A method for purifying inactivated rabies virus vaccine
By layering the composite filler chromatography column and optimizing the eluent method, the problems of low recovery rate and difficulty in impurity removal in the existing rabies vaccine purification were solved, an efficient and simplified virus purification process was achieved, and the antigen recovery rate and purity were improved.
Patent Information
- Application Number
- CN202510812891.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-18
- Publication Date
- 2025-09-30
- Estimated Expiration
- 2045-06-18
AI Technical Summary
Existing rabies vaccine purification methods have problems such as complex processes, low recovery rates, and difficulty in removing host cell proteins and small molecule impurities. In particular, the single use of Capto Core 700 cannot achieve both high recovery rates and high purity.
A layered composite filler chromatography column was used, combined with an optimized eluent purification scheme, and a mixture of Capto Core 700, 4FF, and mesoporous adsorption materials was used as the purification filler. Impurities were removed and virus particles were retained through the synergistic effect of size exclusion and multimodal adsorption.
It achieves efficient removal of impurities such as nucleic acids and host proteins, improves the chromatography purification efficiency of rabies virus liquid, the recovery rate and purity of virus antigens, simplifies the process flow and reduces costs.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for purifying an inactivated rabies virus vaccine. Background Art
[0002] Rabies is a zoonotic infectious disease caused by the rabies virus, with a mortality rate approaching 100%. Currently, rabies vaccines are primarily prepared using Vero cell culture, followed by inactivation and purification to produce inactivated vaccines. However, traditional purification methods (such as ultracentrifugation and sucrose density gradient centrifugation) are complex, have low recovery rates, and are difficult to scale up.
[0003] The rabies vaccine production process primarily involves three key steps: virus culture, inactivation, and purification. Purification technology directly determines the vaccine's safety (residual impurity levels) and efficacy (antigen recovery). Currently, commonly used purification methods in industry include ultracentrifugation, chromatography, membrane filtration, and combinations thereof, but each presents technical bottlenecks.
[0004] Currently, the commonly used purification methods are: (1) Ultracentrifugation: Virus particles are separated by density differences. However, there are the following problems: the process is complex, multiple centrifugations are required, and the recovery rate is low; it is difficult to remove small molecule impurities such as host cell protein HCP and bovine serum albumin BSA. (2) Membrane filtration: Tangential flow filtration is often used for virus concentration, but it is difficult to remove small molecule impurities when used alone. (3) Chromatography: Chromatographic technology (such as size exclusion chromatography and ion exchange chromatography) has gradually become the mainstream method for virus purification due to its high resolution and scalability. However, it is difficult for a single filler to achieve both high recovery and high purity: for example, Capto Core700 has an insufficient HCP removal rate (BSA removal rate is usually <80%), requiring subsequent purification steps; non-specific adsorption leads to virus loss: virus particles easily bind to the filler surface, making elution difficult and affecting the recovery rate.
[0005] In response to the above problems, the present invention proposes a chromatography column with layered filling of composite fillers, combined with a purification scheme of optimized eluent. Summary of the Invention
[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for purifying an inactivated rabies virus vaccine.
[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:
[0008] A method for purifying an inactivated rabies virus vaccine comprises the following steps:
[0009] Step 1: Preparation of rabies virus inactivated ultrafiltration concentrate:
[0010] Step 2: purifying the inactivated rabies virus ultrafiltration concentrate by chromatography;
[0011] The specific steps of the chromatography purification are as follows:
[0012] S1. Chromatography column cleaning: Load the composite filler into the chromatography column, then connect AKTA to the chromatography column, rinse the chromatography column with 4-6CV of the mixed solution, sterilize, and set aside;
[0013] S2. Chromatography operation: Equilibrate the chromatography column with 8-13CV of PBS buffer until the pH is neutral;
[0014] S3. Sample loading: The inactivated rabies virus ultrafiltration concentrate is loaded into the above-mentioned chromatography column, eluted with an eluent, and the eluate corresponding to the absorption peak at an ultraviolet wavelength of 280 nm is collected, which is the purified inactivated rabies virus vaccine;
[0015] S4. Column cleaning: After loading, rinse with PBS buffer until the UV absorption baseline returns to zero;
[0016] The composite filler is at least one of Capto Core 700, 4FF, mesoporous adsorption material, polyacrylamide gel, and porous silica gel.
[0017] Capto Core 700's unique structural design features a non-porous or macroporous inert interior and a porous exterior. This internal structure allows only small impurities, such as host cell proteins, nucleic acids, and endotoxins, to enter and be adsorbed. Viruses, however, are too large to enter the packing pores and instead flow directly out of the column via size exclusion. Capto Core 700 utilizes the synergistic effects of size exclusion and multimodal adsorption to remove multiple impurities in a single step, simplifying the process. Compared to 4FF columns, it offers increased active adsorption capacity for more efficient impurity removal.
[0018] The preparation of the rabies virus inactivated ultrafiltration concentrate comprises the following steps:
[0019] N1. Cell expansion: Vero cells are revived and passaged to a cell factory for large-scale expansion culture. After the cells have expanded to a certain number and grown densely, they are digested and collected to obtain a suspension;
[0020] N2. Virus culture: Inoculate the collected cell suspension with rabies vaccine virus at an MOI of 0.03-0.1 and transfer to a bioreactor for culture;
[0021] N3. Virus harvest: After 2-5 days of culture, add 0.3-0.8% (V / V) trypsin solution to digest the cells, then elute and collect the cells attached to the bioreactor wall with 8-15 mmol / L PBS buffer, pH 7.0, to obtain the virus harvest solution; clarify with a 0.25 μm pore size filter, and then concentrate 40-60 times using a 300 kD pore size membrane package to form a virus concentrate;
[0022] N4. Inactivation: Add β-propiolactone to the virus concentrate at a ratio of 1:3000-5000 (V / V) and inactivate the virus at 3-5°C for 40-60 hours. After inactivation, place the virus at 35-40°C for 2-5 hours to obtain the rabies virus inactivated ultrafiltration concentrate.
[0023] Preferably, the chromatography column model in S1 is any one of XK26 / 70, XK16 / 40, and XK50 / 60.
[0024] Preferably, the mixed solution in S1 consists of 60-75 wt % of a 0.5-2 mol / L NaOH aqueous solution and 20-40 wt % of isopropanol.
[0025] Preferably, the sample loading volume in S3 is 2-10% of the column volume.
[0026] Preferably, the loading flow rate and the elution flow rate in S3 are both 40-50 cm / h.
[0027] Preferably, the eluent in S3 consists of 0.3-0.8 M PBS, 0.4-0.7 M arginine, 0.1-0.6 M NaCl, 0.03-0.1% poloxamer 188, and has a pH of 6.5-7.2.
[0028] Preferably, the bioreactor conditions in N2 are a temperature of 32-37°C, a rotation speed of 50-100 rpm, an oxygen content of 45-55%, and a pH of 7.0-7.6.
[0029] Although the present invention uses Capto Core 700 as a chromatography filler, which has higher antigen virus recovery rate and antigen content, Capto Core 700, as a size exclusion medium, relies on pore size to screen viruses (30-700nm), but virus particles may be retained due to nonspecific adsorption: the interaction between the virus surface charge and the filler microenvironment; at the same time, the virus structure is easily damaged by mechanical shearing during high-speed elution, resulting in a certain loss of antigen virus recovery rate and antigen content. In order to further improve the antigen virus recovery rate and antigen content, the inventors used a layered filler method for optimization during purification.
[0030] The composite filler used in the present invention is a mixture of Capto Core 700, 4FF, and a mesoporous adsorption material. The chromatography column is packed in layers, with the bottom layer consisting of Capto Core 700; the middle layer consisting of 4FF; and the top layer consisting of the mesoporous adsorption material. The top mesoporous adsorption material primarily features a tannic acid-titanium / nickel modified surface, which preferentially adsorbs negatively charged host proteins, such as host cell proteins, nucleic acids, endotoxins, and DNA, through metal ion chelation, reducing competition for viral binding. The chitosan hydrogel coating provides positively charged sites for electrostatic adsorption of nucleic acids and acidic proteins, preventing them from entering the Capto Core 700 layer and interfering with virus recovery. The pore size of bovine serum albumin (BSA) is precisely between 2-10 nm and that of Al-MCM-41, allowing for its removal through physical entrapment. BSA is efficiently captured by the top filler, reducing residual amounts. This addresses the inefficient removal of small amounts of BSA by Capto Core 700, which relies solely on weak surface adsorption. After impurities are pre-removed from the bottom Capto Core 700, viral particles are more easily and efficiently eluted through the size exclusion principle, reducing non-target adsorption. The three work together to play their respective roles. Capto Core 700 separates intact viral particles based on size exclusion, 4FF reduces column pressure and increases throughput through rapid flow, and the mesoporous adsorption material selectively captures host proteins and nucleic acids. The three work together to achieve one-step purification with high recovery rate and low impurity residue.
[0031] Preferably, the preparation method of the mesoporous adsorption material is as follows:
[0032] (1) Al-MCM-41 mesoporous material and water were mixed by ultrasonication, and tannic acid aqueous solution was added under stirring, and the stirring was continued. After the chloride aqueous solution was added, the stirring was continued to obtain tannic acid-modified titanium-doped mesoporous material;
[0033] (2) Dissolve chitosan in acetic acid solution and stir, then dropwise add sodium hydroxide aqueous solution and glutaraldehyde and continue stirring, and filter with filter cloth to obtain chitosan hydrogel;
[0034] (3) Add the tannic acid-modified titanium-doped mesoporous material and the chitosan hydrogel into an oxalic acid solution, heat and stir, and then add into a NaOH aqueous solution and stir to obtain a mesoporous adsorption material.
[0035] Reaction mechanism of mesoporous adsorption materials: The high specific surface area of mesoporous materials can load more metal ions, thereby increasing the adsorption capacity of proteins. Tannic acid contains multiple phenolic hydroxyl groups (such as gallic acid groups), which can be combined with Si-OH on the surface of Al-MCM-41 through hydrogen bonds or electrostatic interactions. At the same time, the catechol groups of tannic acid form stable five-membered ring chelates with titanium and nickel; glutaraldehyde is used as a cross-linking agent to prepare chitosan gel; finally, the carboxylic acid in tannic acid and the hydroxyl group in chitosan are condensed to obtain the mesoporous adsorption material. The rich pore structure of the mesoporous material can increase the virus passage speed and reduce the pressure of the filtration process, thereby realizing a virus purification method with high efficiency and high adsorption capacity for physical removal of impurities such as protein and DNA.
[0036] Furthermore, the preparation method of the mesoporous adsorption material is as follows:
[0037] (1) 7-14 g of Al-MCM-41 mesoporous material and 400-800 mL of water were mixed by ultrasonication, 80-200 mL of 12-300 mg / mL tannic acid aqueous solution was added at a stirring speed of 200-400 rpm, and stirring was continued for 30-60 min. After adding 80-160 mL of 1-2 mg / mL chloride aqueous solution, stirring was continued for 40-90 min, centrifuged, washed, and vacuum dried to obtain tannic acid-modified titanium-doped mesoporous material;
[0038] (2) Dissolve 1-5 g of chitosan in 300-600 mL of acetic acid solution with a mass fraction of 0.3-1 wt%, stir at 500-1000 rpm for 1-3 h, then add 15-300 mL of 3-5 wt% sodium hydroxide aqueous solution and 0.5-3 g of glutaraldehyde dropwise, continue stirring for 1-4 h, and filter with 80-120 mesh filter cloth to obtain chitosan hydrogel;
[0039] (3) Add 8-12 g of the above-mentioned tannic acid-modified titanium-doped mesoporous material and 1-3 g of the above-mentioned chitosan hydrogel into 200-400 mL of 0.1-0.5 M oxalic acid solution, stir at 40-60 ° C and 300-600 rpm for 2-5 h, then add into 400-600 mL of 0.3-0.8 M NaOH aqueous solution, stir at 300-500 rpm for 0.5-2 h at room temperature, wash, and filter with 300-600 mesh filter cloth to obtain a mesoporous adsorption material.
[0040] The chloride is at least one of titanium chloride and nickel chloride; preferably, the chloride is composed of titanium chloride and nickel chloride in a mass ratio of 1:1.
[0041] A rabies virus inactivated vaccine is obtained by using any of the above purification methods.
[0042] The beneficial effects of the present invention are as follows: 1. The present invention provides a method for purifying an inactivated rabies virus vaccine, which adopts a layered packing method for purifying rabies virus, with a high sample loading amount and a fast flow rate, shortening the process time, retaining the effective antigen, and effectively removing impurities such as nucleases, host proteins and DNA, and is suitable for the rapid development and production of inactivated rabies virus vaccines.
[0043] 2. The rabies virus purification method provided by the present invention greatly improves the chromatographic purification efficiency of rabies virus liquid while ensuring high recovery rate and impurity protein removal rate. The rabies virus purification method provided by the present invention is simple, effective, low-cost, and ensures the purity of rabies virus antigens.
[0044] 3. Compared with existing technologies, this invention is the first to utilize Capto Core 700, 4FF, and mesoporous adsorption materials simultaneously as purification fillers for chromatographic purification of inactivated rabies virus ultrafiltrate, significantly improving the recovery rate of viral antigens. Specific tests have shown that residual Vero cell protein levels are as low as 2.37 ng / ml, antigen content can reach 17.32 IU / ml, host protein removal rates can reach 100%, and antigen recovery rates can reach 91.50%, representing significant technological advancements. DETAILED DESCRIPTION
[0045] The above content of the present invention will be further described in detail below in conjunction with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments.
[0046] The sources of some raw materials in this application are introduced. Other raw materials not introduced are commercially available:
[0047] Trypsin was purchased from Shanxi Hongchuang Biotechnology Co., Ltd., product number SXHC-0715-402.
[0048] Al-MCM-41 mesoporous material was purchased from Suzhou Kaifa New Material Technology Co., Ltd., model szbknm201010.
[0049] Chitosan was purchased from Qingdao Haihui Biological Co., Ltd. with a molecular weight of 550,000 g / mol, a degree of deacetylation of 92.3%, and was food grade.
[0050] Example 1
[0051] A method for purifying an inactivated rabies virus vaccine comprises the following steps:
[0052] Step 1: Preparation of rabies virus inactivated ultrafiltration concentrate:
[0053] N1. Cell expansion: Vero cells are revived and passaged to a cell factory for large-scale expansion culture. After the cells have expanded to a certain number and grown densely, they are digested and collected to obtain a suspension;
[0054] N2. Virus culture: Inoculate the collected cell suspension with rabies vaccine at an MOI of 0.05 and transfer the culture to a bioreactor. Control the bioreactor temperature at 35°C, the rotation speed at 80 rpm, the oxygen content at 50%, and the pH at 7.4.
[0055] N3. Virus harvest: After 3 days of culture, add 0.5% (v / v) trypsin solution to digest the cells. Then, elute and collect the cells attached to the bioreactor wall with 10 mmol / L PBS buffer at a pH of 7.0 to obtain the virus harvest solution. Clarify with a 0.25 μm pore size filter cartridge and then concentrate 50 times using a 300 kD pore size membrane package to form a virus concentrate.
[0056] N4. Inactivation: Add β-propiolactone to the virus concentrate at a ratio of 1:4000 (V / V) and inactivate the virus at 4°C for 48 hours. After inactivation, place the concentrate at 37°C for 3 hours to obtain the inactivated rabies virus ultrafiltration concentrate.
[0057] Step 2, chromatography purification: purify the above-mentioned rabies virus inactivated ultrafiltration concentrate by eluting with an eluent, and collect the elution peak with a UV wavelength of 280 nm to obtain a purified solution;
[0058] The specific steps of the chromatography purification are as follows:
[0059] S1. Chromatography column cleaning: The composite filler was loaded into an XK26 / 70 chromatography column with a column bed height of 40 cm. Then, the AKTA was connected to the XK26 / 70 chromatography column. The chromatography column was rinsed with 5 CV of a mixture of 70 wt % 1 mol / L aqueous NaOH solution and 30 wt % isopropanol for 30 min, sterilized, and set aside.
[0060] S2, chromatography operation: the chromatography column was equilibrated with 10 CV of 0.01 mol / L PBS buffer, pH 7.8, until the pH was neutral;
[0061] S3. Loading: The inactivated rabies virus ultrafiltration concentrate was loaded into the above-mentioned chromatography column with a loading volume of 8% of the column volume. The column was eluted with an eluent at a loading flow rate of 46 cm / h and an elution flow rate of 46 cm / h. The eluate was monitored at a UV wavelength of 280 nm and the eluate corresponding to the absorption peak at a UV wavelength of 280 nm was collected to obtain the purified inactivated rabies virus vaccine.
[0062] S4. Column cleaning: After loading, rinse with 0.01 mol / L, pH 7.8 PBS buffer until the UV absorption baseline returns to zero. Replace the cleaning solution and rinse the column with 5 CV of a mixture of 70 wt% 1 mol / L NaOH aqueous solution and 30 wt% isopropanol. The flow rate of the cleaning solution is 2 cm / min.
[0063] The composite filler is 4FF.
[0064] The eluent consisted of 0.5 M PBS, 0.5 M arginine, 0.5 M NaCl, and 0.05% Poloxamer 188, with a pH of 6.8.
[0065] Example 2
[0066] A method for purifying an inactivated rabies virus vaccine comprises the following steps:
[0067] Step 1: Preparation of rabies virus inactivated ultrafiltration concentrate:
[0068] N1. Cell expansion: Vero cells are revived and passaged to a cell factory for large-scale expansion culture. After the cells have expanded to a certain number and grown densely, they are digested and collected to obtain a suspension;
[0069] N2. Virus culture: Inoculate the collected cell suspension with rabies vaccine at an MOI of 0.05 and transfer the culture to a bioreactor. Control the bioreactor temperature at 35°C, the rotation speed at 80 rpm, the oxygen content at 50%, and the pH at 7.4.
[0070] N3. Virus harvest: After 3 days of culture, add 0.5% (v / v) trypsin solution to digest the cells. Then, elute and collect the cells attached to the bioreactor wall with 10 mmol / L PBS buffer at a pH of 7.0 to obtain the virus harvest solution. Clarify with a 0.25 μm pore size filter cartridge and then concentrate 50 times using a 300 kD pore size membrane package to form a virus concentrate.
[0071] N4. Inactivation: Add β-propiolactone to the virus concentrate at a ratio of 1:4000 (V / V) and inactivate the virus at 4°C for 48 hours. After inactivation, place the concentrate at 37°C for 3 hours to obtain the inactivated rabies virus ultrafiltration concentrate.
[0072] Step 2, chromatography purification: purify the above-mentioned rabies virus inactivated ultrafiltration concentrate by eluting with an eluent, and collect the elution peak with a UV wavelength of 280 nm to obtain a purified solution;
[0073] The specific steps of the chromatography purification are as follows:
[0074] S1. Chromatography column cleaning: The composite filler was loaded into an XK26 / 70 chromatography column with a column bed height of 40 cm. Then, the AKTA was connected to the XK26 / 70 chromatography column. The chromatography column was rinsed with 5 CV of a mixture of 70 wt % 1 mol / L aqueous NaOH solution and 30 wt % isopropanol for 30 min, sterilized, and set aside.
[0075] S2, chromatography operation: the chromatography column was equilibrated with 10 CV of 0.01 mol / L PBS buffer, pH 7.8, until the pH was neutral;
[0076] S3. Loading: The inactivated rabies virus ultrafiltration concentrate was loaded into the above-mentioned chromatography column with a loading volume of 8% of the column volume. The column was eluted with an eluent at a loading flow rate of 46 cm / h and an elution flow rate of 46 cm / h. The eluate was monitored at a UV wavelength of 280 nm and the eluate corresponding to the absorption peak at a UV wavelength of 280 nm was collected to obtain the purified inactivated rabies virus vaccine.
[0077] S4. Column cleaning: After loading, rinse with 0.01 mol / L, pH 7.8 PBS buffer until the UV absorption baseline returns to zero. Replace the cleaning solution and rinse the column with 5 CV of a mixture of 70 wt% 1 mol / L NaOH aqueous solution and 30 wt% isopropanol. The flow rate of the cleaning solution is 2 cm / min.
[0078] The composite filler is Capto Core 700.
[0079] The eluent consisted of 0.5 M PBS, 0.5 M arginine, 0.5 M NaCl, and 0.05% Poloxamer 188, with a pH of 6.8.
[0080] Example 3
[0081] A method for purifying an inactivated rabies virus vaccine comprises the following steps:
[0082] Step 1: Preparation of rabies virus inactivated ultrafiltration concentrate:
[0083] N1. Cell expansion: Vero cells are revived and passaged to a cell factory for large-scale expansion culture. After the cells have expanded to a certain number and grown densely, they are digested and collected to obtain a suspension;
[0084] N2. Virus culture: Inoculate the collected cell suspension with rabies vaccine at an MOI of 0.05 and transfer the culture to a bioreactor. Control the bioreactor temperature at 35°C, the rotation speed at 80 rpm, the oxygen content at 50%, and the pH at 7.4.
[0085] N3. Virus harvest: After 3 days of culture, add 0.5% (v / v) trypsin solution to digest the cells. Then, elute and collect the cells attached to the bioreactor wall with 10 mmol / L PBS buffer at a pH of 7.0 to obtain the virus harvest solution. Clarify with a 0.25 μm pore size filter cartridge and then concentrate 50 times using a 300 kD pore size membrane package to form a virus concentrate.
[0086] N4. Inactivation: Add β-propiolactone to the virus concentrate at a ratio of 1:4000 (V / V) and inactivate the virus at 4°C for 48 hours. After inactivation, place the concentrate at 37°C for 3 hours to obtain the inactivated rabies virus ultrafiltration concentrate.
[0087] Step 2, chromatography purification: purify the above-mentioned rabies virus inactivated ultrafiltration concentrate by eluting with an eluent, and collect the elution peak with a UV wavelength of 280 nm to obtain a purified solution;
[0088] The specific steps of the chromatography purification are as follows:
[0089] S1. Chromatography column cleaning: The composite filler was loaded into an XK26 / 70 chromatography column with a column bed height of 40 cm. Then, the AKTA was connected to the XK26 / 70 chromatography column. The chromatography column was rinsed with 5 CV of a mixture of 70 wt % 1 mol / L aqueous NaOH solution and 30 wt % isopropanol for 30 min, sterilized, and set aside.
[0090] S2, chromatography operation: the chromatography column was equilibrated with 10 CV of 0.01 mol / L PBS buffer, pH 7.8, until the pH was neutral;
[0091] S3. Loading: The inactivated rabies virus ultrafiltration concentrate was loaded into the above-mentioned chromatography column with a loading volume of 8% of the column volume. The column was eluted with an eluent at a loading flow rate of 46 cm / h and an elution flow rate of 46 cm / h. The eluate was monitored at a UV wavelength of 280 nm and the eluate corresponding to the absorption peak at a UV wavelength of 280 nm was collected to obtain the purified inactivated rabies virus vaccine.
[0092] S4. Column cleaning: After loading, rinse with 0.01 mol / L, pH 7.8 PBS buffer until the UV absorption baseline returns to zero. Replace the cleaning solution and rinse the column with 5 CV of a mixture of 70 wt% 1 mol / L NaOH aqueous solution and 30 wt% isopropanol. The flow rate of the cleaning solution is 2 cm / min.
[0093] The composite filler is a mixture of Capto Core 700 and 4FF. The chromatography column is packed in layers. The bottom packing layer is Capto Core 700, and its height accounts for 80% of the total packing height of the chromatography column; the top packing layer is composed of 4FF, and its height accounts for 20% of the total packing height of the chromatography column.
[0094] The eluent consisted of 0.5 M PBS, 0.5 M arginine, 0.5 M NaCl, and 0.05% Poloxamer 188, with a pH of 6.8.
[0095] Example 4
[0096] A method for purifying an inactivated rabies virus vaccine comprises the following steps:
[0097] Step 1: Preparation of rabies virus inactivated ultrafiltration concentrate:
[0098] N1. Cell expansion: Vero cells are revived and passaged to a cell factory for large-scale expansion culture. After the cells have expanded to a certain number and grown densely, they are digested and collected to obtain a suspension;
[0099] N2. Virus culture: Inoculate the collected cell suspension with rabies vaccine at an MOI of 0.05 and transfer the culture to a bioreactor. Control the bioreactor temperature at 35°C, the rotation speed at 80 rpm, the oxygen content at 50%, and the pH at 7.4.
[0100] N3. Virus harvest: After 3 days of culture, add 0.5% (v / v) trypsin solution to digest the cells. Then, elute and collect the cells attached to the bioreactor wall with 10 mmol / L PBS buffer at a pH of 7.0 to obtain the virus harvest solution. Clarify with a 0.25 μm pore size filter cartridge and then concentrate 50 times using a 300 kD pore size membrane package to form a virus concentrate.
[0101] N4. Inactivation: Add β-propiolactone to the virus concentrate at a ratio of 1:4000 (V / V) and inactivate the virus at 4°C for 48 hours. After inactivation, place the concentrate at 37°C for 3 hours to obtain the inactivated rabies virus ultrafiltration concentrate.
[0102] Step 2, chromatography purification: purify the above-mentioned rabies virus inactivated ultrafiltration concentrate by eluting with an eluent, and collect the elution peak with a UV wavelength of 280 nm to obtain a purified solution;
[0103] The specific steps of the chromatography purification are as follows:
[0104] S1. Chromatography column cleaning: The composite filler was loaded into an XK26 / 70 chromatography column with a column bed height of 40 cm. Then, the AKTA was connected to the XK26 / 70 chromatography column. The chromatography column was rinsed with 5 CV of a mixture of 70 wt % 1 mol / L aqueous NaOH solution and 30 wt % isopropanol for 30 min, sterilized, and set aside.
[0105] S2, chromatography operation: the chromatography column was equilibrated with 10 CV of 0.01 mol / L PBS buffer, pH 7.8, until the pH was neutral;
[0106] S3. Loading: The inactivated rabies virus ultrafiltration concentrate was loaded into the above-mentioned chromatography column with a loading volume of 8% of the column volume. The column was eluted with an eluent at a loading flow rate of 46 cm / h and an elution flow rate of 46 cm / h. The eluate was monitored at a UV wavelength of 280 nm and the eluate corresponding to the absorption peak at a UV wavelength of 280 nm was collected to obtain the purified inactivated rabies virus vaccine.
[0107] S4. Column cleaning: After loading, rinse with 0.01 mol / L, pH 7.8 PBS buffer until the UV absorption baseline returns to zero. Replace the cleaning solution and rinse the column with 5 CV of a mixture of 70 wt% 1 mol / L NaOH aqueous solution and 30 wt% isopropanol. The flow rate of the cleaning solution is 2 cm / min.
[0108] The eluent consisted of 0.5 M PBS, 0.5 M arginine, 0.5 M NaCl, and 0.05% Poloxamer 188, with a pH of 6.8.
[0109] The composite filler is a mesoporous adsorption material, and the preparation method of the mesoporous adsorption material is as follows:
[0110] (1) 10 g of Al-MCM-41 mesoporous material and 500 mL of water were mixed by ultrasonication, 100 mL of 20 mg / mL tannic acid aqueous solution was added at a stirring speed of 300 rpm, and stirring was continued for 45 min. After adding 100 mL of 1.5 mg / mL chloride aqueous solution, stirring was continued for 60 min, centrifuged, washed, and vacuum dried to obtain tannic acid-modified titanium-doped mesoporous material; the chloride was composed of titanium chloride and nickel chloride in a mass ratio of 1:1;
[0111] (2) Dissolve 4 g of chitosan in 400 mL of acetic acid solution with a mass fraction of 0.5 wt%, stir at 800 rpm for 2 h, then add 20 mL of 4 wt% sodium hydroxide aqueous solution and 1 g of glutaraldehyde, continue stirring for 2 h, and filter with a 100 mesh filter cloth to obtain chitosan hydrogel;
[0112] (3) Add 10 g of the above-mentioned tannic acid-modified titanium-doped mesoporous material and 2 g of the above-mentioned chitosan hydrogel into 300 mL of 0.3 M oxalic acid solution, stir at 50 ° C and 400 rpm for 3 h, then add into 500 mL of 0.5 M NaOH aqueous solution, stir at room temperature and 400 rpm for 1 h, wash, and filter with 500 mesh filter cloth to obtain a mesoporous adsorption material.
[0113] Example 5
[0114] A method for purifying an inactivated rabies virus vaccine comprises the following steps:
[0115] Step 1: Preparation of rabies virus inactivated ultrafiltration concentrate:
[0116] N1. Cell expansion: Vero cells are revived and passaged to a cell factory for large-scale expansion culture. After the cells have expanded to a certain number and grown densely, they are digested and collected to obtain a suspension;
[0117] N2. Virus culture: Inoculate the collected cell suspension with rabies vaccine at an MOI of 0.05 and transfer the culture to a bioreactor. Control the bioreactor temperature at 35°C, the rotation speed at 80 rpm, the oxygen content at 50%, and the pH at 7.4.
[0118] N3. Virus harvest: After 3 days of culture, add 0.5% (v / v) trypsin solution to digest the cells. Then, elute and collect the cells attached to the bioreactor wall with 10 mmol / L PBS buffer at a pH of 7.0 to obtain the virus harvest solution. Clarify with a 0.25 μm pore size filter cartridge and then concentrate 50 times using a 300 kD pore size membrane package to form a virus concentrate.
[0119] N4. Inactivation: Add β-propiolactone to the virus concentrate at a ratio of 1:4000 (V / V) and inactivate the virus at 4°C for 48 hours. After inactivation, place the concentrate at 37°C for 3 hours to obtain the inactivated rabies virus ultrafiltration concentrate.
[0120] Step 2, chromatography purification: purify the above-mentioned rabies virus inactivated ultrafiltration concentrate by eluting with an eluent, and collect the elution peak with a UV wavelength of 280 nm to obtain a purified solution;
[0121] The specific steps of the chromatography purification are as follows:
[0122] S1. Chromatography column cleaning: The composite filler was loaded into an XK26 / 70 chromatography column with a column bed height of 40 cm. Then, the AKTA was connected to the XK26 / 70 chromatography column. The chromatography column was rinsed with 5 CV of a mixture of 70 wt % 1 mol / L aqueous NaOH solution and 30 wt % isopropanol for 30 min, sterilized, and set aside.
[0123] S2, chromatography operation: the chromatography column was equilibrated with 10 CV of 0.01 mol / L PBS buffer, pH 7.8, until the pH was neutral;
[0124] S3. Loading: The inactivated rabies virus ultrafiltration concentrate was loaded into the above-mentioned chromatography column with a loading volume of 8% of the column volume. The column was eluted with an eluent at a loading flow rate of 46 cm / h and an elution flow rate of 46 cm / h. The eluate was monitored at a UV wavelength of 280 nm and the eluate corresponding to the absorption peak at a UV wavelength of 280 nm was collected to obtain the purified inactivated rabies virus vaccine.
[0125] S4. Column cleaning: After loading, rinse with 0.01 mol / L, pH 7.8 PBS buffer until the UV absorption baseline returns to zero. Replace the cleaning solution and rinse the column with 5 CV of a mixture of 70 wt% 1 mol / L NaOH aqueous solution and 30 wt% isopropanol. The flow rate of the cleaning solution is 2 cm / min.
[0126] The composite filler is a mixture of Capto Core 700 and a mesoporous adsorption material. The chromatography column is packed in layers. The bottom packing layer is Capto Core 700, and its height accounts for 80% of the total packing height of the chromatography column; the top packing layer is the mesoporous adsorption material, and its height accounts for 20% of the total packing height of the chromatography column.
[0127] The eluent consisted of 0.5 M PBS, 0.5 M arginine, 0.5 M NaCl, and 0.05% Poloxamer 188, with a pH of 6.8.
[0128] The preparation method of the mesoporous adsorption material is the same as that of Example 4.
[0129] Example 6
[0130] A method for purifying an inactivated rabies virus vaccine comprises the following steps:
[0131] Step 1: Preparation of rabies virus inactivated ultrafiltration concentrate:
[0132] N1. Cell expansion: Vero cells are revived and passaged to a cell factory for large-scale expansion culture. After the cells have expanded to a certain number and grown densely, they are digested and collected to obtain a suspension;
[0133] N2. Virus culture: Inoculate the collected cell suspension with rabies vaccine at an MOI of 0.05 and transfer the culture to a bioreactor. Control the bioreactor temperature at 35°C, the rotation speed at 80 rpm, the oxygen content at 50%, and the pH at 7.4.
[0134] N3. Virus harvest: After 3 days of culture, add 0.5% (v / v) trypsin solution to digest the cells. Then, elute and collect the cells attached to the bioreactor wall with 10 mmol / L PBS buffer at a pH of 7.0 to obtain the virus harvest solution. Clarify with a 0.25 μm pore size filter cartridge and then concentrate 50 times using a 300 kD pore size membrane package to form a virus concentrate.
[0135] N4. Inactivation: Add β-propiolactone to the virus concentrate at a ratio of 1:4000 (V / V) and inactivate the virus at 4°C for 48 hours. After inactivation, place the concentrate at 37°C for 3 hours to obtain the inactivated rabies virus ultrafiltration concentrate.
[0136] Step 2, chromatography purification: purify the above-mentioned rabies virus inactivated ultrafiltration concentrate by eluting with an eluent, and collect the elution peak with a UV wavelength of 280 nm to obtain a purified solution;
[0137] The specific steps of the chromatography purification are as follows:
[0138] S1. Chromatography column cleaning: The composite filler was loaded into an XK26 / 70 chromatography column with a column bed height of 40 cm. Then, the AKTA was connected to the XK26 / 70 chromatography column. The chromatography column was rinsed with 5 CV of a mixture of 70 wt % 1 mol / L aqueous NaOH solution and 30 wt % isopropanol for 30 min, sterilized, and set aside.
[0139] S2, chromatography operation: the chromatography column was equilibrated with 10 CV of 0.01 mol / L PBS buffer, pH 7.8, until the pH was neutral;
[0140] S3. Loading: The inactivated rabies virus ultrafiltration concentrate was loaded into the above-mentioned chromatography column with a loading volume of 8% of the column volume. The column was eluted with an eluent at a loading flow rate of 46 cm / h and an elution flow rate of 46 cm / h. The eluate was monitored at a UV wavelength of 280 nm and the eluate corresponding to the absorption peak at a UV wavelength of 280 nm was collected to obtain the purified inactivated rabies virus vaccine.
[0141] S4. Column cleaning: After loading, rinse with 0.01 mol / L, pH 7.8 PBS buffer until the UV absorption baseline returns to zero. Replace the cleaning solution and rinse the column with 5 CV of a mixture of 70 wt% 1 mol / L NaOH aqueous solution and 30 wt% isopropanol. The flow rate of the cleaning solution is 2 cm / min.
[0142] The composite filler is a mixture of 4FF and mesoporous adsorption material. The chromatography column adopts a layered filling method. The bottom filling layer is composed of 4FF, and its height accounts for 80% of the total filling height of the chromatography column; the top filling layer is a mesoporous adsorption material, and its height accounts for 20% of the total filling height of the chromatography column.
[0143] The eluent consisted of 0.5 M PBS, 0.5 M arginine, 0.5 M NaCl, and 0.05% Poloxamer 188, with a pH of 6.8.
[0144] The preparation method of the mesoporous adsorption material is the same as that of Example 4.
[0145] Example 7
[0146] The method is basically the same as Example 5, except that: the composite filler is a mixture of Capto Core 700 and a mesoporous adsorption material; the chromatography column is packed in layers; the bottom packing layer is Capto Core 700, and its height accounts for 70% of the total packing height of the chromatography column; the top packing layer is a mesoporous adsorption material, and its height accounts for 30% of the total packing height of the chromatography column.
[0147] Example 8
[0148] It is basically the same as Example 5, except that:
[0149] The composite filler is a mixture of Capto Core 700 and a mesoporous adsorption material. The chromatography column is packed in layers. The bottom packing layer is Capto Core 700, and its height accounts for 60% of the total packing height of the chromatography column; the top packing layer is the mesoporous adsorption material, and its height accounts for 40% of the total packing height of the chromatography column.
[0150] Example 9
[0151] It is basically the same as Example 5, except that:
[0152] The composite filler is a mixture of Capto Core 700 and a mesoporous adsorption material. The chromatography column is packed in layers, with the bottom packing layer being Capto Core 700, whose height accounts for 90% of the total packing height of the chromatography column; and the top packing layer being the mesoporous adsorption material, whose height accounts for 10% of the total packing height of the chromatography column.
[0153] Example 10
[0154] It is basically the same as Example 5, except that:
[0155] The composite filler is a mixture of Capto Core 700, 4FF and a mesoporous adsorption material. The chromatography column is packed in layers: the bottom packing layer is Capto Core 700, and its height accounts for 80% of the total packing height of the chromatography column; the middle packing layer is 4FF, and its height accounts for 10% of the total packing height of the chromatography column; and the top packing layer is the mesoporous adsorption material, and its height accounts for 10% of the total packing height of the chromatography column.
[0156] Example 11
[0157] The method is basically the same as Example 10, except that the sample loading volume in S3 is different and is 2% of the column volume.
[0158] Example 12
[0159] The method is basically the same as Example 10, except that the sample loading volume in S3 is different and is 5% of the column volume.
[0160] Example 13
[0161] The method is basically the same as Example 10, except that the sample loading volume in S3 is different and is 7% of the column volume.
[0162] Example 14
[0163] The method is basically the same as Example 10, except that the sample loading volume in S3 is different and is 10% of the column volume.
[0164] Example 15
[0165] The method is basically the same as Example 10, except that: the composite filler is a mixture of Capto Core 700, 4FF and a mesoporous adsorption material; the chromatography column is packed in layers; the bottom packing layer is Capto Core 700, and its height accounts for 80% of the total packing height of the chromatography column; the middle packing layer is the mesoporous adsorption material, and its height accounts for 10% of the total packing height of the chromatography column; the top packing layer is 4FF, and its height accounts for 10% of the total packing height of the chromatography column.
[0166] Example 16
[0167] The method is basically the same as Example 10, except that: the composite filler is a mixture of Capto Core 700, 4FF and a mesoporous adsorption material; the chromatography column is packed in layers, the bottom packing layer is 4FF, and its height accounts for 10% of the total packing height of the chromatography column; the middle packing layer is Capto Core 700, and its height accounts for 80% of the total packing height of the chromatography column; the top packing layer is a mesoporous adsorption material, and its height accounts for 10% of the total packing height of the chromatography column.
[0168] Example 17
[0169] The method is basically the same as Example 10, except that: the composite filler is a mixture of Capto Core 700, 4FF and a mesoporous adsorption material; the chromatography column is packed in layers, the bottom packing layer is 4FF, and its height accounts for 10% of the total packing height of the chromatography column; the middle packing layer is the mesoporous adsorption material, and its height accounts for 10% of the total packing height of the chromatography column; the top packing layer is Capto Core 700, and its height accounts for 80% of the total packing height of the chromatography column.
[0170] Example 18
[0171] The method is basically the same as Example 10, except that: the composite filler is a mixture of Capto Core 700, 4FF and a mesoporous adsorption material; the chromatography column is packed in layers; the bottom packing layer is the mesoporous adsorption material, and its height accounts for 10% of the total packing height of the chromatography column; the middle packing layer is Capto Core 700, and its height accounts for 80% of the total packing height of the chromatography column; and the top packing layer is 4FF, and its height accounts for 10% of the total packing height of the chromatography column.
[0172] Example 19
[0173] The method is basically the same as Example 10, except that: the composite filler is a mixture of Capto Core 700, 4FF and a mesoporous adsorption material; the chromatography column is packed in layers; the bottom packing layer is the mesoporous adsorption material, and its height accounts for 10% of the total packing height of the chromatography column; the middle packing layer is 4FF, and its height accounts for 10% of the total packing height of the chromatography column; and the top packing layer is Capto Core 700, and its height accounts for 80% of the total packing height of the chromatography column.
[0174] Test Case
[0175] The test indicators are tested with reference to the following standards:
[0176] According to the enzyme-linked immunosorbent assay (ELISA) specified in the Appendix to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China, the residual host protein content in human rabies vaccine is detected. The host protein removal rate (%) = (initial host protein residue in the clarified liquid - host protein content in the inactivated liquid) / initial host protein residue in the clarified liquid × 100%.
[0177] Antigen content: The antigen content in human rabies vaccine is detected by enzyme-linked immunosorbent assay (ELISA) as specified in the Appendix of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China.
[0178] Antigen content recovery rate (%) = antigen content in the inactivated solution / antigen content in the clarified solution × 100%.
[0179] Residual Vero Cell Protein: This test was conducted in accordance with Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The results of this test showed that the residual bovine serum albumin in the purified samples after one-step purification was less than 100 ng / dose, meeting the national limit. The results are shown in Table 1.
[0180] Table 1 Test results of various indicators of purified virus
[0181]
[0182] As can be seen from the results in Table 1 above, the type and ratio of composite fillers, as well as the sample loading amount, all have an impact on various viral indices. Comparing Examples 1-5, it can be seen that the composite filler using Capto Core 700, 4FF, and a mesoporous adsorbent material has a better effect than using Capto Core 700, 4FF, or a mesoporous adsorbent material alone. Furthermore, from Examples 5-10, it can be seen that the optimal ratio of Capto Core 700, 4FF, and a mesoporous adsorbent material in the composite filler is 8:1:1; the optimal final sample loading volume is 8%. From Examples 15-19, it can be seen that the order in which the composite fillers Capto Core 700, 4FF, or a mesoporous adsorbent material are layered also affects viral indices. In summary, the present invention selects the best composite filler Capto Core 700, 4FF and mesoporous adsorption material with an optimal ratio of 8:1:1, and stacking Capto Core 700, 4FF and mesoporous adsorption material in sequence from bottom to top has the best effect. The reason is that 4FF is on the top, which has slightly weaker antigen capture, impurities are preferentially adsorbed, and subsequent antigen recovery is affected; Capto Core 700 is on the top, small molecules are not initially blocked, antigens are easily retained, and Capto is not effective on the top layer; finally, the optimal sample volume is 8% for rabies virus purification, so that the residual Vero cell protein is as low as 2.37 ng / ml, the antigen content can reach 17.32 IU / ml, the host protein removal rate can reach 100%, and the antigen content recovery rate can reach 91.50%.
Claims
1. A method for purifying an inactivated rabies virus vaccine, characterized in that: The steps include: Step 1: Preparation of rabies virus inactivated ultrafiltration concentrate: Step 2: purifying the inactivated rabies virus ultrafiltration concentrate by chromatography; The specific steps of the chromatography purification are as follows: S1. Chromatography column cleaning: Load the composite filler into the chromatography column, then connect AKTA to the chromatography column, rinse the chromatography column with 4-6CV of the mixed solution, sterilize, and set aside; S2. Chromatography operation: Equilibrate the chromatography column with 8-13CV of PBS buffer until the pH is neutral; S3. Sample loading: The inactivated rabies virus ultrafiltration concentrate is loaded into the above-mentioned chromatography column, eluted with an eluent, and the eluate corresponding to the absorption peak at an ultraviolet wavelength of 280 nm is collected, which is the purified inactivated rabies virus vaccine; S4. Column cleaning: After loading, rinse with PBS buffer until the UV absorption baseline returns to zero; The composite filler is a mixture of Capto Core 700, 4FF and a mesoporous adsorption material. The chromatography column is packed in layers, with the bottom packing layer being Capto Core 700, whose height accounts for 80% of the total packing height of the chromatography column; the middle packing layer being 4FF, whose height accounts for 10% of the total packing height of the chromatography column; and the top packing layer being the mesoporous adsorption material, whose height accounts for 10% of the total packing height of the chromatography column. The preparation method of the mesoporous adsorption material is as follows: (1) 7-14 g of Al-MCM-41 mesoporous material and 400-800 mL of water were mixed by ultrasonication, 80-200 mL of 12-300 mg / mL tannic acid aqueous solution was added at a stirring speed of 200-400 rpm, and stirring was continued for 30-60 min. After adding 80-160 mL of 1-2 mg / mL chloride aqueous solution, stirring was continued for 40-90 min, centrifuged, washed, and vacuum dried to obtain tannic acid-modified titanium-doped mesoporous material; (2) Dissolve 1-5 g of chitosan in 300-600 mL of acetic acid solution with a mass fraction of 0.3-1 wt%, stir at 500-1000 rpm for 1-3 h, then add 15-300 mL of 3-5 wt% sodium hydroxide aqueous solution and 0.5-3 g of glutaraldehyde dropwise, continue stirring for 1-4 h, and filter with 80-120 mesh filter cloth to obtain chitosan hydrogel; (3) Add 8-12 g of the above-mentioned tannic acid-modified titanium-doped mesoporous material and 1-3 g of the above-mentioned chitosan hydrogel into 200-400 mL of 0.1-0.5 M oxalic acid solution, stir at 40-60 ° C and 300-600 rpm for 2-5 h, then add into 400-600 mL of 0.3-0.8 M NaOH aqueous solution, stir at 300-500 rpm for 0.5-2 h at room temperature, wash, and filter with 300-600 mesh filter cloth to obtain a mesoporous adsorption material.
2. The method for purifying the inactivated rabies virus vaccine according to claim 1, wherein The preparation of the rabies virus inactivated ultrafiltration concentrate comprises the following steps: N1. Cell expansion: Vero cells are revived and passaged to a cell factory for large-scale expansion culture. After the cells have expanded to a certain number and grown densely, they are digested and collected to obtain a suspension; N2. Virus culture: Inoculate the collected cell suspension with rabies vaccine virus at an MOI of 0.03-0.1 and transfer to a bioreactor for culture; N3. Virus harvest: After 2-5 days of culture, add 0.3-0.8% (v / v) trypsin solution to digest the cells, then elute and collect the cells attached to the bioreactor wall with 8-15 mmol / L PBS buffer, pH 7.0, to obtain the virus harvest solution; After clarification with a 0.25 μm pore size filter, the virus was concentrated 40-60 times using a 300 KD pore size membrane package to form a virus concentrate. N4. Inactivation: Add β-propiolactone to the virus concentrate at a ratio of 1:3000-5000 (V / V) and inactivate the virus at 3-5°C for 40-60 hours. After inactivation, place the virus at 35-40°C for 2-5 hours to obtain the rabies virus inactivated ultrafiltration concentrate.
3. The method for purifying the inactivated rabies virus vaccine according to claim 1, wherein The chromatography column model in S1 is any one of XK26 / 70, XK16 / 40, and XK50 / 60.
4. The method for purifying the inactivated rabies virus vaccine according to claim 1, wherein The mixed solution in S1 is composed of 60-75 wt % of a 0.5-2 mol / L NaOH aqueous solution and 20-40 wt % of isopropyl alcohol.
5. The method for purifying the inactivated rabies virus vaccine according to claim 1, wherein The sample loading volume in S3 is 2-10% of the column volume.
6. The method for purifying the inactivated rabies virus vaccine according to claim 1, wherein The loading flow rate and elution flow rate in S3 are both 40-50 cm / h.
7. The method for purifying the inactivated rabies virus vaccine according to claim 1, wherein: The eluent in S3 consists of 0.3-0.8 M PBS, 0.4-0.7 M arginine, 0.1-0.6 M NaCl, and 0.03-0.1% Poloxamer 188, with a pH of 6.5-7.
2.
8. The method for purifying the inactivated rabies virus vaccine according to claim 2, wherein: The N2 The bioreactor conditions are a temperature of 32-37°C, a rotation speed of 50-100 rpm, an oxygen content of 45-55%, and a pH of 7.0-7.6.