution for injection 89 .98%;
[0090] 2. Polymer combinations:
[0091] Poly(lactic acid-hydroxyglycolic acid) (PLGA) 7 %;
[0092] Polyvinylpyrrolidone 2 %;
[0093] Chitosan 1 %;
[0094] 3. Immune booster:
[0095] Imiquimod 0 .02%.
[0096] Example 7
[0097] This embodiment is used to illustrate an animal vaccine containing the diluent
described in the present invention.
[0098] This example is based on the swine fever vaccine, which is prepared as follows:
[0099] The raw materials described in Example 1 are proportionally weighed into the bottom of
the stainless steel high-pressure reactor, placed into the magnet, airtight
After that, the reactor was put into a water bath with magnetic stirring, set the water bath temperature
at 45°C, turn on the single cylinder syringe pump to charge carbon dioxide to the pressure
required for the experiment to 12 MPa, stop stamping, close the reactor valve, keep its internal
pressure unchanged, and set the stirring rate at 800 rpm. After applying supercritical carbon dioxide
treatment in the water bath for 60 minutes, unpressure, open the reactor, remove and filter with a filter
membrane to remove bacteria to obtain diluent.
[0100] Commercially available live swine fever cell vaccine antigens conforming to standard
swine fever vaccines were prepared by diluting them with the above diluents in standard
proportions to form live swine fever vaccine experimental group A.
[0101] Example 8
[0102] This embodiment is used to illustrate an animal vaccine containing the diluent
described in the present invention.
[0103] This example is based on a swine fever vaccine, which is prepared as follows:
[0104] The raw materials described in Example 6 were proportionally weighed and placed in the
bottom of the stainless steel high-pressure reactor, and immune-enhanced
The reactor is put into the water bath with magnetic stirring, set the water bath temperature at 40℃, turn on the
single cylinder syringe pump to charge carbon dioxide to the pressure required for the
experiment 10MPa, stop stamping, close the reactor valve, keep its internal pressure unchanged, set the
stirring rate at 1000 rpm. After the reaction is placed at constant temperature in the water bath for
60 minutes, the pressure is removed, the reactor is opened, and the diluent is obtained by filtering and
de-bacterizing with a filter membrane.
[0105] Commercially available live swine fever cell vaccine antigens conforming to the
standard were prepared as live swine fever vaccine experimental group B by using the above
diluents and diluting them according to the standard proportions.

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[0106] ay n o
Experimental group C of inactivated swine fever vaccine was prepared by dilutingpages
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commercially available antigens conforming to the standard swine fever cell inactivated
vaccine with the above diluent, according to the standard ratio of experimental group B.
[0107] Experimental Example 1
[0108] The present experimental example is used to illustrate the index test of an
animal vaccine containing the treated diluent described in the present invention and is
described in relationtothefollowing:
[0109] 1 . Sterility testing
[0110] 1 0 g of peptone,1 0 0 0 m l o f meat dip,5 g o f sodium chloride,15-20g
of agar, take peptone and sodium chloride, agar into meat dip, dissolve at slight temperature, adjust pH
to weakly alkaline, boil, filter clear, adjust pH so that after sterilization is 7 .2±0 .2 divide
, and sterilize. After
inoculation of the prepared experimental and control groups of vaccine with medium by incubation at
30-35°C for 48 hours, the results were observed to be sterile.

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[0111] 2 . Evaluation of animal immunization
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[0112] The evaluation was as follows: animals were vaccinated without abnormal reactions, blood was
collected at the specified time to isolate the serum, and the serum antibody potency was tested.
[0113] Experimental group A:live swine fever vaccine of embodiment 7 of the present invention;
[0114] Experimental group B:live swine fever vaccine of embodiment 8 of the present invention;
[0115] Experimental group C:in
swine
a
tive
cd fever vaccine of embodiment 8 of the present invention;
[0116] Control group A:th
same
e proportions of the same commercially available antigens were
mixed and prepared as vaccines according to the standard antigen dilutions;
[0117] Control group B:commercially inactivated antigens were mixed and diluted with PBS
buffer in the same proportion as in experimental group C to prepare the vaccine;
[0118] Blank control group: no vaccine was administered.
[0119] a . Animal safety tests
[0120] Two guinea pigs weighing 350-450 g were used for each group of vaccine, and
each was injected subcutaneously with m l o f t h e experimental and control
groups:five mice weighing 18-22 g were used, and each was injected subcutaneously with
0 .5 ml of the experimental and control groups of vaccine. 7 days of continuous
observation were observed, and no abnormal clinical reactions were observed.
[0121] b . Antibody detection
[0122] Immunization of animals according to protocol requirements:
[0123] Twenty-seven piglets weaned at 7-8 weeks of age with 20-30 kg of antibodies
against swine fever were divided into six groups, including five piglets in each of experimental groups
A,B,C and control groups A a n
d
B, and two piglets in the blank group. Each head of the experimental
and control groups was vaccinated intramuscularly with the vaccine provided by the present
invention at a dose of 1 ml per head, and the blank group was not vaccinated and served as a blank
control. All groups were kept in isolation for observation. Blood was collected at 1 week, 2 weeks,3 weeks,4
weeksand5 weeks after immunization, and serum was isolated to determine the potency of swine fever
antibodies in the serum by indirect ELISA test for swine fever virus. The OD values of the live vaccine
group were higher than those of the control group A, andhigherantibodies appeared one
week after immunization, while the OD values of the inactivated vaccine group were much
higher than those of the PBS buffer group and could be maintained at higher levels. The
specific data are shown in Table 1.
[0124] Table 1 Results of immune evaluation of this animal (antibody OD)

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[0125]

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[0126]

[0127] Although, the present invention has been described in detail above with a general
description and specific embodiments, some modifications or improvements can be made on the
basis of the present invention, as will be apparent to those skilled in the art. Therefore, these
modifications or improvements made on the basis of not deviating from the spirit of the present invention
are within the scope of protection claimed by the present invention.

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