CN115678859A - Freezing storage protective agent for virus vaccine semi-finished product, preparation method and application thereof - Google Patents
Freezing storage protective agent for virus vaccine semi-finished product, preparation method and application thereof Download PDFInfo
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- CN115678859A CN115678859A CN202110860765.2A CN202110860765A CN115678859A CN 115678859 A CN115678859 A CN 115678859A CN 202110860765 A CN202110860765 A CN 202110860765A CN 115678859 A CN115678859 A CN 115678859A
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Abstract
The invention provides a virus vaccine semi-finished product cryopreservation protective agent which comprises the following components in percentage by mass: 2 to 15 percent of sucrose, 5 to 25 percent of trehalose, 0.5 to 2 percent of pullulan, 1 to 3 percent of mannitol, 0.1 to 3%L-hydroxyproline, 0.15 to 3 percent of epsilon-polylysine, 0.1 to 5 percent of glycine and the balance of water for injection, wherein the total is 100 percent. The virus vaccine semi-finished product cryopreservation protective agent disclosed by the invention is applied to the existing preservation link of virus vaccine semi-finished products, can better regulate and control the appearance of ice crystals during freezing, reduces virus cryopreservation damage, solves the problem of efficient and stable cryopreservation of the vaccine semi-finished products in the industry, provides more powerful guarantee for the subsequent production of high-quality vaccines, and is simple to prepare, simple and convenient to operate and has a very high application prospect.
Description
Technical Field
The invention relates to the field of biotechnology and microbial products, in particular to a virus vaccine semi-finished product cryopreservation protective agent, a preparation method and application thereof.
Background
The vaccine is a biological product which is made of various pathogenic microorganisms and used for vaccination, and is classified into bacterial vaccine and viral vaccine according to whether the pathogenic microorganisms are bacteria or viruses. In the production and manufacturing link of virus vaccines, the virus is called a vaccine semi-finished product after multiplication and harvest and before freeze-drying, before the finished vaccine is produced, the semi-finished product is usually frozen at-20 ℃ for 1-3 weeks, the loss of virus activity is serious and is reduced by at least 90%, and the vaccine preparation standard is difficult to reach in the later stage, so that the semi-finished product is wasted; in contrast, there was little to no loss of viral activity after frozen storage at temperatures of-70 ℃ or even lower. The reason for this is presumably related to the size of the ice crystals formed, and generally the faster the freezing rate, the finer the shape of the ice crystals formed and the closer the ice crystal distribution is to the original water distribution in the fresh material. The freezing rate is slow and ice crystals tend to form in situ, and water initially migrates more to these ice crystals, forming larger ice bodies. However, in the actual production of vaccines, because the energy consumption of a refrigeration house at the temperature of-70 ℃ is too large, generally, a refrigeration house at the temperature of-20 ℃ is only built in vaccine manufacturing enterprises. Therefore, considering that the ice crystal size is adjusted when the virus vaccine is frozen at the slow speed of-20 ℃ and the virus is protected at the same time, the method is one of effective solving ways for solving the frozen damage of the virus vaccine semi-finished product.
The traditional freezing protective agent comprises glycerol, dimethyl sulfoxide and the like, but the dimethyl sulfoxide has cytotoxicity, and the addition of the glycerol has little effect in a vaccine semi-finished product, so that the development of an effective novel vaccine semi-finished product freezing protective agent is urgently needed in the vaccine manufacturing industry.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a virus vaccine semi-finished product cryopreservation protective agent, a preparation method and application thereof, which can ensure that the virus vaccine semi-finished product is stably preserved for 1 month when being frozen and stored at-20 ℃ for turnover so as to ensure the effective activity of the vaccine semi-finished product and guarantee the subsequent vaccine production and preparation.
The technical scheme adopted by the invention is as follows:
a virus vaccine semi-finished product cryopreservation protective agent comprises the following components in percentage by mass: 2 to 15 percent of sucrose, 5 to 25 percent of trehalose, 0.5 to 2 percent of pullulan, 1 to 3 percent of mannitol, 0.1 to 3%L-hydroxyproline, 0.15 to 3 percent of epsilon-polylysine, 0.1 to 5 percent of glycine and the balance of water for injection, wherein the total is 100 percent.
Polyhydroxy saccharides are substances which can inhibit the freezing denaturation of proteins, such as sucrose, trehalose, pullulan and the like, and can effectively inhibit the growth of ice crystals, thereby reducing the mechanical damage of the ice crystals to viruses; trehalose is a non-reducing disaccharide formed by combining two molecules of glucose through hemiacetal hydroxyl, is common in various organisms such as lower plants, yeasts and insects, is one of common natural low-temperature freezing protective agents, can inhibit the growth and morphology of ice crystals in extreme environments such as low temperature and the like at a certain concentration, and more importantly, can regulate the tolerance of viruses to freezing damage through mechanisms such as increasing the fluidity of a lipid membrane, maintaining the liquid crystal phase of the lipid membrane, resisting oxidation and the like; sucrose can be combined with protein molecules by changing the state of the combined water in the protein, and the freeze denaturation of the protein is inhibited to improve the stability; mannitol can regulate the appearance of ice crystals by lowering the freezing point; l-hydroxyproline is a permeable cryoprotectant, and can be combined with water hydrogen bond to inhibit ice crystal formation at low temperature; the epsilon-polylysine is a cationic polymerization polypeptide, can enhance the membrane permeability by cooperating with substances such as trehalose and the like, and accelerates the permeation of the trehalose into the membrane so as to play a stabilizing role.
A preparation method of a virus vaccine semi-finished product cryopreservation protective agent comprises the following steps:
s1, weighing sucrose, trehalose, pullulan and mannitol according to a mass ratio, dissolving the sucrose, the trehalose, the pullulan and the mannitol in water for injection, fully dissolving the mixture, and heating and sterilizing the mixture at 121 ℃ for 15 to 20min for later use;
s2, weighing L-hydroxyproline, epsilon-polylysine and glycine according to the mass ratio, dissolving the L-hydroxyproline, the epsilon-polylysine and the glycine in water for injection, fully dissolving the L-hydroxyproline, the epsilon-polylysine and the glycine, and filtering the solution for sterilization by 0.22 mu for later use;
and S3, mixing the solutions prepared in the steps S1 and S2 under an aseptic condition, and adjusting the pH of the solution to 7.0-7.5 by using a regulating solution to obtain the virus vaccine semi-finished product cryopreservation protective agent.
Preferably, in the preparation method of the virus vaccine semi-finished product cryopreservation protective agent, the conditioning solution in step S3 is one or two of dilute hydrochloric acid and sodium hydroxide.
An application of a virus vaccine semi-finished product freezing protective agent in virus vaccine semi-finished product preservation.
Preferably, the virus vaccine semi-finished product cryopreservation protective agent is applied to virus vaccine semi-finished product preservation, wherein the ratio of the virus vaccine semi-finished product to the cryopreservation protective agent is 8-5: 1, and mixing uniformly.
Preferably, the virus vaccine semi-finished product cryopreservation protective agent is applied to preservation of virus vaccine semi-finished products, wherein the virus vaccine semi-finished products refer to virus vaccines for animals, and comprise swine fever vaccines, porcine reproductive and respiratory syndrome vaccines and infectious bronchitis vaccines for chickens.
The invention has the advantages that:
(1) The virus vaccine semi-finished product cryopreservation protective agent disclosed by the invention is applied to the existing preservation link of virus vaccine semi-finished products, can better regulate and control the appearance of ice crystals during freezing, reduces virus cryopreservation damage, solves the problem of efficient and stable cryopreservation of the vaccine semi-finished products in the industry, provides more powerful guarantee for the subsequent production of high-quality vaccines, and is simple to prepare, simple and convenient to operate and has a very high application prospect.
(2) The cryopreservation protective agent for the virus vaccine semi-finished product is prepared by matching trehalose, sucrose, pullulan, mannitol, proline, polylysine and glycine, can effectively adjust the appearance of ice crystals, can effectively ensure that the activity loss of the virus vaccine semi-finished product is not more than 0.5 titer within 1 month of cryopreservation at-20 ℃ after the cryopreservation protective agent and the virus vaccine semi-finished product are mixed according to a certain proportion, and provides a powerful technical guarantee for the production of subsequent high-quality vaccines.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the size and appearance of ice crystals formed during the freezing of classical swine fever virus solution before and after the addition of a semi-finished cryoprotectant in example 1 of the present invention.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
Example 1
A hog cholera vaccine semi-finished product cryopreservation protective agent comprises the following components in percentage by mass: 15% sucrose, 5% trehalose, 2% pullulan, 1% mannitol, 1.5% L-hydroxyproline, 1.5% epsilon-polylysine, 3% glycine, and the balance water for injection.
A preparation method of a hog cholera vaccine semi-finished product cryopreservation protective agent comprises the following steps:
s1, weighing sucrose, trehalose, pullulan and mannitol according to a mass ratio, dissolving the sucrose, the trehalose, the pullulan and the mannitol in water for injection, fully dissolving the mixture, and heating and sterilizing the mixture for 15min at 121 ℃ for later use;
s2, weighing L-hydroxyproline, epsilon-polylysine and glycine according to the mass ratio, dissolving the L-hydroxyproline, the epsilon-polylysine and the glycine in water for injection, fully dissolving the L-hydroxyproline, the epsilon-polylysine and the glycine, and filtering the solution for sterilization by 0.22 mu for later use;
and S3, mixing the solutions prepared in the steps S1 and S2 under an aseptic condition, and adjusting the pH of the solution to 7.0-7.5 by using dilute hydrochloric acid and/or sodium hydroxide to obtain the virus vaccine semi-finished product cryopreservation protective agent.
And (3) respectively freezing and preserving the semi-finished product of the swine fever vaccine and the semi-finished product of the swine fever vaccine according to the ratio of 8:1 to obtain a swine fever vaccine semi-finished product solution with a freezing storage protection effect at the temperature of 20 ℃ below zero, and the swine fever vaccine semi-finished product solution is placed at the temperature of 20 ℃ below zero for 1 month, 2 months and 3 months for freezing storage to detect the activity of the swine fever virus, and meanwhile, the swine fever vaccine semi-finished product without any component is also placed at the temperature of 20 ℃ below zero as a reference. Specific results are shown in table 1.
TABLE 1 Virus titer after cryopreservation of classical swine fever virus at-20 ℃ before and after addition of vaccine intermediates
As can be seen from the table 1, the swine fever vaccine semi-finished product prepared by adopting the vaccine semi-finished product cryopreservation protective agent has a significantly better cryopreservation protective effect at-20 ℃ than a group without adding the cryopreservation protective agent, and shows that the virus titer loss is 0.5 titer at 1 month of cryopreservation at-20 ℃, and the titer loss is 1.75 titer in the group without adding the cryopreservation protective agent, and the difference is obvious; and the virus titer of the swine fever vaccine semi-finished product group added with the cryopreservation protective agent is reduced by 1.0 titer after the swine fever vaccine is frozen for 2 months at the temperature of-20 ℃, and the titer of the swine fever vaccine semi-finished product group not added with the cryopreservation protective agent is lost by 2.25 titers; and (3) freezing and storing for 3 months at the temperature of minus 20 ℃, wherein the virus titer of the swine fever vaccine semi-finished product group added with the freezing and storing protective agent is reduced by 1.75 titers, and the virus titer of the group not added with the freezing and storing protective agent is lost by 3 titers, which indicates that the freezing and storing protective agent can enhance the virus stability when the swine fever vaccine semi-finished product is frozen and stored at the temperature of minus 20 ℃.
Fig. 1 shows the size morphology of ice crystals formed when the classical swine fever virus solution is frozen before and after the semi-finished product freezing protective agent is added, and as can be seen from fig. 1, compared with the classical swine fever virus solution without the frozen protective agent, the size of the ice crystals after the freezing is obviously reduced, the particle size is more uniform, and the detection result is basically consistent with the titer detection result of the classical swine fever virus.
Example 2
A cryopreservation protective agent for a semi-finished product of a porcine reproductive and respiratory syndrome vaccine comprises the following components in percentage by mass: 2% sucrose, 25% trehalose, 0.5% pullulan, 2% mannitol, 0.1% L-hydroxyproline, 0.15% epsilon-polylysine, 3% glycine, and the balance water for injection;
a preparation method of a semi-finished product of a porcine reproductive and respiratory syndrome vaccine comprises the following steps:
s1, weighing sucrose, trehalose, pullulan and mannitol according to a mass ratio, dissolving the sucrose, the trehalose, the pullulan and the mannitol in water for injection, fully dissolving the mixture, and heating and sterilizing the mixture for 15min at 121 ℃ for later use;
s2, weighing L-hydroxyproline, epsilon-polylysine and glycine according to the mass ratio, dissolving the L-hydroxyproline, the epsilon-polylysine and the glycine in water for injection, fully dissolving the L-hydroxyproline, the epsilon-polylysine and the glycine, and filtering the solution for sterilization by 0.22 mu for later use;
and S3, mixing the solutions prepared in the steps S1 and S2 under an aseptic condition, and adjusting the pH of the solution to 7.0-7.5 by using dilute hydrochloric acid and/or sodium hydroxide to obtain the virus vaccine semi-finished product cryopreservation protective agent.
Respectively mixing the semi-finished product of the porcine reproductive and respiratory syndrome vaccine with the prepared vaccine cryopreservation protective agent according to the weight ratio of 5:1 to obtain the semi-finished product solution of the porcine reproductive and respiratory syndrome vaccine with the frozen storage protection effect at the temperature of-20 ℃. The semi-finished product of the porcine reproductive and respiratory syndrome vaccine is frozen and stored at the temperature of minus 20 ℃ for 1 month, 2 months and 3 months, then the virus activity of the semi-finished product of the porcine reproductive and respiratory syndrome vaccine is detected, and meanwhile, the semi-finished product of the porcine reproductive and respiratory syndrome vaccine without any component is also placed at the temperature of minus 20 ℃ as a control. Specific results are shown in Table 2.
TABLE 2 Virus titer after cryopreservation of porcine reproductive and respiratory syndrome Virus at-20 ℃ before and after addition of vaccine intermediates
As can be seen from the table 2, the cryopreservation protection effect of the semi-finished product of the porcine reproductive and respiratory syndrome vaccine prepared by adopting the cryopreservation protective agent of the semi-finished product of the vaccine at the temperature of 20 ℃ below zero is remarkably better than that of a group without adding the cryopreservation protective agent, the virus titer loss is 0.42 titer when the semi-finished product of the porcine reproductive and respiratory syndrome vaccine is frozen at the temperature of 20 ℃ below zero for 1 month, and the virus titer loss is 1.25 titer when the semi-finished product of the porcine reproductive and respiratory syndrome vaccine is not added, so that the difference is obvious; and the virus titer of the porcine reproductive and respiratory syndrome vaccine semi-finished product group added with the cryopreservation protective agent is reduced by 1.0 titer after the porcine reproductive and respiratory syndrome vaccine is frozen at-20 ℃ for 2 months, while the titer of the porcine reproductive and respiratory syndrome vaccine semi-finished product group not added with the cryopreservation protective agent is lost by 1.75 titers, which shows that the cryopreservation protective agent can enhance the virus stability when the porcine reproductive and respiratory syndrome vaccine semi-finished product is frozen at-20 ℃.
Example 3
A protective agent for cryopreservation of a semi-finished product of a vaccine for infectious bronchitis of chicken, which comprises the following components in percentage by mass: 7% of sucrose, 15% of trehalose, 1.0% of pullulan, 3% of mannitol, 3%L-hydroxyproline, 3% of epsilon-polylysine, 0.1% of glycine and the balance of water for injection;
a preparation method of a chicken infectious bronchitis vaccine semi-finished product cryopreservation protective agent is characterized by comprising the following steps:
s1, weighing sucrose, trehalose, pullulan and mannitol according to a mass ratio, dissolving the sucrose, the trehalose, the pullulan and the mannitol in water for injection, fully dissolving the mixture, and heating and sterilizing the mixture for 15min at 121 ℃ for later use;
s2, weighing L-hydroxyproline, epsilon-polylysine and glycine according to the mass ratio, dissolving the L-hydroxyproline, the epsilon-polylysine and the glycine in water for injection, fully dissolving the L-hydroxyproline, the epsilon-polylysine and the glycine, and filtering the solution for sterilization by 0.22 mu for later use;
and S3, mixing the solutions prepared in the steps S1 and S2 under an aseptic condition, and adjusting the pH of the solution to 7.0-7.5 by using dilute hydrochloric acid and/or sodium hydroxide to obtain the virus vaccine semi-finished product cryopreservation protective agent.
Respectively freezing and preserving the chicken infectious bronchitis vaccine semi-finished product and the vaccine semi-finished product according to the ratio of 6.5:1, and obtaining the semi-finished product solution of the avian infectious bronchitis vaccine with a frozen storage protection effect at the temperature of-20 ℃. And (3) freezing and storing the semi-finished product at-20 ℃ for 1 month, 2 months and 3 months, detecting the virus activity in the semi-finished product of the avian infectious bronchitis vaccine, and meanwhile, placing the semi-finished product of the avian infectious bronchitis vaccine without any component at-20 ℃ as a control. The specific results are shown in Table 3.
TABLE 3 Virus titer of avian infectious bronchitis viruses after cryopreservation at-20 ℃ before and after addition of vaccine intermediates
As can be seen from Table 3, the protective effect of the chicken infectious bronchitis vaccine semi-finished product prepared by adopting the vaccine semi-finished product cryopreservation protective agent is remarkably better than that of a group without adding the cryopreservation protective agent at the temperature of-20 ℃, the virus titer loss is 0.55 titer when the chicken infectious bronchitis vaccine semi-finished product is cryopreserved for 1 month at the temperature of-20 ℃, and the titer loss is 1.0 titer when the chicken infectious bronchitis vaccine semi-finished product is not added with the cryopreservation protective agent, so that the difference is obvious; and the virus titer of the chicken infectious bronchitis vaccine semi-finished product group added with the cryopreservation protective agent is reduced by 1.0 titer after the chicken infectious bronchitis vaccine semi-finished product is frozen at-20 ℃ for 2 months, while the titer of the chicken infectious bronchitis vaccine semi-finished product group not added with the cryopreservation protective agent is lost by 1.58 titers, which shows that the cryopreservation protective agent can enhance the virus stability when the chicken infectious bronchitis vaccine semi-finished product is frozen at-20 ℃.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (6)
1. The virus vaccine semi-finished product cryopreservation protective agent is characterized by comprising the following components in percentage by mass: 2 to 15 percent of sucrose, 5 to 25 percent of trehalose, 0.5 to 2 percent of pullulan, 1 to 3 percent of mannitol, 0.1 to 3%L-hydroxyproline, 0.15 to 3 percent of epsilon-polylysine, 0.1 to 5 percent of glycine and the balance of water for injection, wherein the total is 100 percent.
2. A preparation method of a virus vaccine semi-finished product cryopreservation protective agent is characterized by comprising the following steps:
s1, weighing sucrose, trehalose, pullulan and mannitol according to a mass ratio, dissolving the sucrose, the trehalose, the pullulan and the mannitol in water for injection, fully dissolving the mixture, and heating and sterilizing the mixture at 121 ℃ for 15 to 20min for later use;
s2, weighing L-hydroxyproline, epsilon-polylysine and glycine according to the mass ratio, dissolving the L-hydroxyproline, the epsilon-polylysine and the glycine in water for injection, fully dissolving the L-hydroxyproline, the epsilon-polylysine and the glycine, and filtering the solution for sterilization by 0.22 mu for later use;
and S3, mixing the solutions prepared in the steps S1 and S2 under an aseptic condition, and adjusting the pH of the solution to 7.0-7.5 by using a regulating solution to obtain the virus vaccine semi-finished product cryopreservation protective agent.
3. The method for preparing the cryopreservation protectant for the virus vaccine semi-finished product according to claim 1, wherein the conditioning solution in the step S3 is one or two of diluted hydrochloric acid and sodium hydroxide.
4. An application of a virus vaccine semi-finished product freezing protective agent in virus vaccine semi-finished product preservation.
5. The use of the cryopreservation agent for virus vaccine intermediates in the preservation of virus vaccine intermediates according to claim 4, wherein the ratio of the cryopreservation agent to the vaccine intermediates is 8-5: 1, and mixing uniformly.
6. The use of the cryopreservation agent for virus vaccine intermediates in the preservation of virus vaccine intermediates according to claim 4, wherein the virus vaccine intermediates are virus vaccines for animals, including hog cholera vaccines, porcine reproductive and respiratory syndrome vaccines and infectious bronchitis vaccines.
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