A freeze-dried human rabies vaccine and preparation method thereof

Abstract

The present invention discloses a freeze-dried human rabies vaccine and a preparation method thereof, and belongs to the technical field of virus vaccines. The method comprises the following steps: (1) cell culture; (2) virus inoculation; (3) virus harvesting; (4) concentration and purification: the harvested virus liquid is clarified by a filter, and then ultrafiltration and concentration is performed through an ultrafiltration membrane to obtain a concentrated solution, which is purified by column chromatography to obtain a chromatographic solution; (5) preparation of a stock solution: the chromatographic solution is inactivated and hydrolyzed to obtain a virus inactivation solution, and human serum albumin is added to the virus inactivation solution to obtain a stock solution; (6) preparation of a semi-finished product: the semi-finished product is prepared by diluting with a stabilizer, and the stabilizer includes raw materials such as human serum albumin, arabinoxylan, L-arginine hydrochloride, casein phosphopeptide, α-nicotinic acid tocopherol and solvent; (7) preparation of a finished product. The rabies vaccine of the present invention has excellent quality, stable immune efficacy, and the liquid is clear after redissolution.

Description

Technical Field

[0001] The invention belongs to the technical field of virus vaccines, and particularly relates to a freeze-dried human rabies vaccine and a preparation method thereof. Background Art

[0002] Rabies is a zoonosis, an acute infectious disease caused by the rabies virus (RABV) that mainly invades the central nervous system, with a mortality rate of almost 100%. Rabies exists on all continents except Antarctica. According to incomplete statistics, at least 59,000 people die from rabies each year, resulting in billions of dollars in economic losses. Rabies cases have been reported throughout the year, with summer and autumn being the peak seasons. Rabies vaccination is crucial as the only effective means of preventing rabies, but the current production of rabies vaccines is far from meeting market demand.

[0003] Chinese patent 2013100096828 discloses a method for preparing a human diploid cell rabies vaccine virus liquid, which is produced by inoculating the fixed PM strain of rabies virus into MRC-5 cells, and includes the following steps: resuscitation and expansion of MRC-5 cells; inoculating the PM strain of rabies virus on the MRC-5 cells; continuing to subculture and expand the MRC-5 cells inoculated with the PM strain; washing the cells and then adding the virus culture medium; harvesting the virus liquid and concentrating it; the method of the invention is simple in process, and the prepared virus liquid has a high antigen content and virus titer, and can be used for large-scale industrial production, but the liquid vaccine is difficult to store and transport, and is prone to reduce the titer. Chinese patent 2006101235211 discloses a method for large-scale production of rabies vaccine, and its process steps are as follows: in a bioreactor with a fixed bed basket stirring system using Fibra-Cel disks as carriers, Vero cells are cultured with a cell proliferation culture medium; when the Vero cells grow to a certain density, the cell maintenance culture medium is replaced, rabies virus is inoculated and the cells are infected; the virus is proliferated; the virus is continuously harvested; ultrafiltration concentration and inactivation are performed; Vero cell DNA is removed; chromatography purification is performed using Sepharose 4 Fast Flow as a filler; human serum albumin and sucrose are added as protective agents to prepare a liquid preparation, or human serum albumin, sucrose, and gelatin are added as protective agents and excipients to prepare a freeze-dried preparation. The inventive method can realize continuous virus harvesting in a bioreactor with a smaller capacity and realize large-scale production, but the vaccine produced by this method still cannot guarantee a stable immune efficacy for a long period of time, and further optimization and improvement are needed. Summary of the invention

[0004] In view of the deficiencies in the above-mentioned prior art, the present invention provides a freeze-dried human rabies vaccine, which uses a bioreactor and microcarrier technology to produce a human diploid cell rabies vaccine, and optimizes the downstream purification, concentration and freeze-drying processes, thereby greatly improving the quality of the vaccine. The freeze-dried human rabies vaccine of the present invention has a stable immune efficacy, and the liquid is clear after reconstitution, and the reconstitution is good. The present invention also provides a method for preparing the above-mentioned freeze-dried human rabies vaccine.

[0005] The technical solution adopted by the present invention is:

[0006] A method for preparing a freeze-dried human rabies vaccine comprises the following steps:

[0007] (1) Cell culture: reviving a certain number of human diploid cells in a working cell bank; digesting the revived monolayer cells with 0.2-0.3wt% trypsin digestion solution, dispersing them evenly, adding cell growth medium, and culturing them at 32-38°C to form a uniform monolayer of cells; subculturing them according to the above method to a certain number of generations, inoculating them into a bioreactor, adding cell growth medium for culturing, and the bioreactor includes microcarriers;

[0008] (2) Virus inoculation: After culturing for 4-10 hours, discard the cell growth medium in the reactor, inoculate the rabies virus strain at an MOI of 0.01-0.1, add the virus growth medium, continue culturing, maintain the set culture parameters, and perform perfusion culture;

[0009] (3) Virus harvesting: After the cells on the microcarriers are cultured into a uniform monolayer, the virus growth liquid in the reactor is discarded, and the remaining virus growth liquid in the reactor is replaced with a cleaning liquid. Then, the maintenance liquid is added, and the culture is continued and the virus liquid is harvested by perfusion. After 10-30 days of harvesting, samples are taken for virus titration experiments and stored at 2-8°C.

[0010] (4) Concentration and purification: The virus liquid harvested above is clarified by a 1-2 μm filter, and then ultrafiltered and concentrated by a 600-1200 kD ultrafiltration membrane to obtain a concentrated solution; the concentrated solution is purified by column chromatography, and the protein peak is collected according to the ultraviolet absorption curve at a wavelength of 280 nm to obtain a chromatographic solution;

[0011] (5) Preparing a stock solution: adding β-propiolactone at a concentration of 0.1-1:4000 to the chromatography solution, inactivating at 2-8°C for 20-25 hours, hydrolyzing at 32-38°C for 1-3 hours after the inactivation to obtain a virus-inactivated solution, and adding human serum albumin at a final concentration of no more than 5 wt% to the virus-inactivated solution to obtain a stock solution;

[0012] (6) Preparation of semi-finished products: according to the antigen content of the stock solution, dilute it with a stabilizer to prepare the semi-finished product;

[0013] (7) Preparation of finished product: Filling and freeze-drying the prepared semi-finished product to obtain the finished freeze-dried human rabies vaccine.

[0014] Preferably, the human diploid cells in step (1) are any one of MRC-5 cells, KMB-17 cells, WI-38 cells, and SV-1 cells.

[0015] Preferably, the stabilizer in step (6), based on 500 parts by weight, comprises the following raw materials: 5-10 parts by weight of human albumin, 7-15 parts by weight of arabinoxylan, 1-5 parts by weight of L-arginine hydrochloride, 0.1-0.5 parts by weight of casein phosphopeptide, 0.1-0.5 parts by weight of α-nicotinic acid tocopherol, and the remainder is a solvent; the solvent is a PBS solution or water with a pH of 7.0-7.8.

[0016] In the process of preparing finished products by freeze-drying semi-finished products, freezing and drying will cause protein denaturation, causing obvious damage to the immune activity of the vaccine. The use of stabilizers can avoid the reduction of the activity of freeze-dried vaccines to a certain extent, and is conducive to improving the stability of the vaccine after freeze-drying, increasing the tolerance temperature of the vaccine, and prolonging the shelf life. Many adverse reactions occur after vaccination in the prior art, which is directly related to the adjuvants used in the vaccine products, especially the stabilizers that are indispensable in freeze-dried vaccines. Among them, dextran may cause some vaccine recipients to have adverse reactions such as fever, chest tightness, anaphylactic shock, urticaria, and skin itching. Gelatin and its derivatives may cause some vaccine recipients to have varying degrees of urticaria, neurogenic edema or asthma, etc., which poses certain safety risks. Therefore, the present invention provides a stabilizer that does not contain the above-mentioned ingredients.

[0017] The present invention selects casein phosphopeptide and alpha-nicotinic acid tocopherol in the stabilizer for compounding, and cooperates with other raw materials, which can significantly enhance the auxiliary dispersion and protective activity of the stabilizer in the drying process. The obtained finished dry powder has a loose appearance, good dispersibility, and uniform particle size, which can further reduce the damage of the freeze-drying process to the activity of the finished product, improve the antigen uptake and cellular immunity level; it can also improve the stability of the vaccine potency during storage, and facilitate transportation and storage; when used, the freeze-dried vaccine has good resolubility and a short dissolution time, which is more conducive to use.

[0018] The synergistic effect of casein phosphopeptide and α-nicotinic acid tocopherol mainly stems from their complementarity in chemical structure and function. Casein phosphopeptide is a phosphate-rich peptide chain with multiple hydrophilic amino acids and phosphate groups in its molecular structure. This makes casein phosphopeptide have excellent hydrophilicity and dispersibility, and can form a stable micelle structure in aqueous solution, effectively protecting protein antigens from physical stress damage during freeze-drying, while preventing protein aggregation and denaturation. On the other hand, α-nicotinic acid tocopherol is composed of niacin and tocopherol (vitamin E), and its molecular structure contains benzene rings and long-chain alkyl groups. It has strong antioxidant properties, can neutralize free radicals, and prevent oxidative degradation of proteins. The carboxyl and phenolic hydroxyl groups of the nicotinic acid part provide further antioxidant protection. When used in combination, the physical protection and dispersing effect of casein phosphopeptide and the chemical protection effect of α-nicotinic acid tocopherol complement each other, and can provide comprehensive protection during the freeze-drying process, thereby maximizing the efficacy and stability of the vaccine and improving the immune efficacy and thermal stability of the vaccine.

[0019] In addition, the present invention preferably uses arabinoxylan as the polysaccharide component. Its multi-functional structural characteristics are conducive to interaction with other components, and can better play a stabilizing and protective role, preventing the vaccine from being affected by external factors such as temperature and oxidation during storage, thereby improving the vaccine's resistance to drying and heat. It can also interact with other various components to reduce the aggregation between vaccine components, thereby further improving the stability and shelf life of the vaccine.

[0020] Preferably, the microcarrier in step (1) is a Cytodex1 microcarrier, and the amount of the microcarrier used is 4-8 g / L.

[0021] Preferably, the cell growth medium in step (1) is M199 culture medium containing 1-8 wt % newborn calf serum.

[0022] Preferably, the culture parameters of the bioreactor in step (1) are: temperature of 32-38°C, pH of 7.0-7.8, dissolved oxygen of not less than 25%, and rotation speed of not more than 100 rpm.

[0023] Preferably, the virus growth medium in step (2) is M199 culture medium containing 1-5 wt% newborn calf serum.

[0024] Preferably, the rabies virus strain in step (2) is any one of the rabies virus Pitman-Moore strain, the rabies virus PV strain, and the rabies virus HEP strain.

[0025] Preferably, the cell density when the virus strain is inoculated in step (2) reaches 0.8×10 7 -2×10 7 cells / mL range.

[0026] Preferably, the perfusion culture in step (2) starts 4-6 hours after virus inoculation, and the perfusion rate is no higher than 5 times the working volume per day.

[0027] Preferably, the cleaning solution in step (3) is any one of PBS solution, DPBS, HBSS, and EBSS with a pH of 7.0-7.8.

[0028] Preferably, the maintenance medium in step (3) is M199 culture medium containing 0.5-1 wt % human albumin.

[0029] Preferably, the perfusion rate in step (3) is no higher than 5 times the working volume per day.

[0030] Preferably, the medium used in the column chromatography in step (4) is the novel composite gel Capto Core 700, the sample loading amount does not exceed 200% of the column volume, the linear flow rate is not higher than 400 cm / h, and the elution is performed using a PBS solution with a pH of 7.0-7.8.

[0031] Preferably, the weight ratio of the stock solution to the stabilizer in step (6) is 1:1-10.

[0032] Preferably, the specification of the finished product in step (7) is 0.5-1 mL / dose, and the potency is 2.5-10 IU / dose.

[0033] The present invention utilizes bioreactor and microcarrier technology to culture human diploid cells and rabies virus, wherein a cell suspension is added to a bioreactor (cell tank) and cultured for 4 hours to 10 hours, and then the cell growth liquid in the tank is discarded, and rabies virus strains are inoculated for culture, and a large amount of high-titer virus harvest liquid is continuously and stably harvested through perfusion, and no antibiotics need to be added during the culture process.

[0034] Bovine serum albumin is the main impurity in the preparation process of rabies vaccine, and the lower the content is, the better. The present invention removes bovine serum albumin impurities through multi-step coordinated control of replacement of growth liquid in bioreactor culture, filtration and ultrafiltration concentration with a suitable pore size membrane, and purification by a new composite gel chromatography, and ultimately makes the residual amount of bovine serum albumin in the finished product far lower than the requirements of the current version III of the Chinese Pharmacopoeia.

[0035] The novel composite chromatography filler used in the present invention has both molecular sieve and affinity chromatography functions. Compared with the traditional molecular sieve chromatography method, it significantly improves the sample processing capacity. When the volume of the chromatography column is the same, the sample processing capacity is increased by nearly 20 times, which effectively saves sample processing time, shortens working hours, and saves costs.

[0036] The finished product of the present invention has an appearance of a white loose body, which is a clear liquid after reconstitution and contains no foreign matter; according to the third part of the current edition of the "Chinese Pharmacopoeia" (2020), the osmotic pressure molar concentration is not higher than 650 mOsmol / kg, the pH is 7.2-8.0, and the moisture content is not higher than 3.00%; the potency is not less than 2.5 IU / dose, and the potency remains not less than 2.5 IU / dose after being placed at 37°C for 28 days; the sterility test is qualified, the abnormal toxicity test is qualified, the bovine serum albumin residue is not higher than 50 ng / dose, and the bacterial endotoxin content is not higher than 25 EU / dose.

[0037] The present invention also provides a freeze-dried rabies vaccine for human use, which is prepared by the above method.

[0038] The beneficial effects of the present invention are:

[0039] 1. The freeze-dried human rabies vaccine prepared by the present invention has a perfect white loose appearance, is easy to reconstitute, and the liquid after reconstitution is clear, free of foreign matter, and has good stability.

[0040] 2. The human diploid cell rabies vaccine produced by the present invention using bioreactor and microcarrier technology has the advantages of high yield and excellent quality. In order to ensure that the vaccine has stable immune efficacy, high safety and minimal side effects, the downstream process is optimized at the same time to greatly improve the quality of the vaccine. DETAILED DESCRIPTION

[0041] The following will be combined with the specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Embodiment 1, a method for preparing a freeze-dried human rabies vaccine, comprising the following steps:

[0043] (1) Cell culture: reviving a certain number of human diploid cells in a working cell bank; digesting the revived monolayer cells with 0.25wt% trypsin digestion solution, dispersing them evenly, adding cell growth medium (M199 culture medium containing 8wt% newborn calf serum), and culturing them at 37°C to form a uniform monolayer of cells; subculturing them according to the above method to a certain number of generations, inoculating them into a bioreactor (adding Cytodex1 microcarriers in the bioreactor, the amount of microcarriers used is 6g / L), adding cell growth medium (M199 culture medium containing 8wt% newborn calf serum) for culturing, and the culture parameters are: temperature of 37°C, pH of 7.2, dissolved oxygen of 50%, and rotation speed of 60rpm; the human diploid cells are MRC-5 cells;

[0044] (2) Virus inoculation: After 10 h of culture, discard the cell growth medium in the reactor, inoculate the Pitman-Moore strain of rabies virus at an MOI of 0.01-0.1, add virus growth medium (M199 culture medium with 3 wt% newborn calf serum), and culture. Maintain the set culture parameters. Start perfusion culture 4 h after inoculation, and the perfusion rate should not exceed 5 times the working volume per day. When the virus strain is inoculated, the cell density reaches 0.8×10 7 -2×10 7 cells / mL range;

[0045] (3) Virus harvesting: After the cells on the microcarriers are cultured into a uniform monolayer, the virus growth liquid in the reactor is discarded, and the remaining virus growth liquid in the reactor is replaced with a cleaning solution (PBS solution with a pH of 7.2). Then, a maintenance solution (M199 culture medium containing 1 wt% human albumin) is added to continue culturing and perfuse the virus liquid. The perfusion rate is no more than 5 times the working volume per day. After 15 days of harvesting, samples are taken for virus titration experiments and stored at 2-8°C.

[0046] (4) Concentration and purification: The virus liquid harvested above is clarified by a 1.2 μm filter and then concentrated by ultrafiltration through a 1000 kD ultrafiltration membrane to obtain a concentrate; the concentrate is purified by column chromatography, the medium is a new composite gel CaptoCore 700, the sample load does not exceed 200% of the column volume, the linear flow rate is not higher than 400 cm / h, and the elution is performed with a PBS solution with a pH of 7.2. According to the ultraviolet absorption curve at a wavelength of 280 nm, the protein peak is collected to obtain a chromatographic solution;

[0047] (5) Preparation of stock solution: Add β-propiolactone at a concentration of 1:4000 to the chromatographic solution, inactivate at 2-8°C for 24 hours, hydrolyze at 37°C for 2 hours to obtain a virus-inactivated solution, add human serum albumin at a final concentration of 1.6 wt% to the virus-inactivated solution to obtain a stock solution;

[0048] (6) Preparing semi-finished products: preparing semi-finished products by diluting the stock solution with a stabilizer according to the antigen content of the stock solution; the weight ratio of the stock solution to the stabilizer is 1:4;

[0049] (7) Preparation of finished product: Filling and freeze-drying the prepared semi-finished product to obtain a finished freeze-dried human rabies vaccine with a specification of 1 mL / dose and a potency of 5.3 IU / dose.

[0050] The stabilizer, based on 500 parts by weight, comprises the following raw materials: 8 parts by weight of human albumin, 10 parts by weight of arabinoxylan, 3 parts by weight of L-arginine hydrochloride, 0.45 parts by weight of casein phosphopeptide, 0.3 parts by weight of α-nicotinic acid tocopherol, and the remainder is a solvent; the solvent is a PBS solution with a pH of 7.2.

[0051] Embodiment 2, a method for preparing a freeze-dried human rabies vaccine, comprising the following steps:

[0052] (1) Cell culture: reviving a certain number of human diploid cells in a working cell bank; digesting the revived monolayer cells with 0.25wt% trypsin digestion solution, dispersing them evenly, adding cell growth medium (M199 culture medium containing 8wt% newborn calf serum), and culturing them at 37°C to form a uniform monolayer of cells; subculturing them according to the above method to a certain number of generations, inoculating them into a bioreactor (adding Cytodex1 microcarriers in the bioreactor, the amount of microcarriers used is 6g / L), adding cell growth medium (M199 culture medium containing 8wt% newborn calf serum) for culturing, and the culture parameters are: temperature of 37°C, pH of 7.2, dissolved oxygen of 50%, and rotation speed of 60rpm; the human diploid cells are MRC-5 cells;

[0053] (2) Virus inoculation: After 10 h of culture, the cell growth medium in the reactor was discarded, and the rabies virus Pitman-Moore strain was inoculated at an MOI of 0.01-0.1. Virus growth medium (M199 culture medium with 3 wt% newborn calf serum) was added for culture. The set culture parameters were maintained. Perfusion culture was started 4 h after virus inoculation, and the perfusion rate was not higher than 5 times the working volume per day. When the virus strain was inoculated, the cell density reached 0.8×10 7 -2×10 7 cells / mL range;

[0054] (3) Virus harvesting: After the cells on the microcarriers are cultured into a uniform monolayer, the virus growth liquid in the reactor is discarded, and the remaining virus growth liquid in the reactor is replaced with a cleaning solution (PBS solution with a pH of 7.2). Then, a maintenance solution (M199 culture medium containing 1 wt% human albumin) is added to continue culturing and perfuse the virus liquid. The perfusion rate is no more than 5 times the working volume per day. After 15 days of harvesting, samples are taken for virus titration experiments and stored at 2-8°C.

[0055] (4) Concentration and purification: The virus liquid harvested above is clarified by a 1.2 μm filter and then concentrated by ultrafiltration through a 1000 kD ultrafiltration membrane to obtain a concentrate; the concentrate is purified by column chromatography, the medium is a new composite gel CaptoCore 700, the sample load does not exceed 200% of the column volume, the linear flow rate is not higher than 400 cm / h, and the elution is performed with a PBS solution with a pH of 7.2. According to the ultraviolet absorption curve at a wavelength of 280 nm, the protein peak is collected to obtain a chromatographic solution;

[0056] (5) Preparation of stock solution: Add β-propiolactone at a concentration of 1:4000 to the chromatographic solution, inactivate at 2-8°C for 24 hours, hydrolyze at 37°C for 2 hours to obtain a virus-inactivated solution, add human serum albumin at a final concentration of 1.6 wt% to the virus-inactivated solution to obtain a stock solution;

[0057] (6) Preparing semi-finished products: preparing semi-finished products by diluting the stock solution with a stabilizer according to the antigen content of the stock solution; the weight ratio of the stock solution to the stabilizer is 1:4;

[0058] (7) Preparation of finished product: Filling and freeze-drying the prepared semi-finished product to obtain a finished freeze-dried human rabies vaccine with a specification of 1 mL / dose and a potency of 5.3 IU / dose.

[0059] The stabilizer, based on 500 parts by weight, comprises the following raw materials: 8 parts by weight of human albumin, 10 parts by weight of trehalose, 3 parts by weight of L-arginine hydrochloride, 0.45 parts by weight of casein phosphopeptide, 0.3 parts by weight of α-nicotinic acid tocopherol, and the remainder is a solvent; the solvent is a PBS solution with a pH of 7.2.

[0060] Embodiment 3, a method for preparing a freeze-dried human rabies vaccine, comprising the following steps:

[0061] (1) Cell culture: reviving a certain number of human diploid cells in a working cell bank; digesting the revived monolayer cells with 0.25wt% trypsin digestion solution, dispersing them evenly, adding cell growth medium (M199 culture medium containing 8wt% newborn calf serum), and culturing them at 37°C to form a uniform monolayer of cells; subculturing them according to the above method to a certain number of generations, inoculating them into a bioreactor (adding Cytodex1 microcarriers in the bioreactor, the amount of microcarriers used is 6g / L), adding cell growth medium (M199 culture medium containing 8wt% newborn calf serum) for culturing, and the culture parameters are: temperature of 37°C, pH of 7.2, dissolved oxygen of 50%, and rotation speed of 60rpm; the human diploid cells are MRC-5 cells;

[0062] (2) Virus inoculation: After 10 h of culture, the cell growth medium in the reactor was discarded, and the rabies virus Pitman-Moore strain was inoculated at an MOI of 0.01-0.1. Virus growth medium (M199 culture medium with 3 wt% newborn calf serum) was added for culture. The set culture parameters were maintained. Perfusion culture was started 4 h after virus inoculation, and the perfusion rate was not higher than 5 times the working volume per day. When the virus strain was inoculated, the cell density reached 0.8×10 7 -2×10 7 cells / mL range;

[0063] (3) Virus harvesting: After the cells on the microcarriers are cultured into a uniform monolayer, the virus growth liquid in the reactor is discarded, and the remaining virus growth liquid in the reactor is replaced with a cleaning solution (PBS solution with a pH of 7.2). Then, a maintenance solution (M199 culture medium containing 1 wt% human albumin) is added to continue culturing and perfuse the virus liquid. The perfusion rate is no more than 5 times the working volume per day. After 15 days of harvesting, samples are taken for virus titration experiments and stored at 2-8°C.

[0064] (4) Concentration and purification: The virus liquid harvested above is clarified by a 1.2 μm filter and then concentrated by ultrafiltration through a 1000 kD ultrafiltration membrane to obtain a concentrate; the concentrate is purified by column chromatography, the medium is a new composite gel CaptoCore 700, the sample load does not exceed 200% of the column volume, the linear flow rate is not higher than 400 cm / h, and the elution is performed with a PBS solution with a pH of 7.2. According to the ultraviolet absorption curve at a wavelength of 280 nm, the protein peak is collected to obtain a chromatographic solution;

[0065] (5) Preparation of stock solution: Add β-propiolactone at a concentration of 1:4000 to the chromatographic solution, inactivate at 2-8°C for 24 hours, hydrolyze at 37°C for 2 hours to obtain a virus-inactivated solution, add human serum albumin at a final concentration of 1.6 wt% to the virus-inactivated solution to obtain a stock solution;

[0066] (6) Preparing semi-finished products: preparing semi-finished products by diluting the stock solution with a stabilizer according to the antigen content of the stock solution; the weight ratio of the stock solution to the stabilizer is 1:4;

[0067] (7) Preparation of finished product: Filling and freeze-drying the prepared semi-finished product to obtain a finished freeze-dried human rabies vaccine with a specification of 1 mL / dose and a potency of 5.3 IU / dose.

[0068] The stabilizer, calculated on a weight basis of 500 parts, comprises the following raw materials: 8 parts by weight of human albumin, 10 parts by weight of arabinoxylan, 3 parts by weight of L-arginine hydrochloride, and the remainder is a solvent; the solvent is a PBS solution with a pH of 7.2.

[0069] Embodiment 4, a method for preparing a freeze-dried human rabies vaccine, comprising the following steps:

[0070] (1) Cell culture: reviving a certain number of human diploid cells in a working cell bank; digesting the revived monolayer cells with 0.25wt% trypsin digestion solution, dispersing them evenly, adding cell growth medium (M199 culture medium containing 8wt% newborn calf serum), and culturing them at 37°C to form a uniform monolayer of cells; subculturing them according to the above method to a certain number of generations, inoculating them into a bioreactor (adding Cytodex1 microcarriers in the bioreactor, the amount of microcarriers used is 6g / L), adding cell growth medium (M199 culture medium containing 8wt% newborn calf serum) for culturing, and the culture parameters are: temperature of 37°C, pH of 7.2, dissolved oxygen of 50%, and rotation speed of 60rpm; the human diploid cells are MRC-5 cells;

[0071] (2) Virus inoculation: After 10 h of culture, the cell growth medium in the reactor was discarded, and the rabies virus Pitman-Moore strain was inoculated at an MOI of 0.01-0.1. Virus growth medium (M199 culture medium with 3 wt% newborn calf serum) was added for culture. The set culture parameters were maintained. Perfusion culture was started 4 h after virus inoculation, and the perfusion rate was not higher than 5 times the working volume per day. When the virus strain was inoculated, the cell density reached 0.8×10 7 -2×10 7 cells / mL range;

[0072] (3) Virus harvesting: After the cells on the microcarriers are cultured into a uniform monolayer, the virus growth liquid in the reactor is discarded, and the remaining virus growth liquid in the reactor is replaced with a cleaning solution (PBS solution with a pH of 7.2). Then, a maintenance solution (M199 culture medium containing 1 wt% human albumin) is added to continue culturing and perfuse the virus liquid. The perfusion rate is no more than 5 times the working volume per day. After 15 days of harvesting, samples are taken for virus titration experiments and stored at 2-8°C.

[0073] (4) Concentration and purification: The virus liquid harvested above is clarified by a 1.2 μm filter and then concentrated by ultrafiltration through a 1000 kD ultrafiltration membrane to obtain a concentrate; the concentrate is purified by column chromatography, the medium is a new composite gel CaptoCore 700, the sample load does not exceed 200% of the column volume, the linear flow rate is not higher than 400 cm / h, and the elution is performed with a PBS solution with a pH of 7.2. According to the ultraviolet absorption curve at a wavelength of 280 nm, the protein peak is collected to obtain a chromatographic solution;

[0074] (5) Preparation of stock solution: Add β-propiolactone at a concentration of 1:4000 to the chromatographic solution, inactivate at 2-8°C for 24 hours, hydrolyze at 37°C for 2 hours to obtain a virus-inactivated solution, add human serum albumin at a final concentration of 1.6 wt% to the virus-inactivated solution to obtain a stock solution;

[0075] (6) Preparing semi-finished products: preparing semi-finished products by diluting the stock solution with a stabilizer according to the antigen content of the stock solution; the weight ratio of the stock solution to the stabilizer is 1:4;

[0076] (7) Preparation of finished product: Filling and freeze-drying the prepared semi-finished product to obtain a finished freeze-dried human rabies vaccine with a specification of 1 mL / dose and a potency of 5.3 IU / dose.

[0077] The stabilizer, calculated on a weight basis of 500 parts, comprises the following raw materials: 8 parts by weight of human albumin, 10 parts by weight of arabinoxylan, 3 parts by weight of L-arginine hydrochloride, 0.75 parts by weight of casein phosphopeptide, and the remainder is a solvent; the solvent is a PBS solution having a pH of 7.2.

[0078] Embodiment 5, a method for preparing a freeze-dried human rabies vaccine, comprising the following steps:

[0079] (1) Cell culture: reviving a certain number of human diploid cells in a working cell bank; digesting the revived monolayer cells with 0.25wt% trypsin digestion solution, dispersing them evenly, adding cell growth medium (M199 culture medium containing 8wt% newborn calf serum), and culturing them at 37°C to form a uniform monolayer of cells; subculturing them according to the above method to a certain number of generations, inoculating them into a bioreactor (adding Cytodex1 microcarriers in the bioreactor, the amount of microcarriers used is 6g / L), adding cell growth medium (M199 culture medium containing 8wt% newborn calf serum) for culturing, and the culture parameters are: temperature of 37°C, pH of 7.2, dissolved oxygen of 50%, and rotation speed of 60rpm; the human diploid cells are MRC-5 cells;

[0080] (2) Virus inoculation: After 10 h of culture, the cell growth medium in the reactor was discarded, and the rabies virus Pitman-Moore strain was inoculated at an MOI of 0.01-0.1. Virus growth medium (M199 culture medium with 3 wt% newborn calf serum) was added for culture. The set culture parameters were maintained. Perfusion culture was started 4 h after virus inoculation, and the perfusion rate was not higher than 5 times the working volume per day. When the virus strain was inoculated, the cell density reached 0.8×10 7 -2×10 7 cells / mL range;

[0081] (3) Virus harvesting: After the cells on the microcarriers are cultured into a uniform monolayer, the virus growth liquid in the reactor is discarded, and the remaining virus growth liquid in the reactor is replaced with a cleaning solution (PBS solution with a pH of 7.2). Then, a maintenance solution (M199 culture medium containing 1 wt% human albumin) is added to continue culturing and perfuse the virus liquid. The perfusion rate is no more than 5 times the working volume per day. After 15 days of harvesting, samples are taken for virus titration experiments and stored at 2-8°C.

[0082] (4) Concentration and purification: The virus liquid harvested above is clarified by a 1.2 μm filter and then concentrated by ultrafiltration through a 1000 kD ultrafiltration membrane to obtain a concentrate; the concentrate is purified by column chromatography, the medium is a new composite gel CaptoCore 700, the sample load does not exceed 200% of the column volume, the linear flow rate is not higher than 400 cm / h, and the elution is performed with a PBS solution with a pH of 7.2. According to the ultraviolet absorption curve at a wavelength of 280 nm, the protein peak is collected to obtain a chromatographic solution;

[0083] (5) Preparation of stock solution: Add β-propiolactone at a concentration of 1:4000 to the chromatographic solution, inactivate at 2-8°C for 24 hours, hydrolyze at 37°C for 2 hours to obtain a virus-inactivated solution, add human serum albumin at a final concentration of 1.6 wt% to the virus-inactivated solution to obtain a stock solution;

[0084] (6) Preparing semi-finished products: preparing semi-finished products by diluting the stock solution with a stabilizer according to the antigen content of the stock solution; the weight ratio of the stock solution to the stabilizer is 1:4;

[0085] (7) Preparation of finished product: Filling and freeze-drying the prepared semi-finished product to obtain a finished freeze-dried human rabies vaccine with a specification of 1 mL / dose and a potency of 5.3 IU / dose.

[0086] The stabilizer, calculated on a weight basis of 500 parts, comprises the following raw materials: 8 parts by weight of human albumin, 10 parts by weight of arabinoxylan, 3 parts by weight of L-arginine hydrochloride, 0.75 parts by weight of α-nicotinic acid tocopherol, and the remainder is a solvent; the solvent is a PBS solution with a pH of 7.2.

[0087] Test Example 1: The finished freeze-dried human rabies vaccine prepared in the above embodiment was evaluated according to the standard of the 2020 edition of the Chinese Pharmacopoeia, including appearance, moisture content, and potency in the thermal stability test (37°C). The results are shown in Table 1.

[0088] Table 1. Improvement in the quality of finished vaccine products in the examples

[0089]

[0090] The freeze-dried human rabies vaccine prepared by the present invention has excellent quality and stable potency. The present invention fully utilizes the differences in physical and chemical properties between the target protein and impurities through the coordinated control of replacement of growth liquid in bioreactor culture, appropriate pore size membrane filtration and ultrafiltration concentration, and new composite gel chromatography purification, and removes bovine serum albumin impurities together, and finally makes the bovine serum albumin residue in the finished product far lower than the requirements of the current version of the third part of the "Chinese Pharmacopoeia". Compared with Examples 3-5, Example 1 selects casein phosphopeptide and α-nicotinic acid tocopherol in the stabilizer for compounding, which can significantly enhance the role of the stabilizer in assisting dispersion and protecting activity during the drying process. The finished dry powder obtained has a loose appearance, good dispersibility, and uniform particle size, which can further reduce the damage to the activity of the finished product during the freeze-drying process, and improve the uptake of antigens and cellular immunity levels; it can also improve the stability of the vaccine potency during storage, and facilitate transportation and storage; when used, the freeze-dried vaccine has good resolubility, a short dissolution time, and is more conducive to use. Compared with Example 2, Example 1 prefers arabinoxylan as the polysaccharide component. Its multi-functional structure is conducive to interaction with other components, and can better play a stabilizing and protective role to prevent the vaccine from being affected by external factors such as temperature and oxidation during storage, thereby improving the vaccine's resistance to drying and heat. It can also interact with other various components to reduce aggregation between vaccine components, thereby further improving the stability and shelf life of the vaccine.

[0091] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for preparing a freeze-dried human rabies vaccine, characterized in that: The steps include: (1) Cell culture: Resuscitate human diploid cells, digest and passage the resuscitated monolayer cells with trypsin digestion solution, add cell growth medium, and culture at 32-38°C to form a uniform monolayer of cells; inoculate into a bioreactor, add cell growth medium for culture, and the bioreactor includes microcarriers; (2) Virus inoculation: After a period of culture, discard the cell growth medium in the reactor, inoculate the rabies virus strain, add the virus growth medium, continue to culture, maintain the set culture parameters, and perform perfusion culture; (3) Virus harvesting: After the cells on the microcarriers are cultured into a uniform monolayer, the virus growth liquid in the reactor is discarded, and the remaining virus growth liquid in the reactor is replaced with a cleaning liquid. Then, a maintenance liquid is added, and the culture is continued and the virus liquid is harvested by perfusion. The harvesting time is 10-30 days. (4) Concentration and purification: The virus liquid harvested above is clarified by a 1-2 μm filter, and then ultrafiltered and concentrated by a 600-1200 kD ultrafiltration membrane to obtain a concentrated solution; the concentrated solution is purified by column chromatography, and the protein peak is collected to obtain a chromatographic solution; (5) Preparing a stock solution: adding β-propiolactone to the chromatography solution for inactivation, hydrolyzing after the inactivation to obtain a virus-inactivated solution, and adding human serum albumin to the virus-inactivated solution to obtain a stock solution; (6) Preparing semi-finished products: preparing semi-finished products by diluting with a stabilizer; the stabilizer comprises the following raw materials: human albumin, arabinoxylan, L-arginine hydrochloride, casein phosphopeptide, α-nicotinic acid tocopherol and a solvent; (7) Preparation of finished product: Filling and freeze-drying the prepared semi-finished product to obtain the finished freeze-dried human rabies vaccine.

2. The method for preparing the freeze-dried human rabies vaccine according to claim 1, characterized in that: The human diploid cells in step (1) are any one of MRC-5 cells, KMB-17 cells, WI-38 cells, and SV-1 cells.

3. The method for preparing the freeze-dried human rabies vaccine according to claim 1, characterized in that: The amount of the microcarrier used in step (1) is 4-8 g / L.

4. The method for preparing the freeze-dried human rabies vaccine according to claim 1, characterized in that: The culture parameters of the bioreactor in step (1) are: temperature of 32-38°C, pH of 7.0-7.8, dissolved oxygen of not less than 25%, and rotation speed of not more than 100 rpm.

5. The method for preparing the freeze-dried human rabies vaccine according to claim 1, characterized in that: The rabies virus strain in step (2) is any one of the rabies virus Pitman-Moore strain, the rabies virus PV strain, and the rabies virus HEP strain.

6. The method for preparing the freeze-dried human rabies vaccine according to claim 1, characterized in that: The perfusion rate in steps (2) and (3) should not exceed 5 times the working volume per day.

7. The method for preparing the freeze-dried human rabies vaccine according to claim 1, characterized in that: The medium used in the column chromatography in step (4) is the composite gel Capto Core 700.

8. The method for preparing the freeze-dried human rabies vaccine according to claim 1, characterized in that: The weight ratio of the stock solution to the stabilizer in step (6) is 1:1-10.

9. The method for preparing the freeze-dried human rabies vaccine according to claim 1, characterized in that: The specification of the finished product in step (7) is 0.5-1 mL / dose, and the potency is 2.5-10 IU / dose.

10. A freeze-dried rabies vaccine for human use, characterized in that: The method is prepared by the method according to any one of claims 1 to 9.

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