CN109432017B - Heat-resistant freeze-drying protective agent for henpox and application thereof - Google Patents
Heat-resistant freeze-drying protective agent for henpox and application thereof Download PDFInfo
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- 238000004108 freeze drying Methods 0.000 title claims abstract description 93
- 239000003223 protective agent Substances 0.000 title claims abstract description 71
- 229960005486 vaccine Drugs 0.000 claims abstract description 45
- 201000006082 Chickenpox Diseases 0.000 claims abstract description 27
- 206010046980 Varicella Diseases 0.000 claims abstract description 27
- 238000002360 preparation method Methods 0.000 claims abstract description 19
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 15
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 15
- 229930195725 Mannitol Natural products 0.000 claims abstract description 15
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 15
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 15
- 239000000594 mannitol Substances 0.000 claims abstract description 15
- 235000010355 mannitol Nutrition 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000008213 purified water Substances 0.000 claims abstract description 10
- 241000700605 Viruses Species 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 12
- 208000000666 Fowlpox Diseases 0.000 claims description 11
- 238000001035 drying Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 238000000859 sublimation Methods 0.000 claims description 7
- 230000008022 sublimation Effects 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 241000700662 Fowlpox virus Species 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 2
- 238000007710 freezing Methods 0.000 claims description 2
- 230000008014 freezing Effects 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 230000001012 protector Effects 0.000 claims 1
- 230000009467 reduction Effects 0.000 abstract description 5
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 4
- 239000000203 mixture Substances 0.000 description 13
- 238000012360 testing method Methods 0.000 description 12
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
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- 238000007689 inspection Methods 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 229960001484 edetic acid Drugs 0.000 description 2
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- 238000005259 measurement Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention discloses a heat-resistant freeze-drying protective agent for chicken pox and application thereof, belonging to the field of preparation and application of heat-resistant freeze-drying protective agent. The invention firstly discloses a chicken pox heat-resistant freeze-drying protective agent which comprises the following components: trehalose, mannitol, polypeptone and purified water. The heat-resistant freeze-drying protective agent for the henpox has a simple formula, effectively saves resources, and is more suitable for large-scale production. The invention further discloses an application of the henpox heat-resistant freeze-drying protective agent in the preparation of a henpox heat-resistant protective agent live vaccine. The invention optimizes the freeze-drying curve in the vaccine preparation, so that the freeze-drying time is shortened to 24 hours, and the efficacy of the prepared vaccine is high. The chicken pox heat-resistant protective agent live vaccine prepared by the invention has high efficacy, can be stored for 36 months at the temperature of 2-8 ℃, has small titer reduction after heat resistance, and has more stable efficacy.
Description
Technical Field
The invention relates to a henpox heat-resistant freeze-drying protective agent, and also relates to application of the henpox heat-resistant freeze-drying protective agent in preparation of a henpox heat-resistant protective agent live vaccine, belonging to the field of preparation and application of the henpox heat-resistant freeze-drying protective agent.
Background
The research of China on the aspect of the heat-resistant freeze-drying protective agent starts later, but now, certain achievements are obtained in the research, and a chicken pox heat-resistant freeze-drying protective agent is developed, and the heat-resistant freeze-drying protective agent generally comprises the following components: 3% of gelatin, 5-6% of lactose, 2% of EDTA (ethylene diamine tetraacetic acid), 5% of PVP (K90), 2% of sorbitol, 1% of sodium glutamate, 2% of beef peptone, 1% of bovine serum albumin and 0.1% of Vc; the preparation is a dry matter component, and the residual parts in percentage are distilled water. Respectively sterilizing the effective components of the substances which can be autoclaved and do not resist high temperature of the protective agent, and then mixing to obtain the freeze-dried heat-resistant protective agent; when in use, the virus antigen solution is added into the virus antigen solution according to the proportion of 1:1, and then the virus antigen solution is frozen and dried after being subpackaged. The product is stored at 2-8 deg.C for 24 months without obvious reduction of virus titer, and is placed at 37 deg.C for 10 days, EID50About 0.4 titer drop, which takes about 28 hours for complete lyophilization.
Aiming at the defects of complex formula, long freeze-drying time and the like existing in the processes, the formula and the freeze-drying curve of the heat-resistant freeze-drying protective agent are improved, so that the formula is simpler, the freeze-drying time is shortened, resources are saved, and the method is more suitable for large-scale production.
Disclosure of Invention
The invention aims to solve the first technical problem of providing the heat-resistant chicken pox freeze-drying protective agent, which has a simple formula and is suitable for large-scale production; the chicken pox heat-resistant protective agent prepared by applying the chicken pox heat-resistant freeze-drying protective agent has high live vaccine efficacy, only reduces the titer by about 0.2 after heat resistance, and can be stored for 36 months at the temperature of 2-8 ℃;
the second technical problem to be solved by the invention is to provide the application of the chicken pox heat-resistant freeze-drying protective agent in the preparation of live vaccines of the chicken pox heat-resistant protective agent.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention firstly discloses a chicken pox heat-resistant freeze-drying protective agent which comprises the following components: trehalose, mannitol, polypeptone and purified water.
Furthermore, in each 100ml of the protective agent, the content of each component is as follows: 1-3g of trehalose, 1-3g of mannitol, 1-5g of polypeptone and the balance of purified water.
Preferably, the content of each component in each 100ml of the protective agent is as follows: 2g of trehalose, 2g of mannitol, 5g of polypeptone and the balance of purified water.
The invention further discloses a method for preparing the henpox heat-resistant freeze-drying protective agent, which comprises the following steps: weighing the components according to the proportion, mixing uniformly, and sterilizing under high pressure to obtain the traditional Chinese medicine. Wherein the autoclaving is carried out at 116 ℃ for 25 minutes.
The components of the chicken pox heat-resistant freeze-drying protective agent are preliminarily screened according to the physicochemical characteristics of the chicken pox virus, and trehalose, mannitol and polypeptone are determined as the effective components of the protective agent according to the result of effect detection after freeze-drying. The invention further optimizes the proportion of each component by an orthogonal test method, and determines the optimal formula proportion of the freeze-drying protective agent by comparing indexes such as physical properties, residual moisture, vacuum degree, virus loss before and after freeze-drying and the like. The result shows that each 100ml of the protective agent contains 2g of trehalose, 2g of mannitol, 5g of polypeptone and the balance of purified water, and the protective agent is the optimal protective agent for the chicken pox virus antigen, the physical properties, the residual moisture and the vacuum degree of the protective agent are all qualified, the virus loss after freeze-drying is minimum (the titer is highest), and the titer which is reduced after heat resistance at 37 ℃ is minimum.
The invention further discloses application of the chicken pox heat-resistant freeze-drying protective agent in preparation of a live vaccine of the chicken pox heat-resistant protective agent.
The invention also discloses a preparation method of the chicken pox heat-resistant protective agent live vaccine, which comprises the following steps: the fowl pox virus liquid and the fowl pox heat-resistant freeze-drying protective agent are uniformly mixed and freeze-dried to obtain the chicken pox virus protective agent.
Wherein, the volume ratio of the fowl pox virus liquid to the fowl pox heat-resistant freeze-drying protective agent is 1:1 and mixing. The chicken pox virus liquid can be obtained by adopting SPF chicken embryo culture or cell culture multiplication chicken pox virus, and the concrete method is known by the person skilled in the art. The freeze-drying lyophilization profile is selected from:
prefreezing at-40 deg.c for 3 hr, first stage sublimation at-40 deg.c to-15 deg.c for 28 hr, and second stage drying at-15 deg.c to-28 deg.c for 17 hr; or,
pre-freezing at-30 ℃ for 4 hours, sublimating at-30 ℃ to-15 ℃ for 18 hours in the first stage, and drying at-15 ℃ to 22 ℃ for 14 hours in the second stage; or,
prefreezing at-45 deg.c for 3 hr, first stage sublimation at-30 deg.c to-15 deg.c for 14 hr, and second stage drying at-15 deg.c to 25 deg.c for 7 hr.
Preferably, the freeze-drying profile of the freeze-drying is: prefreezing at-45 deg.c for 3 hr, first stage sublimation at-30 deg.c to-15 deg.c for 14 hr, and second stage drying at-15 deg.c to 25 deg.c for 7 hr. For a total period of 24 hours.
According to the performance characteristics of a freeze-drying machine, the optimized formula of the heat-resistant freeze-drying protective agent for the henpox, the biological characteristics and biochemical indexes of freeze-dried viruses and other factors, 3 groups of freeze-drying curves are set so as to optimize the best freeze-drying curve suitable for production. The results show that the lyophilization profile used: prefreezing at-45 deg.c for 3 hr, first stage sublimation at-30 deg.c to-15 deg.c for 14 hr, second stage drying at-15 deg.c to 25 deg.c for 7 hr; the whole freeze-drying process only needs 24 hours, the freeze-drying time is short, and the virus titer is reduced little after the vaccine is heat-resistant at 37 ℃, and only 0.25 titer is reduced.
In order to verify the production performance of the optimized freeze-drying curve, 10 batches of vaccines are respectively prepared by utilizing the optimized freeze-drying curve and the original freeze-drying curve, and the efficacy comparison result of the vaccines shows that the efficacy of the vaccine prepared by the optimized freeze-drying curve is obviously higher than that of the original freeze-drying curve.
The invention also discloses the fowl pox heat-resistant protective agent live vaccine prepared by the preparation method. The chicken pox heat-resistant protective agent live vaccine prepared by the invention has high efficacy, the vaccine can be stored for 36 months at the temperature of 2-8 ℃, the virus titer is reduced by about 0.2 titer after the heat resistance at the temperature of 37 ℃, and the efficacy of the vaccine is more stable.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the heat-resistant freeze-drying protective agent for the henpox has a simple formula, effectively saves resources, and is more suitable for large-scale production. The invention optimizes the freeze-drying curve in the vaccine preparation, shortens the freeze-drying time to 24 hours and has high vaccine preparation efficiency. The chicken pox heat-resistant protective agent live vaccine prepared by the invention can be stored for 36 months at the temperature of 2-8 ℃, the titer reduction after heat resistance is less, the efficacy of the vaccine is more stable, and the safety is good.
Definitions of terms to which the invention relates
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The terms "vaccine" or "vaccine composition" are used interchangeably to refer to a pharmaceutical composition that includes at least one immunogenic composition that induces an immune response in an animal. The vaccine or vaccine composition may protect the animal from disease or possible death due to infection and may or may not include one or more additional components that enhance the immunological activity of the active component. The vaccine or vaccine composition may additionally comprise further components typical for vaccines or vaccine compositions, including for example adjuvants or immunomodulators. The immunologically active components of the vaccine may include the fully live organism in its original form or as an attenuated organism in a modified live vaccine, or an organism inactivated by suitable means in a killed or inactivated vaccine, or a subunit vaccine comprising one or more immunogenic components of the virus, or a genetically engineered, mutated or cloned vaccine prepared by methods known to those skilled in the art. The vaccine or vaccine composition may comprise one or more than one of the above components at the same time.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 screening of Heat-resistant Freeze-drying protective agent for fowlpox and optimization of Freeze-drying Curve
1. Preparation of heat-resistant freeze-drying protective agent for henpox
Preparation of solution A: PVP (K30) 5%, trehalose 3%, mannitol 1% and polypeptone 4% are evenly mixed, and then the mixture is sterilized by high pressure for standby (sterilization is carried out at 116 ℃ for 25 minutes).
And (3) preparation of a solution B: mixing three protective agents of Medium1990.3%, vitamin C (Vc) 0.3%, and glycine 0.2%, sterilizing 0.22um, and keeping.
In the preparation of the solution A or the solution B, the percentage is calculated by g/ml, and the rest components are purified water.
Mixing the solution A and the solution B in a ratio (volume ratio) of 1:1 to obtain the heat-resistant chicken pox freeze-drying protective agent.
The heat-resistant freeze-drying protective agent for chicken pox and the chicken pox virus liquid are prepared into vaccine according to the proportion (volume ratio) of 1:1, and after being uniformly mixed, the mixture is subpackaged, capped and sent into a freeze-drying box for freeze-drying. The freeze-drying curve is: the product is pre-frozen for 4 hours at the temperature of minus 30 ℃, sublimed for the first time at the temperature of minus 30 ℃ to minus 15 ℃ for 18 hours, and dried for the second time at the temperature of minus 15 ℃ to 22 ℃ for 14 hours. For a total period of 36 hours.
The results of the test after lyophilization are shown in Table 1.
TABLE 1 Freeze-drying product test results
2. The protective agent is primarily screened according to the physicochemical characteristics of the virus
Since solution B is sensitive to heat, autoclaving cannot be performed, and ultrafiltration costs are high. The effect on lyoprotection after omission of solution B was tested. The results of the test are shown in Table 2.
Table 2 freeze-dried product test results
The above results show that the protective effect of the protective agent after omitting solution B after lyophilization is reduced, but the magnitude is not large.
3. Optimization of liquid A ingredients
Adjusting the component of the solution A:
group 1 PVP (K30) 2%, trehalose 2%, mannitol 2%;
group 2 PVP (K30) 2%, mannitol 2%, polypeptone 2%;
trehalose 2%, mannitol 2%, polypeptone 2% of group 3;
group 4 PVP (K30) 2%, trehalose 2%, polypeptone 2%;
firstly, fixing the proportion (2 percent in terms of g/ml) and optimizing the components; the balance of each group was purified water. The results of the efficacy test after the freeze-drying of the prepared vaccine are shown in Table 3.
TABLE 3 Effect test results after lyophilization
The results show that the efficacy test for the vaccine prepared by the group 3 formulation is best.
4. Optimizing the component ratio
The components of the group 3 formulation with the best efficacy test were selected: trehalose, mannitol and polypeptone are subjected to orthogonal test method (three factors and three levels) to prepare 9 groups of protective agents, and a control group 1 and 10 groups (table 4 and table 5) are prepared to respectively prepare vaccines for freeze-drying. And performing primary screening by comparing indexes such as physical properties, residual water, vacuum degree, virus loss before and after freeze-drying and the like to determine the optimal formula proportion of the freeze-drying protective agent.
TABLE 4 screening ratios of different protective Agents
Table 510 different combinations of group protectants
And (3) performing character inspection, safety inspection, efficacy inspection, residual moisture measurement, vacuum degree measurement and aging resistance test on the 10 groups of freeze-dried finished products. And screening out all qualified protective agent formulas of the effect test, the safety test and the aging resistance test according to the results. The results of the freeze-dried product are shown in Table 6. According to the results, the 5 th group of protective agents were screened to have all acceptable physical properties, residual moisture and vacuum, and to have minimal virus loss (highest titer) after lyophilization. The invention determines the protective agent of the group 5 as the optimal protective agent of the chicken pox virus antigen, and the content of each component is as follows: in 100ml of protective agent, 2g of trehalose, 2g of mannitol, 5g of polypeptone and the balance of purified water.
TABLE 6 test results for lyophilized products
As can be seen from the above table, formulation No. 5 showed the least titer reduction, 0.21 titer reduction, by the initial screening of the 9-group formulation.
5. Optimization of freeze-drying curve of formula of qualified product protective agent
According to the performance characteristics of the freeze-drying machine, the type of the protective agent, the biological characteristics of the freeze-dried virus, biochemical indexes and other factors, 3 groups of freeze-drying curves are preliminarily set so as to optimize the optimal freeze-drying curve suitable for production.
The product is pre-frozen for 3 hours at the temperature of minus 40 ℃, sublimated for the first time to be minus 40 ℃ to minus 15 ℃ and for 28 hours, and dried for the second time to be minus 15 ℃ to 28 ℃ and for 17 hours. The whole process lasts for 48 hours.
② the product is pre-frozen for 4 hours at-30 ℃, sublimed for the first time at-30 ℃ to-15 ℃ for 18 hours, and dried for the second time at-15 ℃ to 22 ℃ for 14 hours. For a total period of 36 hours.
③ prefreezing the product at minus 45 ℃ for 3 hours, sublimating for the first time at minus 30 ℃ to minus 15 ℃ for 14 hours, and drying for the second time at minus 15 ℃ to 25 ℃ for 7 hours. For a total period of 24 hours.
The optimized protective agent formula freeze-drying curve optimization results are shown in table 7.
TABLE 7 optimized protectant formulation lyophilization curve optimization results
The results show that the freeze-drying curve of group 3 is the best freeze-drying curve, not only the freeze-drying time is short, and the whole freeze-drying process only needs 24 hours, but also the virus titer is reduced little after the heat resistance at 37 ℃, and is reduced by 0.25 titer.
In order to verify the production performance of the new lyophilization curve, 10 batches of vaccines were prepared according to the present invention using the optimized lyophilization curve of group 3 and the original lyophilization curve, and the efficacy comparison results of the vaccines are shown in table 8. Original freeze-drying curve: the product is pre-frozen for 4 hours at the temperature of minus 30 ℃, sublimed for the first time at the temperature of minus 30 ℃ to minus 15 ℃ for 18 hours, and dried for the second time at the temperature of minus 15 ℃ to 22 ℃ for 14 hours. For a total period of 36 hours.
Table 8 comparison of the potency of two lyophilization profiles for vaccine preparation (10)X)
As can be seen from the above table, the optimized lyophilization profile produced vaccines with significantly higher potency than the original lyophilization profile.
Claims (9)
1. The heat-resistant freeze-drying protective agent for the chicken pox is characterized by comprising the following components: trehalose, mannitol, polypeptone and purified water; in each 100ml of the protective agent, the contents of the components are as follows: 1-3g of trehalose, 1-3g of mannitol, 1-5g of polypeptone and the balance of purified water.
2. The henpox heat-resistant freeze-drying protective agent according to claim 1, which is characterized in that the contents of the components are as follows: 2g of trehalose, 2g of mannitol and 5g of polypeptone.
3. A method for producing the fowlpox heat-resistant lyoprotectant of claim 1 or 2, comprising: weighing the components according to the proportion, mixing uniformly, and sterilizing under high pressure to obtain the traditional Chinese medicine.
4. The use of the fowlpox heat-resistant lyoprotectant according to claim 1 or 2 in the preparation of a live vaccine of fowlpox heat-resistant protectant.
5. A preparation method of a fowl pox heat-resistant protective agent live vaccine is characterized by comprising the following steps: the fowl pox virus liquid and the fowl pox heat-resistant freeze-drying protective agent of claim 1 or 2 are uniformly mixed and freeze-dried to obtain the chicken pox virus protective agent.
6. The method of claim 5, wherein the freeze-dried lyophilization profile is selected from the group consisting of:
prefreezing at-40 deg.c for 3 hr, first stage sublimation at-40 deg.c to-15 deg.c for 28 hr, and second stage drying at-15 deg.c to-28 deg.c for 17 hr; or,
pre-freezing at-30 ℃ for 4 hours, sublimating at-30 ℃ to-15 ℃ for 18 hours in the first stage, and drying at-15 ℃ to 22 ℃ for 14 hours in the second stage; or,
prefreezing at-45 deg.c for 3 hr, first stage sublimation at-30 deg.c to-15 deg.c for 14 hr, and second stage drying at-15 deg.c to 25 deg.c for 7 hr.
7. The method of claim 6, wherein the freeze-drying has a freeze-drying profile of: prefreezing at-45 deg.c for 3 hr, first stage sublimation at-30 deg.c to-15 deg.c for 14 hr, and second stage drying at-15 deg.c to 25 deg.c for 7 hr.
8. The method of claim 5, wherein: the volume ratio of the fowl pox virus liquid to the fowl pox heat-resistant freeze-drying protective agent is 1:1 and mixing.
9. A live vaccine of a fowl pox protector prepared by the process according to any one of claims 5 to 8.
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