AU2021107276A4 - Purification process of chicken embryo allantoic fluid of tetravalent influenza virus, tetravalent influenza virus split vaccine and preparation method thereof - Google Patents
Purification process of chicken embryo allantoic fluid of tetravalent influenza virus, tetravalent influenza virus split vaccine and preparation method thereof Download PDFInfo
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Abstract
The embodiment of the present disclosure provides a purification process of a chicken embryo
allantoic fluid of a tetravalent influenza virus split vaccine, a preparation method of the
tetravalent influenza virus split vaccine, and related products The purification process includes
the following steps: 1) filtration clarification by using a 40 m filter element; 2) centrifugation
by using a disk centrifuge; 3) ultrafiltration concentration; 4) zonal centrifugation; 5)
ultrafiltration sugar removal; 6) chromatographic purification; 7) virus lysis and inactivation; 8)
ultrafiltration purification (for removing a lysing agent and an inactivating agent); and 9) sterile
filtration for obtaining a monovalent stock solution. According to the implementations of the
present disclosure, a higher antigen recovery rate, a hemagglutinin antigen with a higher purity, a
lower ovalbumin content and a lower miscellaneous protein content, and an improved yield and
effective rate of the chicken embryo influenza vaccines can be achieved.
Description
Purification Process of Chicken Embryo Allantoic Fluid of Tetravalent
Influenza Virus, Tetravalent Influenza Virus Split Vaccine and Preparation
Method Thereof
Technical Field The present disclosure relates to the technical field of influenza vaccines, and particularly relates to a purification process of a chicken embryo allantoic fluid of a tetravalent influenza virus split vaccine, a tetravalent influenza virus split vaccine and a preparation method thereof.
Background Art Tetravalent influenza virus split vaccines are basically prepared by chicken embryo proliferation culture. A virus is proliferated and cultured in the chicken embryo, and a chicken embryo allantoic fluid is obtained and purified to obtain a monovalent stock solution which is mixed to obtain a tetravalent influenza virus split vaccine. In the prior art, the purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus generally includes the steps of clarification, inactivation, ultrafiltration concentration, lysis, centrifugation purification, chromatographic purification, and filtration sterilization. A monovalent stock solution of a tetravalent influenza virus split vaccine basically complying with the requirements of Pharmacopoeiacan be obtained by using the existing purification process of a chicken embryo allantoic fluid of the tetravalent influenza virus.
Summary of the Invention
A technical problem to be solved by the present disclosure is to provide a purification process of a chicken embryo allantoic fluid of a tetravalent influenza virus split vaccine, so as to achieve effects of obtaining a higher antigen recovery rate, a hemagglutinin antigen with a higher purity, a lower ovalbumin content, and a lower miscellaneous protein content, and improving a yield and an effective rate of the chicken embryo influenza vaccines.
To solve the above technical problem, the present disclosure discloses the following technical solutions:
a purification process of a chicken embryo allantoic fluid of a tetravalent influenza virus split vaccine, characterized by including the following steps: filtration clarification: Harvesting chicken embryo allantoic fluid, adding sodium citrate solution at a final concentration of 0.125 mol/L, mixing uniformly and thoroughly, and filtering by using a 40 mfilter element; disk centrifugation: performing continuous flow centrifugation on the filtered chicken embryo allantoic fluid by using a disk centrifuge, and collecting a supernatant; performing ultrafiltration concentration, zonal centrifugation, ultrafiltration sugar removal, and chromatographic purification on the supernatant to obtain a purified liquid; virus lysis and inactivation: adding a virus lysing agent into the purified liquid for lysis, and then adding an inactivating agent for inactivation; and ultrafiltration purification: performing ultrafiltration washing filtration on the inactivated liquid to remove the inactivating agent and the lysing agent, and performing sterile filtration to obtain a monovalent stock solution.
As some possible implementations, in the step of disk centrifugation, a rotating speed of the disk centrifuge is controlled at 8,500 to 9,000 rpm, and a loading speed is controlled at 70 to 150 L/h.
As some possible implementations, the zonal centrifugation specifically includes:
pumping 100 ml of phosphate buffer solution (pH 7.4), 600 ml of ultrafiltrated and concentrated liquid, 500 ml of 30% sucrose solution, and 470 ml of 55% sucrose solution in sequence from a side hole at a flow rate of 50 ml/min at 2,000 rpm by using a sucrose density gradient centrifugation method, centrifuging the at 30,000 rpm and 4°C for 180 min after loading the sample, pumping 58% sucrose solution from the side hole; and collecting a centrifuged and purified liquid by monitoring a wavelength of ultraviolet and a concentration of sucrose during the period.
As some possible implementations, the ultrafiltration sugar removal specifically includes:
performing ultrafiltration sugar removal by using a 300KD ultrafiltration membrane package, performing 0 to 3 times of concentration, and performing washing filtration by using a phosphate buffer solution (pH 7.4) with a volume of 8 to 10 times to control a protein content at 3,000 to 4,000 [g/ml.
As some possible implementations, the chromatographic purification specifically includes: taking a 4FF gel as a medium and balancing by using a phosphate buffer solution (pH 7.4), loading a sample for 2 column volumes at a loading flow rate of 48 to 55 cm/h, eluting by using a phosphate buffer solution (pH 7.4) at a elution rate of 48 to 55 cm/h, and collecting a chromatographically purified liquid at an UV280 elution peak.
As some possible implementations, the ultrafiltration concentration specifically includes:
performing 30 to 50 times of ultrafiltration concentration on the supernatant by using a 300KD ultrafiltration membrane package, and performing washing filtration with a volume of 10 times.
As some possible implementations, the ultrafiltration purification specifically includes:
performing 6 to 10 times of ultrafiltration washing filtration on the inactivated liquid by using a 30KD ultrafiltration membrane package to remove the lysing agent and inactivating agent.
As some possible implementations, in the step of viral lysis and inactivation:
the lysing agent is Triton X-100 at a final concentration of 0.5%, and the lysis is performed in a shaking table at 2 to 8°C for 1 hour;
the inactivating agent is formaldehyde at a final concentration of 200 [g, and the inactivation is performed at room temperature for 60 to 72 hours.
Accordingly, the present disclosure further discloses a preparation method of a tetravalent influenza virus split vaccine using the above purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus split vaccine.
Accordingly, the present disclosure further discloses a tetravalent influenza virus vaccine prepared by the above method.
The present disclosure has the following beneficial effects:
according to the technical solutions of 1) filtration clarification by using a 40 m filter element; 2) centrifugation by using a disk centrifuge; 3) ultrafiltration concentration; 4) zonal centrifugation; 5) ultrafiltration sugar removal; 6) chromatographic purification; 7) virus lysis and inactivation; 8) ultrafiltration purification (for removing a lysing agent and an inactivating agent); and 9) sterile filtration for obtaining a monovalent stock solution used in the embodiments of the present disclosure, the monovalent stock solution of the tetravalent influenza virus split vaccine is prepared by a process of one-step chromatography, two-step centrifugation and three-step ultrafiltration, thereby achieving the technical effects of obtaining a higher antigen recovery rate, a hemagglutinin antigen with a higher purity, a lower ovalbumin content, and a lower miscellaneous protein content, and improving a yield and an effective rate of the chicken embryo influenza vaccines.
Detailed Description of the Invention To make the objectives, technical solutions and advantages of the present disclosure clearer, the present disclosure will be further described in detail below in conjunction with specific embodiments. The exemplary implementations and descriptions thereof of the present disclosure are merely used to explain the present disclosure, but are not intended to limit the present disclosure.
An embodiment of a purification method of a chicken embryo allantoic fluid of a tetravalent influenza virus provided in the present disclosure includes the following steps:
filtration clarification: the chicken embryo allantoic fluid is harvested, the sodium citrate solution is added at a final concentration of 0.125 mol/L, mixed uniformly and thoroughly, thenfiltered through a 40 m filter element;
disk centrifugation: continuous flow centrifugation is performed on the filtered chicken embryo allantoic fluid by using a disk centrifuge, and a supernatant is collected;
ultrafiltration concentration, zonal centrifugation, ultrafiltration sugar removal, and chromatographic purification are performed on the supernatant to obtain a purified liquid;
virus lysis and inactivation: a virus lysing agent is added into the purified liquid for lysis, and then an inactivating agent is added for inactivation; and
ultrafiltration purification: ultrafiltration washing filtration is performed on the inactivated liquid to remove the inactivating agent and the lysing agent, and sterile filtration is performed to obtain a monovalent stock solution.
As some possible implementations, in the step of disk centrifugation, a rotating speed of the disk centrifuge is controlled at 8,500 to 9,000 rpm, and a loading speed is controlled at 70 to 150 L/h.
As some possible implementations, the zonal centrifugation specifically includes:
100 ml of phosphate buffer solution (pH 7.4), 600 ml of ultrafiltrated and concentrated liquid,
500 ml of 30% sucrose solution, and 470 ml of 55% sucrose solution are pumped in sequence from a side hole at a flow rate of 50 ml/min at 2,000 rpm by using a sucrose density gradient centrifugation method, and centrifuge at 30,000 rpm and 4°C for 180 min after loading the sample, 58% sucrose solution is pumped from the side hole, and a centrifuged and purified liquid is collected by monitoring a wavelength of ultraviolet and a concentration of sucrose during the period.
As some possible implementations, the ultrafiltration sugar removal specifically includes:
the ultrafiltration sugar removal is performed by using a 300KD ultrafiltration membrane package, 0 to 3 times of concentration is performed, and washing filtration is performed by using a phosphate buffer solution (pH 7.4) with a volume of 8 to 10 times to control a protein content at 3,000 to 4,000 [g/ml.
As some possible implementations, the chromatographic purification specifically includes:
taking a 4FF gel as a medium and balancing by using a phosphate buffer solution (pH 7.4), a sample is loaded for 2 column volumes at a loading flow rate of 48 to 55 cm/h, elution is performed by using a phosphate buffer solution (pH 7.4) at a elution rate of 48 to 55 cm/h, and a chromatographically purified liquid is collected at an UV280 elution peak.
As some possible implementations, the ultrafiltration concentration specifically includes:
30 to 50 times of ultrafiltration concentration is performed on the supernatant by using a 300KD ultrafiltration membrane package, and washing filtration is performed with a volume of 10 times.
As some possible implementations, the ultrafiltration purification specifically includes:
6 to 10 times of ultrafiltration washing filtration is performed on the inactivated liquid by using a 30KD ultrafiltration membrane package to remove the lysing agent and inactivating agent.
As some possible implementations, in the step of viral lysis and inactivation:
the lysing agent is Triton X-100 at a final concentration of 0.5%, and the lysis is performed in a shaking table at 2 to 8°C for 1 hour;
the inactivating agent is formaldehyde at a final concentration of 200 [g, and the inactivation is performed at room temperature for 60 to 72 hours.
In the preparation method of a tetravalent influenza virus split vaccine provided in the embodiments of the present disclosure, the purification process of chicken embryo allantoic fluid can adopt the purification process of the tetravalent influenza virus split vaccine chicken embryo allantoic fluid as described in the previous embodiments, and the remaining processes can be substantially the same as the prior art. A tetravalent influenza virus split vaccine provided in the embodiments of the present disclosure can be prepared by the preparation method described in the previous embodiments.
To further describe the technical means and effects adopted in the embodiments for achieving the expected objectives of the present disclosure, the specific steps of the purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus split vaccine of the embodiments will be described in detail below through example data.
Embodiment
Filtration clarification: the chicken embryo allantoic fluid was harvested, the sodium citrate solution was added at a final concentration of 0.125 mol/L, and mixed uniformly and thoroughly, and then filtered through a 40 mfilter element.
Centrifugation by using a disk centrifuge: continuous flow centrifugation was performed on the filtered chicken embryo allantoic fluid by using a disk centrifuge at a rotating speed of 8,500 to 9,000 rpm and a loading speed of 70 to 150 L/h, precipitates were removed according to an actual situation, and a supernatant was collected.
Ultrafiltration concentration: 30 to 50 times of ultrafiltration concentration was performed on the supernatant by using a 300KD ultrafiltration membrane package, and then washing filtration was performed with a volume of 10 times.
Zonal centrifugation: 100 ml of phosphate buffer solution (pH 7.4), 600 ml of ultrafiltrated and concentrated liquid, 500 ml of 30% sucrose solution, and 470 ml of 55% sucrose solution were pumped in sequence from a side hole at a flow rate of 50 ml/min at 2,000 rpm by using a sucrose density gradient centrifugation method, centrifuged at 30,000 rpm and 4°C for 180 min after loading the sample, 58% sucrose solution was pumped from the side hole, and a centrifuged and purified liquid was collected by monitoring a wavelength of ultraviolet and a concentration of sucrose during the period.
Ultrafiltration sugar removal: the ultrafiltration sugar removal was performed by using a 300KD ultrafiltration membrane package, 0 to 3 times of concentration was performed, washing filtration was performed by using a phosphate buffer solution (pH 7.4) with a volume of 8 to 10 times, the liquid subjected to ultrafiltration sugar removal was sampled, and protein content detection was performed on the sample to control a protein content at 3,000 to 4,000 [g/ml; chromatographic purification: taking a 4FF gel as a medium and balancing by using a phosphate buffer solution (pH 7.4), a sample is loaded for 2 column volumes at a loading flow rate of 48 to 55 cm/h, the elution was performed by using a phosphate buffer solution (pH 7.4) at an elution rate of 48 to 55 cm/h, and a chromatographically purified liquid was collected at an UV280 elution peak.
Virus lysis and inactivation: a lysing agent Triton X-100 at afinal concentration of 0.5% was added into the purified viral liquid for virus lysis in a shaking table at 2 to 8°C for 1 hour; formaldehyde at a final concentration of 200 g was added for inactivation at room temperature for 60 to 72 h, the liquid was sampled for an inactivation test, and at the same time, an electron microscope sample was prepared for electron microscope detection.
Ultrafiltration purification (for removing the lysing agent and the inactivating agent): 6 to 10 times of ultrafiltration washing filtration was performed on the inactivated liquid by using a 30KD ultrafiltration membrane package to remove the lysing agent and the inactivating agent.
Sterile filtration: ultrafiltrated and purified liquid was filtered by using a 0.2 m sterilization filter to obtain a monovalent stock solution.
Table 1 Hemagglutination titer/ recovery rate of hemagglutinin
Process step Lot 1 Lot 2 Lot 3
Clarification 95.60% 66.89% 68.89%
Ultrafiltration 73.00% 72.98% 72.98% concentration
Centrifugalsugar 222.22% 222.22% 222.22% removal
Chromatographic 40.00% 62.50% 43.33% purification
Total recovery rate 62.03% 67.80% 48.41%
Table 2 Removal rate of miscellaneous protein
Process step Lot 1 Lot 2 Lot 3
Clarification N/A 36.09% 35.14%
Ultrafiltration 51.70% 41.10% 58.48% concentration
Centrifugalsugar 98.10% 98.80% 97.26% removal
Chromatographic 21.10% 45.10% 56.00% purification
Table 3 Removal rate of ovalbumin
Process step Lot 1 Lot 2 Lot 3
Ultrafiltration N/A 99.90% N/A concentration
Centrifugalsugar 99.90% 95.50% 100.00% removal
Chromatographic 93.60% 96.60% 98.91% purification
Total recovery rate 93.51% 92.16% 98.91%
The monovalent stock solution of the tetravalent influenza virus split vaccine prepared by the process of one-step chromatography, two-step centrifugation and three-step ultrafiltration of the present disclosure had a higher antigen recovery rate, a hemagglutinin antigen with a higher purity, a lower ovalbumin content, and a lower miscellaneous protein content, and a yield and an effective rate of the chicken embryo influenza vaccines were improved.
The above content is a further detailed description of the present disclosure in conjunction with the specific preferred implementations, and it should not be considered that the specific implementations of the present disclosure are limited to these descriptions. Those of ordinary skill in the art can further make several simple deductions or substitutions without departing from the concept of the present disclosure, and these deductions or substitutions shall be within the scope of protection of the present disclosure.
Claims (10)
1. A purification process of a chicken embryo allantoic fluid of a tetravalent influenza virus, characterized by comprising the following steps:
filtration clarification: Harvesting the chicken embryo allantoic fluid, adding a sodium citrate solution at a final concentration of 0.125 mol/L, mixing uniformly and thoroughly, and filtering by using a 40 mfilter element;
disk centrifugation: performing continuous flow centrifugation on the filtered chicken embryo allantoic fluid by using a disk centrifuge, and collecting a supernatant;
performing ultrafiltration concentration, zonal centrifugation, ultrafiltration sugar removal, and chromatographic purification on the supernatant to obtain a purified liquid;
virus lysis and inactivation: adding a virus lysing agent into the purified liquid for lysis, and then adding an inactivating agent for inactivation; and
ultrafiltration purification: performing ultrafiltration washing filtration on the inactivated liquid to remove the inactivating agent and the lysing agent, and performing sterile filtration to obtain a monovalent stock solution.
2. The purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus according to claim 1, characterized in that in the step of disk centrifugation, a rotating speed of the disk centrifuge is controlled at 8,500 to 9,000 rpm, and a loading speed is controlled at 70 to 150 L/h.
3. The purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus split vaccine according to claim 2, characterized in that the zonal centrifugation specifically comprises:
pumping 100 ml of phosphate buffer solution (pH 7.4), 600 ml of ultrafiltrated and concentrated liquid, 500 ml of 30% sucrose solution, and 470 ml of 55% sucrose solution in sequence from a side hole at a flow rate of 50 ml/min at 2,000 rpm by using a sucrose density gradient centrifugation method, centrifuging at 30,000 rpm and 4°C for 180 min after loading the sample, pumping 58% sucrose solution from the side hole, and collecting a centrifuged and purified liquid by monitoring a wavelength of ultraviolet and a concentration of sucrose during the period.
4. The purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus according to claim 3, characterized in that the ultrafiltration sugar removal specifically comprises:
performing ultrafiltration sugar removal by using a 300KD ultrafiltration membrane package, performing 0 to 3 times of concentration, and performing washing filtration by using a phosphate buffer solution (pH 7.4) with a volume of 8 to 10 times to control a protein content at 3,000 to 4,000 [g/ml.
5. The purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus according to claim 3, characterized in that the chromatographic purification specifically comprises:
taking a 4FF gel as a medium and balancing by using a phosphate buffer solution (pH 7.4), loading a sample for 2 column volumes at a loading flow rate of 48 to 55 cm/h, eluting by using a phosphate buffer solution (pH 7.4) at an elution rate of 48 to 55 cm/h, and collecting a chromatographically purified liquid at an UV280 elution peak.
6. The purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus according to claim 3, characterized in that the ultrafiltration concentration specifically comprises:
performing 30 to 50 times of ultrafiltration concentration on the supernatant by using a 300KD ultrafiltration membrane package, and then performing washing filtration with a volume of 10 times.
7. The purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus according to claim 3, characterized in that the ultrafiltration purification specifically comprises:
performing 6 to 10 times of ultrafiltration washing filtration on the inactivated liquid by using a KD ultrafiltration membrane package to remove the lysing agent and inactivating agent.
8. The purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus according to claim 3, characterized in that in the step of virus lysis and inactivation:
the lysing agent is Triton X-100 at a final concentration of 0.5%, and the lysis is performed in a shaking table at 2 to 8°C for 1 hour; the inactivating agent is formaldehyde at a final concentration of 200 g, and the inactivation is ) performed at room temperature for 60 to 72 hours.
9. A preparation method of a tetravalent influenza virus split vaccine, characterized by using the purification process of the chicken embryo allantoic fluid of the tetravalent influenza virus according to any one of claims I to 8.
10. A tetravalent influenza virus split vaccine, characterized by being prepared by the method according to claim 9.
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| CN115992101B (en) * | 2023-03-22 | 2023-07-28 | 深圳市卫光生物制品股份有限公司 | Preparation method of influenza virus split vaccine stock solution |
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| CN101633911A (en) * | 2008-07-22 | 2010-01-27 | 大连雅立峰生物制药有限公司 | Purification technology applicable to mass production of human-used avian influenza vaccine |
| CN102133399B (en) * | 2011-03-09 | 2013-01-16 | 浙江天元生物药业有限公司 | Novel process for preparing influenza virus split vaccine |
| WO2015017673A1 (en) * | 2013-08-01 | 2015-02-05 | Medimmune, Llc | Methods for producing influenza vaccine compositions |
| CN103520716A (en) * | 2013-09-06 | 2014-01-22 | 成都康华生物制品有限公司 | Preparation method for avian influenza virus split vaccine |
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| CN108159411A (en) * | 2018-01-11 | 2018-06-15 | 江苏中慧元通生物科技有限公司 | A kind of influenza virus subunit vaccine purification process and its application |
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| CN111068048A (en) * | 2019-12-19 | 2020-04-28 | 江苏金迪克生物技术有限公司 | Preparation method of tetravalent influenza virus split vaccine |
| CN111420044B (en) * | 2020-05-11 | 2021-04-09 | 中逸安科生物技术股份有限公司 | Tetravalent influenza virus subunit vaccine and preparation method thereof |
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