CN114767842A - Freeze-dried preparation of recombinant BCG vaccine and preparation method and application thereof - Google Patents
Freeze-dried preparation of recombinant BCG vaccine and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 33
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 17
- 229930006000 Sucrose Natural products 0.000 claims abstract description 17
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims abstract description 17
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- 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 16
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- 208000008128 pulmonary tuberculosis Diseases 0.000 description 1
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- 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
- A61K47/02—Inorganic compounds
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- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- 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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- 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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- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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Abstract
The invention provides a recombinant BCG vaccine freeze-dried preparation which comprises the following components in parts by weight: 0.05 part of recombinant bacillus calmette-guerin, 401-3 parts of dextran-401, 2.5-3.5 parts of cane sugar, 0.2-0.5 part of sodium glutamate, 0.2-0.7 part of potassium chloride, 3-4 parts of trehalose and 0.015 part of tween-800.005. The freeze-dried preparation of the recombinant BCG vaccine has high viable count, stable property and easy storage, and has good application value as a vaccine for preventing tuberculosis.
Description
Technical Field
The invention belongs to the field of biological products, and particularly relates to a lyophilized preparation of a recombinant BCG vaccine, a preparation method and application thereof.
Background
Calmette and Guerin, a strain of Mycobacterium bovis from tuberculosis-bearing cattle, was isolated by culturing on a medium containing glycerol, potato and oxgall for a total of 231 passages over 13 years to obtain an attenuated strain of Mycobacterium bovis. The vaccine prepared from the bacillus calmette-guerin thallus obtained by the bacillus calmette-guerin cultivation: the BCG vaccine (BCG) can effectively prevent severe tuberculosis of children such as tuberculous meningitis and miliary tuberculosis, and reduce the mortality rate of tuberculosis of children.
However, the research finds that the immunity protection of the BCG on the pulmonary tuberculosis varies from 0% to 80%, the difference of the protection effect is obvious, the protection is not ideal enough, but the immunogenicity and the safety of the BCG serving as a vaccine are verified in the using process of more than half a century. Therefore, research on recombinant bcg has attracted more and more attention, and the recombinant bcg is a vaccine prepared from recombinant bacteria which rely on bcg as a recipient bacterium, introduce a target gene by using a genetic engineering technology, and express a target antigen at a high level to induce humoral immunity and cellular immunity to various diseases, thereby enhancing immunogenicity and protective efficacy. For example, Chinese patent application No. CN201780089047.1 discloses a recombinant BCG bacterium that expresses phoPR in high amount in Japanese strain of BCG, and has significantly enhanced immunogenicity and protective efficacy, and the same safety as the parent strain.
The stability of BCG vaccine has important influence on its clinical effectiveness, and most of the methods of preservation of BCG vaccine are freeze-dried, for example, patent No. CN111588859B discloses a freeze-dried vaccine containing dextran, sucrose, sodium glutamate, potassium chloride and trehaloseThe protecting agent and Bacillus Calmette-Guerin are BCG D2A BCG vaccine freeze-dried preparation prepared from a PB302 strain. However, factors affecting the stability of bcg relate to various aspects such as the strain itself, a lyoprotectant, a culture medium and the like, and since the difference exists between the strains of the bcg and the rBCG, the physiological activity and the physicochemical property of the rBCG are different, so that the lyoprotectant or the freeze-drying process for preparing the bcg freeze-dried preparation is difficult to be directly applied to the rBCG.
In order to ensure the stability and effectiveness of the recombinant BCG, the development of a recombinant BCG freeze-dried preparation with good stability and high efficacy is of great significance.
Disclosure of Invention
The invention aims to provide a freeze-drying protective agent for recombinant BCG vaccine and application thereof.
The invention provides a recombinant BCG vaccine freeze-dried preparation which comprises the following components in parts by weight:
0.05 part of recombinant bacillus calmette-guerin, 401-3 parts of dextran-401, 2.5-3.5 parts of cane sugar, 0.2-0.5 part of sodium glutamate, 0.2-0.7 part of potassium chloride, 3-4 parts of trehalose and 0.015 part of tween-800.005.
Further, the composition comprises the following components in parts by weight: 0.05 part of recombinant bacillus calmette-guerin, 0.05 part of dextran-403 parts, 3-3.5 parts of cane sugar, 0.2-0.5 part of sodium glutamate, 0.5 part of potassium chloride, 3-3.5 parts of trehalose and 800.01 parts of tween-E;
preferably, the composition comprises the following components in parts by weight: 0.05 part of recombinant bacillus calmette-guerin, 0.05 part of dextran-403 parts, 3 parts of cane sugar, 0.4 part of sodium glutamate, 0.5 part of potassium chloride, 3.5 parts of trehalose and 800.01 parts of tween-800.
Furthermore, it is prepared by mixing and freeze-drying the recombinant bacillus calmette-guerin with a stabilizer;
the stabilizer is an aqueous solution containing the dextran-40, sucrose, sodium glutamate, potassium chloride, trehalose and tween-80, and the pH value of the aqueous solution is 7.3-7.5.
Further, the process of the above lyophilization is as follows:
(a) pre-freezing: the temperature is kept between 40 ℃ below zero and 45 ℃ below zero for 2 to 3 hours;
(b) sublimation drying: drying for 12-16 hours at the temperature of minus 20-minus 10 ℃ and the vacuum pressure of 8-10 Pa;
(c) desorption and drying: drying for 6-12 hours at the temperature of 32-35 ℃ and the vacuum pressure of 0.5-10 Pa.
Furthermore, the recombinant BCG is BCG which over-expresses PhoP and PhoR proteins; preferably, the recombinant bcg overexpressing PhoP and PhoR proteins is a recombinant bcg as described in patent application No. CN 201780089047.1.
The invention also provides a preparation method of the recombinant BCG vaccine freeze-dried preparation, which comprises the following steps:
(1) dextran-40, sucrose, sodium glutamate, potassium chloride, trehalose and tween-80 are added with water for injection to prepare a solution, wherein the mass concentration of the dextran is 1-3%;
(2) adding the recombinant bacillus calmette-guerin to the solution in the step (1) to form a uniform solution,
(3) freeze-drying the uniform solution obtained in the step (2), wherein the freeze-drying process comprises the following steps: pre-freezing for 2-3 hours at the temperature of-40 to-45 ℃; then drying for 12-16 hours at the temperature of-20 to-10 ℃ and the vacuum pressure of 8-10 Pa; and finally, drying for 6-12 hours at the temperature of 34-36 ℃ and the vacuum pressure of 0.5-10 Pa.
Further, the step (1) also comprises the step of adding alkali to adjust the pH value of the solution to 7.3-7.5; preferably, the base is sodium hydroxide;
and/or step (1) further comprises the step of sterilizing the solution; preferably, the sterilization is performed for 30-60 minutes at the temperature of 115-120 ℃ and under the pressure of 0.15-0.20 MPa.
Further, the step (2) of adding the recombinant BCG bacteria into the solution in the step (1) comprises the steps of firstly diluting the recombinant BCG bacteria with the solution in the step (1) to prepare a stock solution, and then adding the solution in the step (1) to dilute the stock solution until the mass concentration of the recombinant BCG bacteria is 0.05%.
Further, the homogeneous solution in the step (3) is filled in a penicillin bottle for freeze-drying.
The invention also provides the application of the recombinant BCG vaccine freeze-dried preparation in medicaments for preventing tuberculosis.
The invention has the beneficial effects that: the invention provides a recombinant BCG vaccine freeze-dried preparation prepared from recombinant BCG bacteria for over-expressing PhoP and PhoR proteins, wherein a used stabilizer can effectively reduce or prevent the damage of the freeze-drying process to the recombinant BCG bacteria, so that the vitality and the biological activity of the recombinant BCG bacteria are effectively ensured, and the recombinant BCG vaccine has high viable count, good dispersibility and good stability and has good clinical application value.
The terms of the present invention: "Bacillus Calmette-Guerin" refers to attenuated Mycobacterium bovis, including its cultured and passaged thallus.
The BCG vaccine is live vaccine prepared from attenuated mycobacterium bovis, and has effects in enhancing macrophage activity, activating T lymphocyte, and enhancing cellular immunity.
It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
The raw materials and equipment used in the invention are known products, and are obtained by purchasing products sold in the market.
The source of the recombinant BCG bacteria is as follows: the recombinant BCG bacterium is the recombinant BCG bacterium disclosed in patent application 201780089047.1 (BCG-Japan/PhoPR), and the primordial seed (BCG-3 strain) thereof was introduced from Toronto university of Canada. The main seed lot and the working seed lot were prepared by the biological technology limited company of the Chengdu Yongding industry.
Example 1 preparation of lyophilized formulations of recombinant BCG vaccine of the present invention
Preparing a stabilizer: the following components are added into the water for injection in mass fraction: dextran-403.0%, cane sugar 3.0%, sodium glutamate 0.2%, potassium chloride 0.5%, trehalose 3.5%, and tween-800.01%, adjusting pH to 7.3-7.5 with 10% sodium hydroxide solution, and sterilizing at 116 deg.C and 0.16MPa for 50 min.
(II) passage of production strain
1. And (3) recovering working seeds: the recombinant BCG working seeds are inoculated with a Roche medium and cultured for 2-3 weeks at 38 ℃.
2. Passage sutong culture medium: the Roseburia species are inoculated into Souton's medium and cultured at an appropriate temperature (38 ℃) for an appropriate period of time (2-3 weeks).
(III) stock solution preparation
1. Producing a mycoderm and culturing: the production strain is used for carrying out 2 times of passages on the sutong culture medium with proper inoculation amount, and after the sutong culture medium is cultured for proper time (8-10 days) at proper temperature (38 ℃), a sutong culture medium mycoderm is obtained and used for stock solution preparation.
2. Preparing stock solution: collecting the cultured mycoderm, and grinding on a low-temperature oscillator; then diluting the solution to a certain concentration by using a stabilizer to obtain the recombinant BCG vaccine stock solution.
(IV) preparing a semi-finished product
Diluting the stock solution with stabilizer to 0.5mg/mL to obtain the semi-finished product of the recombinant BCG vaccine.
(V) filling and freeze-drying
1. Packaging with penicillin bottles, wherein the filling amount is as follows: 0.5 ml/bottle.
2. Freeze-drying
The recombinant BCG vaccine freeze-drying process comprises the following steps:
a, pre-freezing: the lowest temperature is-40-45 ℃, and the temperature is maintained for 2-3 hours after the lowest temperature is reached;
b, sublimation drying: the final temperature is-20 to-10 ℃, the operation time is 12 to 16 hours, and the vacuum pressure is controlled to be 8 to 10 Pa;
c, desorption and drying: and finally, controlling the temperature to be 32-35 ℃, controlling the vacuum pressure to be 0.5-10 Pa, and operating for 6-12 hours.
EXAMPLE 2 preparation of lyophilized formulations of rBCG
Preparation of the stabilizer: the following components are added into the water for injection in percentage by mass: dextran-402.0%, cane sugar 3.5%, sodium glutamate 0.4%, potassium chloride 0.5%, trehalose 3.5%, Tween-800.01%, adjusting pH to 7.3-7.5 with 10% sodium hydroxide solution, and sterilizing at 116 deg.C and 0.16MPa for 50 min.
The remaining process steps were the same as in example 1.
Comparative examples 1,
Preparation of a tween-free stabiliser: the following components are added into the water for injection in percentage by mass: dextran-403.0%, sucrose 3.0%, sodium glutamate 0.2%, potassium chloride 0.5%, and trehalose 3.5%; then adjusting the pH value to 7.3-7.5 by using 10% sodium hydroxide solution, and sterilizing for 50 minutes at 116 ℃ under the condition of 0.16 MPa.
The remaining process steps were the same as in example 1.
Comparative examples 2,
The following components are added into the water for injection in mass fraction: dextran-402.0%, sucrose 5.5%, sodium glutamate 0.2%, potassium chloride 0.5%, trehalose 2.0%; then adjusting the pH value to 7.3-7.5 by using a 10% sodium hydroxide solution, and sterilizing for 50 minutes at 116 ℃ under the condition of 0.16 MPa.
The remaining process steps were the same as in example 1.
Comparative examples 3,
The following components are added into the water for injection in percentage by mass: dextran-401.0%, sucrose 6.5%, sodium glutamate 0.2%, potassium chloride 0.5%, and trehalose 2.0%; then adjusting the pH value to 7.3-7.5 by using a 10% sodium hydroxide solution, and sterilizing for 50 minutes at 116 ℃ under the condition of 0.16 MPa.
The remaining process steps were the same as in example 1.
The beneficial effects of the present invention are demonstrated by the following experimental examples.
Experimental example 1 evaluation of quality of the preparation of the present invention
The viable cell count, physical appearance, and properties of moisture, thermal stability, etc. before and after freeze-drying of the example and comparative example preparations were evaluated and compared, and the results are shown in table 1.
TABLE 1
As can be seen from the results in the table, the lyophilized preparation of rBCG added with the stabilizer of specific composition of the present invention (examples 1 and 2) has a viable bacteria ratio of 15-25% and a final viable bacteria number of not less than 30X 106CFU/mg; the product has good thermal stability; the preparation is favorable for dispersion of the recombinant bacillus calmette-guerin, the consistency of the viable count of the samples at the front and rear sections of the subpackaging of the same batch of products is good, the preparation effectively reduces bacterial aggregation, and the uniformity of the viable count of the products is excellent; each performance is obviously superior to that of the comparative example.
The results prove that the recombinant BCG vaccine freeze-dried preparation which has good dispersion effect, high viable count, stable property and easy storage can be prepared only under the specific components and dosage of the stabilizer.
In conclusion, the invention provides the lyophilized preparation of the rBCG vaccine, which has high viable count, stable property and easy storage and has good application value as a vaccine for preventing tuberculosis.
Claims (10)
1. The recombinant BCG vaccine freeze-dried preparation is characterized by comprising the following components in parts by weight:
0.05 part of recombinant bacillus calmette-guerin, 401-3 parts of dextran-401, 2.5-3.5 parts of cane sugar, 0.2-0.5 part of sodium glutamate, 0.2-0.7 part of potassium chloride, 3-4 parts of trehalose and 0.015 part of tween-800.005.
2. The lyophilized formulation of claim 1, comprising the following components in parts by weight: 0.05 part of recombinant bacillus calmette-guerin, 3-3.5 parts of dextran-403, 3-3.5 parts of cane sugar, 0.2-0.5 part of sodium glutamate, 0.5 part of potassium chloride, 3-3.5 parts of trehalose and 800.01 parts of tween-E;
preferably, the composition comprises the following components in parts by weight: 0.05 part of recombinant bacillus calmette-guerin, 0.05 part of dextran-403 part, 3 parts of cane sugar, 0.4 part of sodium glutamate, 0.5 part of potassium chloride, 3.5 parts of trehalose and 800.01 parts of tween-800.
3. The lyophilized formulation according to claim 1 or 2, which is prepared by mixing and lyophilizing rBCG with a stabilizer;
the stabilizer is an aqueous solution containing the dextran-40, sucrose, sodium glutamate, potassium chloride, trehalose and tween-80, and the pH value of the aqueous solution is 7.3-7.5.
4. The lyophilized formulation according to claim 3, wherein the lyophilization process is as follows:
(a) pre-freezing: the temperature is kept between 40 ℃ below zero and 45 ℃ below zero for 2 to 3 hours;
(b) sublimation drying: drying for 12-16 hours at the temperature of minus 20-minus 10 ℃ and the vacuum pressure of 8-10 Pa;
(c) desorption and drying: drying for 6-12 hours at the temperature of 32-35 ℃ and the vacuum pressure of 0.5-10 Pa.
5. The lyophilized formulation of claim 1 or 2, wherein the recombinant bcg is a bcg that overexpresses PhoP and PhoR proteins; preferably, the bcg which overexpresses PhoP and PhoR proteins is a recombinant bcg as described in patent application No. CN 201780089047.1.
6. The method for preparing the lyophilized preparation of rBCG according to any one of claims 1 to 5, comprising the steps of:
(1) dextran-40, sucrose, sodium glutamate, potassium chloride, trehalose and tween-80 are added with water for injection to prepare a solution, wherein the mass concentration of the dextran is 1-3%;
(2) adding the recombinant bacillus calmette-guerin into the solution obtained in the step (1) to form a uniform solution;
(3) and (3) freeze-drying the uniform solution obtained in the step (2), wherein the freeze-drying process comprises the following steps: pre-freezing for 2-3 hours at the temperature of-40 to-45 ℃; then drying for 12-16 hours at the temperature of-20 to-10 ℃ and the vacuum pressure of 8-10 Pa; and finally, drying for 6-12 hours at the temperature of 32-35 ℃ and the vacuum pressure of 0.5-10 Pa.
7. The method according to claim 6, wherein the step (1) further comprises the step of adjusting the pH of the solution to 7.3 to 7.5 by adding a base; preferably, the base is sodium hydroxide;
and/or step (1) further comprises the step of sterilizing the solution; preferably, the sterilization is performed for 30-60 minutes under the conditions of 115-120 ℃ and 0.15-0.20 MPa.
8. The method according to claim 6, wherein the step (2) of adding BCG into the solution of step (1) comprises diluting the BCG preparation stock solution with the solution of step (1) and adding the solution of step (1) for dilution until the concentration of BCG is 0.05%.
9. The method of claim 6, wherein the homogeneous solution of step (3) is lyophilized by filling in vials.
10. The use of a lyophilized formulation of rBCG according to any one of claims 1-6 in the preparation of a medicament for the prevention of tuberculosis.
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| CN116077634A (en) * | 2022-12-01 | 2023-05-09 | 安徽智飞龙科马生物制药有限公司 | Dispersion and freeze-drying method and stock solution, semi-finished product and finished product of BCG bacteria for treatment |
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