CN116510025B - Freeze-drying protective agent for improving titer stability of varicella virus vaccine and preparation method thereof - Google Patents
Freeze-drying protective agent for improving titer stability of varicella virus vaccine and preparation method thereof Download PDFInfo
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- 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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- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16734—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 relates to a freeze-drying protective agent for improving titer stability of varicella virus vaccine and a preparation method thereof, wherein the freeze-drying protective agent comprises the following components in initial concentration in freeze-drying stock solution: trehalose 70-90g/l, mannitol 20-40g/l, human serum albumin 0.4-0.6g/l, PB solution 0.01M, wherein the pH of the freeze-drying protective agent is 7.0-7.6. The protective agent provided by the application does not contain gelatin or 199 solution, and simultaneously reduces the use amount of human serum albumin, so that the endotoxin content of the vaccine is greatly reduced, the irritation and harm to human bodies are greatly reduced, and better safety is provided for the vaccine; meanwhile, the protective agent disclosed by the invention is simple and clear in components, so that the protective agent has the advantages of improving the working efficiency and reducing the cost, and is more suitable for industrialization. In the freeze-drying process, trehalose and mannitol in the protective agent can stabilize the cell membrane structure and protect vaccine viruses from being damaged by environmental stress such as heat, freezing and the like.
Description
Technical Field
The invention relates to the technical field of varicella virus vaccines, in particular to a freeze-drying protective agent for improving titer stability of varicella virus vaccines and a preparation method thereof.
Background
Varicella is a highly infectious disease caused by varicella-zoster virus, is a common highly infectious disease in childhood, is mainly transmitted through air droplets and contact, and after primary infection is recovered, latent infection is commonly present, and the latent virus is reactivated to cause herpes zoster. Varicella is characterized clinically by the appearance of a whole body of skin mucosa with eruptions, maculopapules, pimples, herpes and crusts, which are all present and widely spread worldwide, seriously endangering human health. There is no specific drug for treating varicella, and immunization is the only effective preventive method.
Varicella-zoster virus (VZV), which is the only natural host of the virus, is the causative agent of both varicella and shingles. At present, no specific drug treatment is available for VZV infection, varicella-zoster immunoglobulin and anti-herpesvirus drugs are very expensive, so that the use of varicella attenuated live vaccine for common childhood immunization has become an important means and trend for reducing or even cutting off varicella epidemic in countries around the world. VZV is an enveloped virus that is extremely unstable and extremely sensitive to pH, temperature, and protectants. When the pH is less than 6.2 or more than 7.8, the infectivity is lost, and the bacteria are usually stored at-65 ℃ to-70 ℃ and quickly lost under the condition of no stabilizing agent.
There are also some problems to be solved urgently in varicella vaccines. The varicella vaccine on the market in China initially contains gelatin and human serum albumin, and allergic reaction is easy to cause after the varicella vaccine is inoculated; meanwhile, gelatin component in the protective agent is also a main source of vaccine endotoxin, and can cause heat reaction of an inoculator, so that removal of gelatin from the protective agent has become common in industry, for example, chinese patent publication No. CN 101537186A, CN102657870A, CN104188923A, CN105582534A and the like discloses a vaccine freeze-drying protective agent without gelatin or gelatin and human blood protein component, and varicella vaccine is taken as a preparation example. In addition, 2017, ma Rui and the like report that the quality standard shallow analysis of the varicella attenuated live vaccine of the Chinese pharmacopoeia shows that the detection of the virus content (titer) of the varicella attenuated live vaccine by the quality standard shallow analysis of the varicella attenuated live vaccine of the Chinese pharmacopoeia, the quality standard shallow analysis of the varicella attenuated live vaccine by the varicella attenuated live vaccine of the varicella virus has certain difference in the aspects of selection and operation of control points and judgment of results. As no international reference for viral titres is currently available, WHO recommends the establishment of own viral titre references for each country. As for the limitation requirements of the titer, although no limitation was imposed on WHO, BP, EP except that ChP prescribes a specific limit (stock solution. Gtoreq.4.0 l g PFU/ml, finished product. Gtoreq.3.6 l g PFU/ml), WHO prescribes a lower limit of the titer of the finished product virus in each country. In particular, BP and EP prescribe that at least 3 vaccines are used for respectively measuring virus titer, each reference is titrated repeatedly for 3 times, so that the vaccine virus titer is comprehensively calculated, a statistical analysis method is introduced in result judgment to set the condition for establishing the test (the 95% reliable interval of the estimated virus content of the 3 repeated results of the reference is more than +/-0.3 l log PFU; the virus titer of the reference deviates from the set value by more than 0.5l log PFU, the test is not established), and the accuracy and reliability of the detection result are ensured by avoiding the systematic error of the test through the quality control of the reference. The WHO protocol also suggests that at least 5 vaccines are preferably used to determine viral activity separately and the vaccine virus content is determined by titration of a reference. In addition, in 2022, chen Zhen and other published viral titers are also reported in the evaluation, and the method for measuring the viral titer of varicella attenuated live vaccines in the Chinese pharmacopoeia can meet the requirement of ensuring the effectiveness of the vaccines.
Disclosure of Invention
The freeze-drying protective agent for improving the titer stability of varicella virus vaccine provided by the embodiment of the invention comprises the following components in initial concentration: trehalose 70-90g/l, mannitol 20-40g/l, human serum albumin 0.4-0.6g/l, PB solution 0.01M, wherein the pH of the freeze-drying protective agent is 7.0-7.6.
Preferably, the composition further comprises 1-5g/l of propyl-beta-cyclodextrin and 0.1-0.2g/l of phospholipid.
Preferably, 0.5-2g/l of magnesium sulfate is also included.
Preferably, the ingredients included and their initial concentrations in the lyophilized stock solution are: 80-85g/l of trehalose, 30-35g/l of mannitol, 0.5-0.6g/l of human serum albumin, 0.01M of PB solution, 1-2g/l of hydroxypropyl-beta-cyclodextrin, 0.15-0.2g/l of phospholipid and 0.8-1.2g/l of magnesium sulfate.
Also provided is a method for preparing a lyoprotectant for improving titer stability of varicella virus vaccine, comprising the following steps:
step S1, weighing 70-90g/l of trehalose, 20-40g/l of mannitol and 0.4-0.6g/l of human serum albumin, and fully dissolving the substances in a PB solution of 0.01M;
and S2, regulating the pH value to 7.0-7.6, measuring the osmotic pressure to be 300-500mosm, and sterilizing and preserving after constant volume.
Preferably, step S1 includes:
uniformly mixing 1-5g/l of propyl-beta-cyclodextrin and 0.1-0.2g/l of phospholipid to prepare a phospholipid-propyl-beta-cyclodextrin mixture;
heating the PB solution to 40-50 ℃, adding the phospholipid-propyl-beta-cyclodextrin mixture into the heated PB solution, and stirring until the mixture is completely dissolved;
then adding 70-90g/l trehalose, 20-40g/l mannitol and 0.4-0.6g/l human serum albumin into the dissolved solution.
Preferably, in step S1, the phospholipid-propyl- β -cyclodextrin mixture and the dodecyl polyoxyethylene ether are added to the heated PB solution and stirred until completely dissolved; in the step S2, the pH is firstly adjusted to 7.8-8.0, and then freeze drying is carried out at the temperature of minus 20 ℃ to minus 15 ℃; then adjusting pH to 7.0-7.6 at 2-8deg.C, measuring osmotic pressure to 300-500mosm, sterilizing, and preserving.
Preferably, after preparing the phospholipid-propyl-beta-cyclodextrin mixture, the method further comprises: stirring and mixing the phospholipid-propyl-beta-cyclodextrin mixture and glycolic acid in a solvent to fully mix the mixture; and (3) using trichloroacetic acid as a catalyst, and carrying out esterification reaction on hydroxyl part of glycolic acid and hydroxyl part of the phospholipid-propyl-beta-cyclodextrin mixture in the reaction to generate a hydroxylated phospholipid-propyl-beta-cyclodextrin mixture.
Preferably, in step S1, the hydroxylated phospholipid-propyl- β -cyclodextrin mixture and magnesium sulfate are added to the heated PB solution.
Preferably, in step S2, after the constant volume, the bacteria are removed by a 0.22 μm filter and stored at 2-8deg.C.
The technical effects are as follows:
(1) The protective agent provided by the application does not contain gelatin or 199 solution, and simultaneously reduces the use amount of human serum albumin, so that the endotoxin content of the vaccine is greatly reduced, the irritation and harm to human bodies are greatly reduced, and better safety is provided for the vaccine; meanwhile, the protective agent disclosed by the invention is simple and clear in components, so that the protective agent has the advantages of improving the working efficiency and reducing the cost, and is more suitable for industrialization. In the freeze-drying process, trehalose and mannitol in the protective agent can stabilize the cell membrane structure and protect vaccine viruses from being damaged by environmental stress such as heat, freezing and the like. Meanwhile, the low-dose human serum albumin can reduce the endotoxin content of the vaccine, so that the safety of the vaccine is improved.
(2) The propyl-beta-cyclodextrin has a hydrophobic inner cavity and a hydrophilic outer surface, when the propyl-beta-cyclodextrin contacts the phospholipid, the hydrophobic inner cavity of the propyl-beta-cyclodextrin is combined with the hydrophobic group of the phospholipid, and the hydrophilic outer surface is exposed to the solution, so that a liposome-like complex is formed, and the complex can encapsulate trehalose and mannitol to prevent the inactivation and degradation of the trehalose and mannitol in the freeze-drying process, thereby improving the stability of the vaccine. In addition, the propyl-beta-cyclodextrin can be combined with proteins in the vaccine to form a stable compound, so that the stability and the protection effect of the vaccine are improved, trehalose and mannitol form a protection film together with the propyl-beta-cyclodextrin, and biomolecules in the vaccine, such as proteins and nucleic acids, are protected from damage caused by environmental stress, such as oxidation, heat, cold and the like, so that the stability and the protection effect of the vaccine are improved.
(3) The magnesium sulfate can be used as an auxiliary component, has a certain protection effect, and improves the stability of the protective agent. Meanwhile, the pH value of the protective agent can be regulated by the magnesium sulfate, so that the protective agent is kept in a proper range, and the stability of the protective agent and the effect of protecting vaccines are enhanced. The magnesium sulfate can also be combined with phospholipid to form stable magnesium phosphate salt, so that the stability of the phospholipid in a protective agent is enhanced, viruses in the vaccine are protected from being influenced by freezing, the magnesium sulfate and the propyl-beta-cyclodextrin form a compound, the capacity of wrapping vaccine components by the propyl-beta-cyclodextrin is improved, the magnesium sulfate, trehalose and mannitol form an ionic crosslinking structure, the stability is enhanced, the wrapping effect on the trehalose and mannitol can be further improved, the inactivation and degradation of the trehalose and the mannitol in the freeze-drying process are prevented, and the stability of the vaccine is further improved.
(4) And (2) heating the PB solution to promote the mixture of the phospholipid and the propyl-beta-cyclodextrin to be fully dissolved, and adding dodecyl polyoxyethylene ether in the step S1 to enhance the solubility of the mixture of the phospholipid and the propyl-beta-cyclodextrin, so that the uniformity and the stability of the freeze-drying protective agent are improved. Freeze-drying at-20deg.C to-15deg.C, wherein water is frozen to form ice crystals during freeze-drying process, so that phospholipid molecules are arranged into lamellar structure, and propyl-beta-cyclodextrin is inserted into bilayer structure of phospholipid molecules. This process is very important for protecting the stability of vaccine titers, as it can reduce the impact of water molecules on vaccine particles, preventing them from undergoing changes in thermodynamic instability.
(5) After hydroxylation, the hydrophilicity of the phospholipid-propyl-beta-cyclodextrin mixture is enhanced, so that the solubility in water is improved, and the hydroxylated phospholipid-propyl-beta-cyclodextrin mixture, magnesium sulfate, trehalose and mannitol further form a cross-linked structure, so that the stability is enhanced.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims thereof as well as the appended drawings.
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a schematic flow chart of a method for preparing a freeze-drying protective agent with high titer stability of varicella virus vaccine according to an embodiment of the invention.
Detailed Description
The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.
The freeze-drying protective agent for improving the titer stability of varicella virus vaccine provided by the embodiment of the invention comprises the following components in initial concentration: trehalose 70-90g/l, mannitol 20-40g/l, human serum albumin 0.4-0.6g/l, PB solution 0.01M, wherein the pH of the freeze-drying protective agent is 7.0-7.6.
The protective agent provided by the application does not contain gelatin or 199 solution, and simultaneously reduces the use amount of human serum albumin, so that the endotoxin content of the vaccine is greatly reduced, the irritation and harm to human bodies are greatly reduced, and better safety is provided for the vaccine; meanwhile, the protective agent disclosed by the invention is simple and clear in components, so that the protective agent has the advantages of improving the working efficiency and reducing the cost, and is more suitable for industrialization. In the freeze-drying process, trehalose and mannitol in the protective agent can stabilize the cell membrane structure and protect vaccine viruses from being damaged by environmental stress such as heat, freezing and the like. Meanwhile, the low-dose human serum albumin can reduce the endotoxin content of the vaccine, so that the safety of the vaccine is improved.
Preferably, the composition further comprises 1-5g/l of propyl-beta-cyclodextrin and 0.1-0.2g/l of phospholipid.
The propyl-beta-cyclodextrin has a hydrophobic inner cavity and a hydrophilic outer surface, when the propyl-beta-cyclodextrin contacts the phospholipid, the hydrophobic inner cavity of the propyl-beta-cyclodextrin is combined with the hydrophobic group of the phospholipid, and the hydrophilic outer surface is exposed to the solution, so that a liposome-like complex is formed, and the complex can encapsulate trehalose and mannitol to prevent the inactivation and degradation of the trehalose and mannitol in the freeze-drying process, thereby improving the stability of the vaccine. In addition, the propyl-beta-cyclodextrin can be combined with proteins in the vaccine to form a stable compound, so that the stability and the protection effect of the vaccine are improved, trehalose and mannitol form a protection film together with the propyl-beta-cyclodextrin, and biomolecules in the vaccine, such as proteins and nucleic acids, are protected from damage caused by environmental stress, such as oxidation, heat, cold and the like, so that the stability and the protection effect of the vaccine are improved.
Preferably, 0.5-2g/l of magnesium sulfate is also included.
The magnesium sulfate can be used as an auxiliary component, has a certain protection effect, and improves the stability of the protective agent. Meanwhile, the pH value of the protective agent can be regulated by the magnesium sulfate, so that the protective agent is kept in a proper range, and the stability of the protective agent and the effect of protecting vaccines are enhanced. The magnesium sulfate can also be combined with phospholipid to form stable magnesium phosphate salt, so that the stability of the phospholipid in a protective agent is enhanced, viruses in the vaccine are protected from being influenced by freezing, the magnesium sulfate and the propyl-beta-cyclodextrin form a compound, the capacity of wrapping vaccine components by the propyl-beta-cyclodextrin is improved, the magnesium sulfate, trehalose and mannitol form an ionic crosslinking structure, the stability is enhanced, the wrapping effect on the trehalose and mannitol can be further improved, the inactivation and degradation of the trehalose and the mannitol in the freeze-drying process are prevented, and the stability of the vaccine is further improved.
Preferably, the ingredients included and their initial concentrations in the lyophilized stock solution are: 80-85g/l of trehalose, 30-35g/l of mannitol, 0.5-0.6g/l of human serum albumin, 0.01M of PB solution, 1-2g/l of hydroxypropyl-beta-cyclodextrin, 0.15-0.2g/l of phospholipid and 0.8-1.2g/l of magnesium sulfate.
As shown in FIG. 1, the invention also provides a preparation method of the freeze-drying protective agent for improving the titer stability of the varicella virus vaccine, which comprises the following steps:
step S1, weighing 70-90g/l of trehalose, 20-40g/l of mannitol and 0.4-0.6g/l of human serum albumin, and fully dissolving the substances in a PB solution of 0.01M;
and S2, regulating the pH value to 7.0-7.6, measuring the osmotic pressure to be 300-500mosm, and sterilizing and preserving after constant volume.
Wherein, PB solution 0.01M solution configuration is identified in Table 1 below
TABLE 1
Preferably, step S1 includes:
uniformly mixing 1-5g/l of propyl-beta-cyclodextrin and 0.1-0.2g/l of phospholipid to prepare a phospholipid-propyl-beta-cyclodextrin mixture;
heating the PB solution to 40-50 ℃, adding the phospholipid-propyl-beta-cyclodextrin mixture into the heated PB solution, and stirring until the mixture is completely dissolved;
then adding 70-90g/l trehalose, 20-40g/l mannitol and 0.4-0.6g/l human serum albumin into the dissolved solution.
Preferably, in step S1, the phospholipid-propyl- β -cyclodextrin mixture and the dodecyl polyoxyethylene ether are added to the heated PB solution and stirred until completely dissolved; in the step S2, the pH is firstly adjusted to 7.8-8.0, and then freeze drying is carried out at the temperature of minus 20 ℃ to minus 15 ℃; then adjusting pH to 7.0-7.6 at 2-8deg.C, measuring osmotic pressure to 300-500mosm, sterilizing, and preserving.
And (2) heating the PB solution to promote the mixture of the phospholipid and the propyl-beta-cyclodextrin to be fully dissolved, and adding dodecyl polyoxyethylene ether in the step S1 to enhance the solubility of the mixture of the phospholipid and the propyl-beta-cyclodextrin, so that the uniformity and the stability of the freeze-drying protective agent are improved. Freeze-drying at-20deg.C to-15deg.C, wherein water is frozen to form ice crystals during freeze-drying process, so that phospholipid molecules are arranged into lamellar structure, and propyl-beta-cyclodextrin is inserted into bilayer structure of phospholipid molecules. This process is very important for protecting the stability of vaccine titers, as it can reduce the impact of water molecules on vaccine particles, preventing them from undergoing changes in thermodynamic instability.
Preferably, after preparing the phospholipid-propyl-beta-cyclodextrin mixture, the method further comprises: stirring and mixing the phospholipid-propyl-beta-cyclodextrin mixture and glycolic acid in a solvent to fully mix the mixture; and (3) using trichloroacetic acid as a catalyst, and carrying out esterification reaction on hydroxyl part of glycolic acid and hydroxyl part of the phospholipid-propyl-beta-cyclodextrin mixture in the reaction to generate a hydroxylated phospholipid-propyl-beta-cyclodextrin mixture.
After hydroxylation, the hydrophilicity of the phospholipid-propyl-beta-cyclodextrin mixture is enhanced, so that the solubility in water is improved, and the hydroxylated phospholipid-propyl-beta-cyclodextrin mixture, magnesium sulfate, trehalose and mannitol further form a cross-linked structure, so that the stability is enhanced.
Preferably, in step S1, the hydroxylated phospholipid-propyl- β -cyclodextrin mixture and magnesium sulfate are added to the heated PB solution.
Preferably, in step S2, after the constant volume, the bacteria are removed by a 0.22 μm filter and stored at 2-8deg.C.
The freeze-drying protective agent for improving the titer stability of varicella virus vaccine and the preparation method thereof provided by the invention are further described below with reference to examples.
Example 1
The following substances (g/l) were weighed: trehalose 70, mannitol 40 and human serum albumin 0.5 are fully dissolved in a filtered and sterilized 0.01M PBS solution (800 mL), the pH value is adjusted to 7.0 by using 0.2M sodium dihydrogen phosphate, the osmotic pressure is measured to be within the range of 500mosm, and after the osmotic pressure is measured to be constant, a 0.22 mu M filter is sterilized and stored at the temperature of 2 ℃.
Example 2
The following substances (g/l) were weighed: trehalose 80, mannitol 30 and human serum albumin 0.5 are fully dissolved in a filtered and sterilized 0.01M PBS solution (800 mL), the pH value is adjusted to 7.6 by using 0.2M sodium dihydrogen phosphate, the osmotic pressure is measured to be in the range of 300mosm, and after the osmotic pressure is measured to be constant, a 0.22 mu M filter is sterilized and stored at 8 ℃.
Example 3
The following substances (g/l) were weighed: trehalose 90, mannitol 20 and human serum albumin 0.5 are fully dissolved in a filtered and sterilized 0.01M PBS solution (800 mL), the pH value is adjusted to 7.2 by using 0.2M sodium dihydrogen phosphate, the osmotic pressure is measured to be in the range of 400mosm, and after the osmotic pressure is measured to be constant, a 0.22 mu M filter is sterilized and stored at 5 ℃.
Example 4
The following substances (g/l) were weighed: trehalose 70, mannitol 40 and human serum albumin 0.4 are fully dissolved in a filtered and sterilized 0.01M PBS solution (800 mL), the pH value is adjusted to 7.6 by using 0.2M sodium dihydrogen phosphate, the osmotic pressure is measured to be in the range of 400mosm, and after the osmotic pressure is fixed, a 0.22 mu M filter is sterilized and stored at 5 ℃.
Example 5
The following substances (g/l) were weighed: trehalose 70, mannitol 40 and human serum albumin 0.6 are fully dissolved in a filtered and sterilized 0.01M PBS solution (800 mL), the pH value is adjusted to 7.6 by using 0.2M sodium dihydrogen phosphate, the osmotic pressure is measured to be in the range of 400mosm, and after the osmotic pressure is fixed, a 0.22 mu M filter is sterilized and stored at 5 ℃.
Example 6
The following substances (g/l) were weighed: trehalose 70, mannitol 40, human serum albumin 0.5, propyl-beta-cyclodextrin 1g/l and phospholipid 0.2g/l.
Uniformly mixing propyl-beta-cyclodextrin and phospholipid to prepare a phospholipid-propyl-beta-cyclodextrin mixture;
heating the PB solution to 40 ℃, adding the phospholipid-propyl-beta-cyclodextrin mixture into the heated PB solution, and stirring until the mixture is completely dissolved;
then adding trehalose, mannitol and human serum albumin into the dissolved solution.
The pH was adjusted to 7.6 with 0.2M sodium dihydrogen phosphate, the osmotic pressure was measured to be in the range of 400mosm, the volume was fixed, and then the membrane was sterilized with a 0.22 μm filter and stored at 5 ℃.
Example 7
The following substances (g/l) were weighed: trehalose 70, mannitol 40, human serum albumin 0.5, propyl-beta-cyclodextrin 5g/l and phospholipid 0.1g/l.
Uniformly mixing propyl-beta-cyclodextrin and phospholipid to prepare a phospholipid-propyl-beta-cyclodextrin mixture;
heating the PB solution to 50 ℃, adding the phospholipid-propyl-beta-cyclodextrin mixture into the heated PB solution, and stirring until the mixture is completely dissolved;
then adding trehalose, mannitol and human serum albumin into the dissolved solution.
Adjusting pH to 7.6 with 0.2M sodium dihydrogen phosphate, measuring osmotic pressure in 400mosm, sterilizing with 0.22 μm filter, and preserving at 5deg.C.
Example 8
The following substances (g/l) were weighed: trehalose 70, mannitol 40, human serum albumin 0.5, propyl-beta-cyclodextrin 5g/l, phospholipid 0.1g/l, magnesium sulfate 0.5g/l.
Uniformly mixing propyl-beta-cyclodextrin and phospholipid to prepare a phospholipid-propyl-beta-cyclodextrin mixture;
heating PB solution to 50 ℃, adding a phospholipid-propyl-beta-cyclodextrin mixture and dodecyl polyoxyethylene ether into the heated PB solution, and stirring until the mixture is completely dissolved;
then adding trehalose, mannitol and human serum albumin into the dissolved solution.
Firstly adjusting the pH to 8.0, and then freeze-drying at-20 ℃; then adjusting pH to 7.6 at 6deg.C, measuring osmotic pressure to 400mosm, sterilizing, and preserving
Example 9
The following substances (g/l) were weighed: trehalose 70, mannitol 40, human serum albumin 0.5, propyl-beta-cyclodextrin 5g/l, phospholipid 0.1g/l, magnesium sulfate 2g/l.
Uniformly mixing propyl-beta-cyclodextrin and phospholipid to prepare a phospholipid-propyl-beta-cyclodextrin mixture;
heating PB solution to 50 ℃, adding a phospholipid-propyl-beta-cyclodextrin mixture and dodecyl polyoxyethylene ether into the heated PB solution, and stirring until the mixture is completely dissolved;
then adding trehalose, mannitol and human serum albumin into the dissolved solution.
Firstly, regulating the pH value to 7.8, and then, freeze-drying at the temperature of minus 15 ℃; then adjusting pH to 7.6 at 8deg.C, measuring osmotic pressure to 400mosm, sterilizing, and preserving
Example 10
The following substances (g/l) were weighed: trehalose 70, mannitol 40, human serum albumin 0.5, propyl-beta-cyclodextrin 5g/l, phospholipid 0.1g/l, magnesium sulfate 1g/l.
Uniformly mixing propyl-beta-cyclodextrin and phospholipid to prepare a phospholipid-propyl-beta-cyclodextrin mixture, and stirring and mixing the phospholipid-propyl-beta-cyclodextrin mixture and glycolic acid in a solvent to fully mix the mixture; using trichloroacetic acid as a catalyst, and carrying out esterification reaction on hydroxyl part of glycolic acid and hydroxyl part of the phospholipid-propyl-beta-cyclodextrin mixture in the reaction to generate a hydroxylated phospholipid-propyl-beta-cyclodextrin mixture;
heating the PB solution to 50 ℃, adding the hydroxylated phospholipid-propyl-beta-cyclodextrin mixture and magnesium sulfate into the heated PB solution, and stirring until the PB solution is completely dissolved;
then adding trehalose, mannitol and human serum albumin into the dissolved solution.
Firstly, regulating the pH value to 7.8, and then, freeze-drying at the temperature of minus 20 ℃; then adjusting pH to 7.6 at 8deg.C, measuring osmotic pressure to 300-500mosm, sterilizing, and preserving
Comparative example the following substances (g/l) were weighed: 70 g of sucrose, 8g of gelatin, 10 g of human serum albumin, 0.5g of arginine, 0.5g of sodium glutamate, 0.5g of urea and 199 of the comprehensive culture medium 1, fully dissolving the substances in a proper amount of sterilized water for injection, then fixing the volume, simultaneously adjusting the pH value to be in a range of 7.2-7.8, finally sterilizing and filtering, and preserving at 2-8 ℃.
And (3) testing: taking human embryo lung diploid cells as a culture medium, preparing varicella virus harvest, mixing the varicella virus harvest with a proper amount of the protective agent prepared in the embodiment 1, freezing and thawing below-70 ℃ or digesting the varicella virus harvest by using EDTA, centrifuging the harvested virus at 1500 revolutions and 15 min, and re-dissolving and precipitating the virus seed by using the freeze-drying protective agent.
The harvested virus harvest is split-packed into 2mL penicillin bottles according to the loading amount of 0.58mL, and is put into a freeze-drying box after half plugs are added, so as to prepare freeze-drying.
The vaccine obtained according to the step 2 is pre-frozen to-45 ℃ in a freeze-drying box, the temperature is kept for 2 hours, then the vacuum is pumped to 0.1mbar, the temperature of the partition board is raised to-28 ℃ to start sublimation once, and the temperature is controlled for 15 hours at 33 ℃, thus the freeze-drying is completed.
And (3) performing titer detection on the vaccine before and after freeze-drying, and simultaneously detecting osmotic pressure, pH, water content, stability at 2-8 ℃ and stability at 37 ℃ of the vaccine after freeze-drying.
The vaccine samples of examples 1 to 10 and the control test example were subjected to osmotic pressure, water content, pH, drop in titer before and after lyophilization, stability at 2 to 8℃and stability at 37℃respectively, and the specific results were as follows:
evaluation of the varicella vaccine osmotic pressure index prepared by the lyoprotectant of the present invention
And respectively carrying out osmotic pressure measurement on vaccine part samples prepared in the test examples and the control test examples according to the method provided by three annex V H of Chinese pharmacopoeia. The results show that the vaccine osmotic pressure of the protective agent is in the range of 280-400m0sm and is far lower than that of the vaccine of the control freeze-drying protective agent. The results are shown in Table 2.
Table 2.
As can be seen from the data in Table 2, compared with the varicella vaccine prepared by the existing freeze-drying protective agent for the vaccine containing gelatin, the varicella vaccine prepared by the freeze-drying protective agent for the vaccine containing only trehalose, human serum albumin and mannitol has the advantages that the vaccine titer, the thermal stability test, the residual moisture content and the like are all achieved or are not inferior to those of the varicella vaccine prepared by the existing freeze-drying protective agent for the vaccine, the titer consistency is achieved, the test examples are in accordance with European Union pharmacopoeia regulations, and the varicella vaccine has absolute advantages over the existing vaccine, and has the advantages of improving the safety of the vaccine and reducing the production cost due to the optimization of the formula of the freeze-drying protective agent.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (7)
1. A lyoprotectant for improving titer stability of varicella virus vaccine, comprising the following components in initial concentration in a lyophiled stock solution: trehalose 70-90g/l, mannitol 20-40g/l, human serum albumin 0.4-0.6g/l, PB solution 0.01M, wherein the pH of the freeze-drying protective agent is 7.0-7.6; also comprises 1-5g/l propyl-beta-cyclodextrin and 0.1-0.2g/l phosphatide.
2. The lyoprotectant of claim 1, further comprising 0.5-2g/l magnesium sulfate.
3. The lyoprotectant of claim 2, wherein the components and their initial concentrations in the lyophile stock solution are: trehalose 80-85g/l, mannitol 30-35g/l, human serum albumin 0.5-0.6g/l, PB solution 0.01M, propyl-beta-cyclodextrin 1-2g/l, phospholipid 0.15-0.2g/l and magnesium sulfate 0.8-1.2g/l.
4. A method for preparing a freeze-dried protective agent for improving titer stability of varicella virus vaccine, which is characterized by comprising the following steps:
step S1, uniformly mixing 1-5g/l of propyl-beta-cyclodextrin and 0.1-0.2g/l of phospholipid to prepare a phospholipid-propyl-beta-cyclodextrin mixture;
heating the PB solution to 40-50 ℃, adding the phospholipid-propyl-beta-cyclodextrin mixture into the heated PB solution, and stirring until the mixture is completely dissolved;
then adding 70-90g/l trehalose, 20-40g/l mannitol and 0.4-0.6g/l human serum albumin into the dissolved solution;
and S2, regulating the pH value to 7.0-7.6, measuring the osmotic pressure to be 300-500mosm, and sterilizing and preserving after constant volume.
5. The preparation method according to claim 4, wherein in step S1, the phospholipid-propyl- β -cyclodextrin mixture and the dodecyl polyoxyethylene ether are added to the heated PB solution and stirred until completely dissolved; in the step S2, the pH is firstly adjusted to 7.8-8.0, and then freeze drying is carried out at the temperature of minus 20 ℃ to minus 15 ℃; then adjusting pH to 7.0-7.6 at 2-8deg.C, measuring osmotic pressure to 300-500mosm, sterilizing, and preserving.
6. A method for preparing a freeze-dried protective agent for improving titer stability of varicella virus vaccine, which is characterized by comprising the following steps:
step S1, uniformly mixing 1-5g/l of propyl-beta-cyclodextrin and 0.1-0.2g/l of phospholipid to prepare a phospholipid-propyl-beta-cyclodextrin mixture;
stirring and mixing the phospholipid-propyl-beta-cyclodextrin mixture and glycolic acid in a solvent to fully mix the mixture; using trichloroacetic acid as a catalyst, and carrying out esterification reaction on hydroxyl part of glycolic acid and hydroxyl part of the phospholipid-propyl-beta-cyclodextrin mixture in the reaction to generate a hydroxylated phospholipid-propyl-beta-cyclodextrin mixture;
adding the hydroxylated phospholipid-propyl-beta-cyclodextrin mixture and magnesium sulfate into the heated PB solution, and stirring until the mixture is completely dissolved;
then adding 70-90g/l trehalose, 20-40g/l mannitol and 0.4-0.6g/l human serum albumin into the dissolved solution;
and S2, regulating the pH value to 7.0-7.6, measuring the osmotic pressure to be 300-500mosm, and sterilizing and preserving after constant volume.
7. The process according to claim 6, wherein in step S2, the medium is sterilized with a 0.22 μm filter and stored at 2 to 8 ℃.
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