CN113583801A

CN113583801A - Virus density gradient centrifugation purification device

Info

Publication number: CN113583801A
Application number: CN202010368232.8A
Authority: CN
Inventors: 安祺; 刘珍珍; 田大勇
Original assignee: Beijing Saierfusen Biotechnology Co ltd; Shanghai Qingsai Biotechnology Co ltd
Current assignee: Beijing Saierfusen Biotechnology Co ltd; Shanghai Qingsai Biotechnology Co ltd
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2021-11-02

Abstract

The invention relates to a virus density gradient centrifugal purification device. Specifically, the invention provides a virus density gradient centrifugation and purification device, which comprises a loop pipeline and a continuous flow density gradient centrifuge, wherein the continuous flow density gradient centrifuge comprises a continuous flow density gradient centrifugal rotor. The virus density gradient centrifugal purification device can be used for simply, conveniently and timely removing air bubbles in the continuous flow density gradient centrifugal rotor in the sucrose density gradient purification process, and particularly can be used for removing the air bubbles in the sucrose density gradient, so that the vibration of the rotor and the distribution of the sucrose density gradient in the rotor caused by the air bubbles are avoided, and a high-quality virus vaccine can be prepared.

Description

Virus density gradient centrifugation purification device

Technical Field

The invention relates to the field of virus purification, in particular to a virus density gradient centrifugal purification device.

Background

The virus is a pathogenic agent and seriously harms the health of human bodies. At present, vaccination is an effective means for disease vaccine, so the quality of the vaccine is very important, and the quality of the vaccine mainly depends on the purification effect of the product.

The purification method is one of the key factors determining the quality of the vaccine. Currently, there are two main methods for rabies vaccine (RABV) purification: 1. chromatography techniques; 2. sucrose density gradient centrifugation technique. The former is suitable for the production of a product with passage cells as a production matrix and without cytopathy, but has larger shearing force and is not beneficial to the maintenance of antigen integrity; the latter has wide application range and small shearing force. The main immunogen of RABV is G protein, the immunogenicity of which has structural dependence, namely only the G protein forms a stable tripolymer structure, the organism can be efficiently induced to generate neutralizing antibody; the G protein is present as a trimer on the surface of RABV virions. However, in the process of purifying the virus vaccine by using the continuous flow density gradient centrifuge, the continuous flow density gradient centrifuge can generate bubbles, and the bubbles can cause the vibration of a rotor and influence the distribution of sucrose density gradient in the rotor, thereby influencing the final purification effect and causing the quality reduction or failure of the produced vaccine.

Therefore, there is a need in the art to develop a purification device that improves the quality of viral vaccines.

Disclosure of Invention

The invention aims to provide a virus density gradient centrifugal purification device which can simply, conveniently and timely remove air bubbles in a continuous flow density gradient centrifugal rotor and is used for improving the quality of virus vaccines.

The invention also aims to provide a rabies virus density gradient centrifugation purification method, which is used for purifying to obtain the virus vaccine with high quality.

In a first aspect of the invention, there is provided a virus density gradient centrifugation purification apparatus comprising an annular conduit and a continuous flow density gradient centrifuge, said continuous flow density gradient centrifuge comprising a continuous flow density gradient centrifuge rotor 1;

a first pipe orifice 2, a third pipe orifice 3, a second pipe orifice 4 and a fourth pipe orifice 5 are sequentially arranged on the annular pipe, a first valve 6 is arranged on the pipe between the first pipe orifice and the third pipe orifice, a second valve 7 is arranged on the pipe between the third pipe orifice and the second pipe orifice, a third valve 8 is arranged on the pipe between the second pipe orifice and the fourth pipe orifice, and a fourth valve 9 is arranged on the pipe between the fourth pipe orifice and the first pipe orifice;

the first pipe orifice is connected with the upper end of the continuous flow density gradient centrifugal rotor through a first pipe 10, the second pipe orifice is connected with the lower end of the continuous flow density gradient centrifugal rotor through a second pipe 11, the third pipe orifice is connected with a first bottle body 13 through a third pipe 12, and the fourth pipe orifice is connected with a second bottle body 15 through a fourth pipe 14;

a fifth valve 16 is arranged on the first pipeline, and a sixth valve 17 is arranged on the second pipeline;

the third pipeline is provided with a liquid conveying device 18.

In another preferred embodiment, the continuous flow density gradient centrifuge rotor is configured to spin and centrifuge around a central axis of the continuous flow density gradient centrifuge rotor, and the continuous flow density gradient centrifuge rotor is provided with an inner cavity for containing material, the inner cavity has a volume of V0, a material inlet is provided at the bottom (lower end) and a material outlet is provided at the top (upper end), the material inlet is communicated with the second pipe 11, and the material outlet is communicated with the first pipe 10.

In another preferred embodiment, the first pipe orifice is communicated with the upper end of the continuous flow density gradient centrifugal rotor through a first pipe 10.

In another preferred embodiment, the second pipe orifice is communicated with the lower end of the continuous flow density gradient centrifugal rotor through a second pipe 11.

In another preferred embodiment, the third pipe 12 and/or the fourth pipe 14 are provided with a filter.

In another preferred example, the first nozzle, the third nozzle, the second nozzle and the fourth nozzle sequentially surround the annular pipeline.

In another preferred embodiment, the first pipe orifice, the third pipe orifice, the second pipe orifice and the fourth pipe orifice are sequentially arranged on the annular pipe in a clockwise manner.

In another preferred embodiment, the first orifice is located between the third orifice and the fourth orifice.

In another preferred embodiment, the second orifice is located between the third orifice and the fourth orifice.

In another preferred embodiment, the third nozzle is located between the first nozzle and the second nozzle.

In another preferred embodiment, the fourth orifice is located between the first orifice and the second orifice.

In another preferred example, the pipelines between each two adjacent nozzles in the first nozzle 2, the third nozzle 3, the second nozzle 4 and the fourth nozzle 5 are the same.

In another preferred example, the pipelines are the same, including that the lengths of the pipelines and the pipe diameters of the pipelines are the same.

In another preferred example, the annular pipeline is a diamond pipeline.

In another preferred example, the first nozzle 2, the third nozzle 3, the second nozzle 4 and the fourth nozzle 5 are respectively positioned at four diamond corners of the diamond-shaped pipeline.

In another preferred example, the valve is a liquid valve.

In another preferred embodiment, the third pipeline is provided with a seventh valve.

In another preferred embodiment, the fourth pipeline is provided with an eighth valve.

In another preferred embodiment, the first bottle body is a sample bottle.

In another preferred example, the second bottle body is a receiving bottle.

In another preferred example, the liquid delivery device is a bidirectional delivery device.

In another preferred example, the liquid delivery device is a peristaltic pump.

In another preferred example, the peristaltic pump is a bidirectional delivery peristaltic pump.

In another preferred embodiment, the connection is solid or detachable.

In another preferred embodiment, the connection is a solid connection or a detachable connection.

In another preferred example, the first pipeline is provided with a detector.

In another preferred embodiment, the detector is selected from the group consisting of: ultraviolet detector, cane sugar detector.

In another preferred embodiment, the density gradient centrifugation is sucrose density gradient centrifugation.

In a second aspect of the invention, there is provided a use of a virus density gradient centrifugation purification apparatus as described in the first aspect of the invention for purifying rabies virus-containing material by density gradient centrifugation.

In another preferred embodiment, the rabies virus-containing material is a supernatant obtained by centrifugal purification of a virus culture solution.

In a third aspect of the invention, there is provided a rabies virus density gradient centrifugation purification method, comprising the steps of:

(1) providing a continuous flow density gradient centrifuge rotor configured for rotational centrifugation about a central axis of the continuous flow density gradient centrifuge rotor and having an interior chamber for containing material, the interior chamber having a volume of V0, a material inlet at the bottom and a material outlet at the top;

(2) injecting a buffer solution into the interior cavity of the continuous flow density gradient centrifuge rotor through the feed inlet, followed by a volume of 55-65% (wt%) sucrose solution V1;

(3) centrifuging the continuous flow density gradient centrifuge rotor at T1 to form a sucrose gradient solution within the lumen of the continuous flow density gradient centrifuge rotor; wherein, T1 is 3000-7000 rpm;

(4) injecting rabies virus-containing material into the inner cavity from the material inlet at a flow rate of F1 under the centrifugal condition of T2, wherein F1 is 90-240 ml/min; wherein T2 is 30000-40000 rpm;

(5) after the injection operation of the material containing the rabies viruses is finished, under the centrifugal condition of T2, the buffer solution is injected into the inner cavity from the material inlet at the flow rate of F2, so that the rabies viruses are enriched in the sucrose gradient solution, and a gradient centrifugal layer rich in the rabies viruses is formed;

wherein F2 is 90-240 ml/min;

(6) slowing down the centrifugation speed of the continuous flow density gradient centrifugation rotor so that the rotation speed is 0 rpm;

(7) collecting the gradient centrifugation layer enriched in rabies virus from the bottom material inlet of the continuous flow density gradient centrifugation rotor, thereby obtaining purified rabies virus.

In another preferred embodiment, the method further comprises providing a rabies virus-containing material.

In another preferred example, F2/F1 is 0.9-1.1.

In another preferred embodiment, V1/V0 is 0.4-0.6.

In another preferred embodiment, the continuous flow density gradient centrifuge rotor is cylindrical.

In another preferred embodiment, the material inlet is arranged in the central area of the bottom.

In another preferred embodiment, the material outlet is arranged in the central area of the top.

In another preferred embodiment, the V0 of the centrifugal rotor is 2-5L, preferably 3-3.4L.

In another preferred embodiment, the buffer solution is PBS buffer.

In another preferred embodiment, the pH of the PBS buffer is 7.2-7.6.

In another preferred embodiment, the concentration of the PBS buffer is 0.005-0.02 mM.

In another preferred example, in the step (2), the volume of the buffer solution to be injected is 0.8 to 1.2 times of the volume of V0.

In another preferred example, in step (2), the buffer solution volume fills the inner cavity of the continuous flow density gradient centrifuge rotor.

In another preferred example, in the step (2), the speed of injecting the sucrose solution into the inner cavity of the continuous flow density gradient centrifugal rotor is 80-120 ml/min.

In another preferred embodiment, in step (3), the rotation speed T1 of the centrifugation is 4000-6000rpm, preferably 4500-5500 rpm.

In another preferred embodiment, in the step (4), the volume Vx of the rabies virus-containing material is 0.25-62.5 times of that of V0.

In another preferred example, the rotation speed T1 is less than T2.

In another preferred example, in the step (4), the buffer solution enters from the material inlet of the continuous flow density gradient centrifugal rotor when the rotating speed is from T1 to T2.

In another preferred example, in the step (4), the time from T1 to T2 is 25-35 min.

In another preferred example, in the step (4), the rotation speed is increased at a constant speed from T1 to T2.

In another preferred embodiment, T2 is 33000-37000rpm, more preferably 34000-36000 rpm.

In another preferred embodiment, F1 is 200ml/min for 100-.

In another preferred embodiment, in the step (5), after the loading is finished, the centrifugation is continued for 30-90min, preferably 60min, at T2.

In another preferred example, in the step (5), the time for continuing the centrifugation at T2 is calculated from the time when the buffer solution is injected into the inner cavity from the material inlet at the flow rate F2.

In another preferred example, in the step (5), the buffer solution is continuously injected into the inner cavity from the material inlet.

In another preferred embodiment, the operation time of step (5) is 40-80min, preferably 50-70min, more preferably 55-65 min.

In another preferred embodiment, F2 is 200ml/min for 100-.

In another preferred example, in step (6), the speed is decreased to 0 revolution at a speed of 600-.

In another preferred example, in the step (7), the method further comprises the steps of: and (3) detecting the ultraviolet absorbance of the collected centrifuged material, thereby determining the gradient centrifugal layer (namely virus gradient liquid) rich in rabies virus.

In another preferred embodiment, the rabies virus-containing material is prepared by a method comprising the following steps:

(W1) providing primary chicken embryo fibroblasts;

(W2) inoculating rabies virus in said rabies virus-containing material and performing virus culture, thereby obtaining a rabies virus-containing culture;

(W3) isolating a rabies virus-containing supernatant from said culture;

(W4) pretreating the supernatant, thereby obtaining a pretreated supernatant;

(W5) concentrating the pretreated supernatant, thereby obtaining a virus concentrate;

(W6) inactivating said viral supernatant or concentrate to obtain said rabies virus-containing material.

In another preferred embodiment, the pretreatment is clarification and centrifugation.

In another preferred example, in the step (W6), the inactivation treatment uses beta-propiolactone.

In another preferred example, in the step (W5), the concentration treatment is performed by a factor of 1 to 30 times, preferably 3 to 20 times, more preferably 5 to 12 times, and still more preferably 10 times.

In another preferred example, in step (W3), the separating the harvest supernatant comprises one or more separating the harvest supernatant.

In another preferred embodiment, the concentration of sucrose in the gradient centrifugal layer rich in rabies virus is 2% to 5% to 50% by weight, preferably 30% to 45% by weight.

In another preferred embodiment, the method is performed using a virus density gradient centrifugation purification apparatus according to the first aspect of the invention.

In another preferred embodiment, the method comprises the steps of:

1 the rabies virus-containing material;

2. preparing a sucrose density gradient;

2.1, when the continuous flow density gradient centrifugal rotor 1 is at 0rpm, adding a buffer solution into a first bottle body 13, closing a first valve 6 and a third valve 8, opening a conveying device 18, and allowing the buffer solution to flow out to a second bottle body 15 through a third pipeline 12, a pipeline between a third pipe orifice 3 and a second pipe orifice 4, a second pipeline 11, the continuous flow density gradient centrifugal rotor 1, a first pipeline 10, a pipeline between the first pipe orifice 2 and a fourth pipe orifice 5, and a fourth pipeline 14 in sequence to allow the buffer solution to fill the continuous flow density gradient centrifugal rotor 1 and the whole pipeline system;

2.2, opening the first valve 6 and the third valve 8, closing the fifth valve 16 and the sixth valve 17, opening the conveying device 18, and allowing the buffer solution in the first bottle body 13 to flow out of the fourth pipe 14 to the second bottle body 15 through the third pipe 12, the pipe between the third pipe orifice 3 and the second pipe orifice 4 and the fourth pipe orifice 5, the pipe between the third pipe orifice 3 and the first pipe orifice 2 and the fourth pipe orifice 5;

2.3, opening a fifth valve 16 and a sixth valve 17, closing a first valve 6 and a third valve 8, starting a conveying device 18, and allowing the buffer solution to flow out to a second bottle 15 through a third pipeline 12, a pipeline between a third pipe orifice 3 and a second pipe orifice 4, a second pipeline 11, a continuous flow density gradient centrifugal rotor 1, a first pipeline 10, a pipeline between the first pipe orifice 2 and a fourth pipe orifice 5, and a fourth pipeline 14 in sequence;

2.4, opening the first valve 6 and the third valve 8, closing the second valve 7 and the fourth valve 9, starting the conveying device 18 (peristaltic pump), and allowing the buffer solution to flow out of the second bottle body 15 through the third pipeline 12, the pipeline between the third pipe orifice 3 and the first pipe orifice 2, the first pipeline 10, the continuous flow density gradient centrifugal rotor 1, the second pipeline 11, the pipeline between the second pipe orifice 4 and the fourth pipe orifice 5, and the fourth pipeline 14 in sequence;

2.5, opening the second valve 7 and the fourth valve 9, replacing the slow solution in the first bottle 13 with 55-65% (wt%) of sucrose solution, closing the fifth valve 16 and the sixth valve 17, starting the conveying device 18, allowing the sucrose solution in the first bottle 13 to sequentially pass through the third pipeline 12, and then respectively flowing out of the fourth pipeline 14 to the second bottle 15 through the third pipeline 3, the pipeline between the second pipeline 4 and the fourth pipeline 5, and the pipeline between the third pipeline 3 and the pipeline between the first pipeline 2 and the fourth pipeline 5;

2.6, opening a fifth valve 16 and a sixth valve 17, closing the first valve 6 and the third valve 8, opening a conveying device 18, and allowing a sucrose solution to enter the continuous flow density gradient centrifugal rotor 1 through a third pipeline 12, a pipeline between the third pipe orifice 3 and the second pipe orifice 4 and the second pipeline 11 in sequence, so that the sucrose solution enters the continuous flow density gradient centrifugal rotor 1 and is contacted with a buffer solution in the rotor 1, wherein the volume of the sucrose solution entering the continuous flow density gradient centrifugal rotor is V1;

2.7, opening the first valve 6 and the third valve 8, closing the sixth valve 17, and performing rotation centrifugation on the continuous flow density gradient centrifugal rotor 1 at the speed of T1 so that the buffer solution and the sucrose solution in the continuous flow density gradient centrifugal rotor 1 form a longitudinal density gradient, wherein during the centrifugal rotation of the continuous flow density gradient centrifugal rotor 1, gas dissolved in the buffer solution and the sucrose solution is generated during the rotation of the continuous flow density gradient centrifugal rotor 1, and generated bubbles appear at the upper end and the lower end of the continuous flow density gradient centrifugal rotor 1;

2.8, keeping the continuous flow density gradient centrifugal rotor 1 rotating for centrifugation at T1, replacing 55-65% (wt%) of sucrose solution in the first bottle 13 with buffer solution, closing the fifth valve 16 and the sixth valve 17, opening the conveying device 18, allowing the buffer solution in the first bottle 13 to sequentially pass through the third pipeline 12, and then to respectively flow out of the fourth pipeline 14 to the second bottle 15 through the third pipeline 3, the pipeline between the second pipeline 4 and the fourth pipeline 5, and the pipeline between the third pipeline 3 and the pipeline between the first pipeline 2 and the fourth pipeline 5;

2.9 removing the air bubbles at the lower end and the upper end of the continuous flow density gradient centrifugal rotor 1 in sequence;

2.9.1 removing bubbles from the lower end of the continuous flow density gradient centrifuge rotor 1, the specific steps are as follows:

keeping the continuous flow density gradient centrifugal rotor 1 rotating and centrifuging at T1, opening the fifth valve 16 and the sixth valve 17, closing the second valve 7 and the fourth valve 9, opening the conveying device 18, and allowing the buffer solution to flow out to the second bottle 15 through the third pipeline 12, the pipeline between the third pipe orifice 3 and the first pipe orifice 2, the first pipeline 10, the continuous flow density gradient centrifugal rotor 1, the second pipeline 11, the pipeline between the second pipe orifice 4 and the fourth pipe orifice 5, and the fourth pipeline 14 in sequence, so as to push out and remove air bubbles at the lower end of the density gradient centrifugal rotor 1;

2.9.2 removal of air bubbles from the upper end of the continuous flow density gradient centrifuge rotor 1, the specific steps are as follows:

keeping the continuous flow density gradient centrifugal rotor 1 rotating at T1, opening the second valve 7 and the fourth valve 9, closing the first valve 6 and the third valve 8, opening the conveying device 18, and allowing the buffer solution to flow out to the second bottle 15 through the third pipeline 12, the pipeline between the third nozzle 3 and the second nozzle 4, the second pipeline 11, the continuous flow density gradient centrifugal rotor 1, the first pipeline 10, the pipeline between the first nozzle 2 and the fourth nozzle 5, and the fourth pipeline 14 in sequence, thereby pushing out and removing air bubbles at the upper end of the density gradient centrifugal rotor 1;

3. sample introduction:

3.1, increasing the centrifugal speed of the continuous flow density gradient centrifugal rotor 1 from T1 to T2, keeping the continuous flow density gradient centrifugal rotor 1 rotating for centrifugation at T2, replacing the buffer solution in the first bottle 13 with the material containing rabies virus, opening the first valve 6 and the third valve 8, closing the fifth valve 16 and the sixth valve 17, starting the conveying device 18, and allowing the material containing rabies virus in the first bottle 13 to sequentially pass through the third pipeline 12, then respectively pass through the third pipeline 3, the pipeline between the second pipeline 4 and the fourth pipeline 5, the pipeline between the third pipeline 3 and the first pipeline 2 and the fourth pipeline 5, and then flow out of the fourth pipeline 14 to the second bottle 15;

3.2 keeping the continuous flow density gradient centrifugal rotor 1 rotating for centrifugation at T2, opening a fifth valve 16 and a sixth valve 17, closing a first valve 6 and a third valve 8, opening a conveying device 18, enabling the rabies virus-containing material to sequentially pass through a third pipeline 12, a pipeline between a third pipe orifice 3 and a second pipe orifice 4 and a second pipeline 11, and enter the continuous flow density gradient centrifugal rotor 1 from the lower end of the continuous flow density gradient centrifugal rotor 1, wherein the sampling speed of the rabies virus-containing material is F1;

3.3 keeping the continuous flow density gradient centrifugal rotor 1 rotating for centrifugation at T2, opening the first valve 6 and the third valve 8, closing the fifth valve 16 and the sixth valve 17, replacing the rabies virus-containing material in the first bottle 13 with a buffer solution, starting the conveying device 18, allowing the buffer solution in the first bottle 13 to sequentially pass through the third pipeline 12, and then respectively pass through the third pipeline 3, the pipeline between the second pipeline 4 and the fourth pipeline 5, the pipeline between the third pipeline 3 and the first pipeline 2 and the fourth pipeline 5, and then flow out of the fourth pipeline 14 to the second bottle 15;

3.4 opening a fifth valve 16 and a sixth valve 17, closing a first valve 6 and a third valve 8, keeping the continuous flow density gradient centrifugal rotor 1 rotating for centrifugation at T2, simultaneously, during the centrifugation process of the continuous flow density gradient centrifugal rotor 1, opening a conveying device 18, allowing a buffer solution to enter the continuous flow density gradient centrifugal rotor 1 from the lower end of the continuous flow density gradient centrifugal rotor 1 through a third pipeline 12, a pipeline between a third pipe orifice 3 and a second pipe orifice 4 and a second pipeline 11 in sequence, wherein the inlet speed of the buffer solution is F2, the continuous flow density gradient centrifugal rotor 1 rotating for centrifugation at T2, and the rabies virus-containing material is distributed in different longitudinal sucrose gradient layers in the continuous flow density gradient centrifugal rotor 1 due to different molecular weights;

4. collecting a sample:

and closing the sixth valve 17, slowing down the continuous flow density gradient centrifugal rotor 1 to 0 revolution, forming a transverse density gradient by the longitudinal density gradient in the continuous flow density gradient centrifugal rotor 1, and collecting the gradient centrifugal layer rich in the rabies virus so as to obtain the purified rabies virus.

In another preferred example, in the step 4, the collecting method includes the steps of: placing the empty first bottle body 13, opening the sixth valve 17, starting the conveying device 18 for reverse conveying, wherein air can enter the continuous flow density gradient centrifugal rotor 1 through the fourth pipeline 14, the pipeline between the fourth pipe orifice 5 and the first pipe orifice 2 and the first pipeline 10, the gradient sample in the continuous flow density gradient centrifugal rotor 1 can sequentially flow into the first bottle body 13 through the second pipeline 11, the pipeline between the second pipe orifice 4 and the third pipe orifice 3 and the third pipeline 12, and the purified rabies virus is obtained by continuously replacing the new first bottle body 13.

In a fourth aspect of the invention, there is provided a purified rabies virus produced by the method of the third aspect of the invention.

In another preferred embodiment, the rabies virus is selected from the group consisting of AVO1, CVS, ERA, Kelev, HEP-FLURY, Nishigahara RCEH, Ontario fox, Ontario skunk, Pasteur/PV, Pittman Moore (PM), Street Alabama Dufferin (SAD) B19, SAD-Bern, ERA, SAG2, Vnukovo-32, Eth2003, strain RC-HL, Nishigahara, SHBRV-18, SRV9, Ni-CE, Flury-LEP, and Kissling rabies virus strains.

In a fifth aspect of the invention, there is provided a vaccine composition comprising: (a) a pharmaceutically acceptable carrier; and (b) purified rabies virus as described in the fourth aspect of the invention.

In another preferred embodiment, the vaccine composition further comprises: (c) an adjuvant.

In a sixth aspect of the invention, there is provided the use of a purified rabies virus according to the fourth aspect of the invention for the preparation of a vaccine against rabies virus.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

The drawings are for illustration purposes only and are not to be construed as limiting the invention; for the purpose of better illustrating the embodiments, certain features of the drawings may be omitted, enlarged or reduced, and do not represent the size of an actual product; it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted; the same or similar reference numerals correspond to the same or similar parts; the terms describing positional relationships in the drawings are for illustrative purposes only and are not to be construed as limiting the patent.

Fig. 1 is a schematic structural diagram of a virus density gradient centrifugation purification device, wherein each number in fig. 1 represents: 1 is a continuous flow density gradient centrifugal rotor, 2 is a first pipe orifice, 3 is a third pipe orifice, 4 is a second pipe orifice, 5 is a fourth pipe orifice, 6 is a first valve, 7 is a second valve, 8 is a third valve, 9 is a fourth valve, 10 is a first pipeline, 11 is a second pipeline, 12 is a third pipeline, 13 is a first bottle, 14 is a fourth pipeline, 15 is a second bottle, 16 is a fifth valve, 17 is a sixth valve and 18 is a liquid conveying device.

FIG. 2 is a SDS-PAGE image of samples after density gradient centrifugation purification, wherein 4-15 represent 4-15 sample collection vials.

FIG. 3 is a western blot assay of samples after density gradient centrifugation purification, where 4-15 represent 4-15 sample collection vials.

FIG. 4 is an SDS-PAGE image of purified samples of virus harvest at 200ml/min flow rate, wherein 24-36 represents 24-36 th sample collection bottles.

FIG. 5 is a Western Blot of purified samples of virus harvest injected at 200ml/min flow rate, where 24-36 represent 24-36 sample collection vials.

FIG. 6 is an overlay of absorbance curves for 3 batches of samples.

FIG. 7 is an overlay of absorbance curves for 3 batches of samples.

FIG. 8 is a SDS-PAGE and Western Blot plot of rabies virus harvest at different fold concentrations, wherein 25-36 represents the 25 th-36 th sample collection vial; the left is sample 1 without concentration, the right is sample 2 concentrated 10 times, the upper is SDS-PAGE and the lower is western blot.

FIG. 9 is an SDS-PAGE image of purified samples of virus harvest at 250ml/min flow rate, wherein 24-36 represents 24-36 th sample collection bottles.

FIG. 10 is a western blot of samples after viral harvest was injected at 250ml/min, where 24-36 represent the 24 th-36 th sample collection vial.

FIG. 11 is an SDS-PAGE image of purified samples of virus harvest at 50ml/min flow rate, where 24-36 represents the 24 th-36 th sample collection bottle.

FIG. 12 is a western blot of samples after virus harvest was purified by injection at a flow rate of 50ml/min, where 24-36 represents the 24 th-36 th sample collection vial

Fig. 13 is a density gradient layer formed after purification was completed for two different initial sucrose concentrations.

Figure 14 is a graph of the resulting concentration profiles formed for the 3 batch 60% (wt%) sucrose solution protocol.

FIG. 15 is a SDS-PAGE of samples after density gradient centrifugation purification in a 30% + 60% (wt%) sucrose concentration protocol, where 2-13 represent 2-13 sample collection vials.

FIG. 16 is a western blot test chart of samples after density gradient centrifugation purification in a 30% + 60% (wt%) sucrose concentration protocol, where 2-13 represents the 2 nd-13 th sample collection vial.

Detailed Description

The present inventors have made extensive and intensive studies and have unexpectedly developed a virus density gradient centrifugal purification apparatus. The virus density gradient centrifugal purification device can be used for simply, conveniently and timely removing bubbles in a continuous flow density gradient centrifugal rotor in a sucrose density gradient purification virus process, particularly removing the bubbles in the sucrose density gradient, so that the vibration of the rotor and the distribution of the sucrose density gradient in the rotor caused by the bubbles are avoided, and a high-quality virus vaccine can be prepared. The invention also provides a rabies virus density gradient centrifugal purification method, which is a process method for efficiently purifying the material for producing the rabies virus based on continuous flow sucrose density gradient centrifugation, has high recovery rate for the rabies virus, and can efficiently remove impurities possibly introduced in the rabies virus process, including various impurities (including protein impurities, nucleic acid impurities and the like) from chicken embryo fibroblasts, so that the purified rabies virus can meet the vaccine sanitary standard. In addition, the rabies virus density gradient centrifugal purification method can be carried out by using the virus density gradient centrifugal purification device to prepare high-quality vaccines, and the virus density gradient centrifugal purification device is simple in structure, convenient to operate and suitable for industrial production. The present invention has been completed based on this finding.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the terms "comprising," "including," and "containing" are used interchangeably and include not only open-ended definitions, but also semi-closed and closed-ended definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".

As used herein, the terms "upper", "lower", "top" and "bottom" and the like refer to orientations or positional relationships based on those shown in the drawings, which are created and simplified for the purpose of describing the present invention, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus are not to be construed as limiting the present invention.

As used herein, the terms "first," "second," "third," "fourth," "fifth," and "sixth," etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defined as "first," "second," "third," "fourth," "fifth," and "sixth," may explicitly or implicitly include one or more of the features.

As used herein, the terms "connected" and "contiguous" are used interchangeably and are to be construed broadly and may be either fixedly connected or removably connected or integrally connected.

As used herein, the term "continuous flow density gradient centrifuge" is a density gradient centrifuge that is continuously fed at one end and continuously discharged at the other end. The continuous flow density gradient centrifuge has the main working part of a continuous flow density gradient centrifugal rotor, the continuous flow density gradient centrifuge is ultra-high speed centrifuge equipment, and in the process of high-speed operation, air bubbles cannot exist in the rotor, and the air bubbles can cause the vibration of the continuous flow density gradient centrifugal rotor and influence the distribution of density gradients (such as sucrose density gradients) in the rotor, so that the final purification effect is influenced, and therefore, the air bubbles in the rotor need to be removed before sample introduction into the continuous flow density gradient centrifuge.

As used herein, the terms "continuous flow density gradient centrifuge rotor" and "rotor" are used interchangeably, the "top of the continuous flow density gradient centrifuge rotor" and "upper end of the continuous flow density gradient centrifuge rotor" and the "bottom of the continuous flow density gradient centrifuge rotor" and "lower end of the continuous flow density gradient centrifuge rotor" are used interchangeably.

As used herein, the term "density gradient centrifugation" refers to the formation of a continuous or discontinuous density gradient in a centrifuge tube with a medium, placing a cell suspension or homogenate on top of the medium, and layering and separating the cells by the action of gravity or centrifugal force fields.

The term "wt%" means weight percent, and concentrations used in the present invention are weight percent unless otherwise specified.

In the present invention, the virus used is subjected to inactivation treatment, which may be carried out using an inactivating agent commonly used in the art. Such as formaldehyde, glutaraldehyde or beta-propiolactone, and the like.

Rabies virus

In the present invention, the rabies virus may be from any source as long as it is propagated in cells sensitive to rabies virus. Typically, rabies virus strains are used, which have been established from primary isolates, and known rabies virus strains include, but are not limited to: AVO1, CVS, ERA, Kelev, HEP-FLURY, Nishigahara RCEH, Ontario fox, Ontario skunk, Pasteur/PV, Pittman Moore (PM), Street Alabama Dufferin (SAD) B19, SAD-Bern, ERA, SAG2, Vnukovo-32, Eth2003, strain RC-HL, Nishigahara, SHBRV-18, SRV9, Ni-CE, Flury-LEP, and Kissling rabies virus strains. Strains of particular importance for vaccine production are PV, Pitman-Moore, CVS, Flury LEP, Flury HEP, Kelev and ERA. Two particularly useful strains are the Pitman-Moore strain used in VEROLAB (or Wistar rabies PM/WI38-1503-3M strain) and the Flury LEP strain used in RABIPUR. These very virulent strains are used to produce inactivated vaccines against rabies. Attenuated rabies virus strains can also be used, with the aim of producing live vaccines against rabies. For example the SAD Bern strain or SAD B19 strain or strains derived therefrom, for example the SAG1 and SAG2 strains derived from the SAD Bern strain due to the presence of point mutations on the glycoprotein G of the rabies virus (EP350398, EP 583998). EP1253197 also describes some other more stable attenuated rabies strains having one or more point mutations in the phosphoprotein P and glycoprotein G of the rabies virus.

Virus density gradient centrifugal purification device

For ease of illustration, the virus density gradient centrifugation purification apparatus of the present invention is further described below with reference to FIG. 1, which should be understood not to limit the scope of the present invention.

The invention provides a virus density gradient centrifugation purification device, which comprises an annular pipeline and a continuous flow density gradient centrifuge, wherein the continuous flow density gradient centrifuge comprises a continuous flow density gradient centrifugal rotor 1;

the third pipeline is provided with a liquid conveying device 18.

In particular, the virus density gradient centrifugation purification device is as described in the first aspect of the invention.

Rabies virus density gradient centrifugal purification method

The invention also provides a rabies virus density gradient centrifugal purification method, which comprises the following steps:

wherein F2 is 90-240 ml/min;

In particular, the rabies virus density gradient centrifugation purification method is as described above in the second aspect of the invention.

In a preferred embodiment, the rabies virus density gradient centrifugal purification method is performed by using the virus density gradient centrifugal purification device.

Purified rabies virus and vaccine compositions

The invention also provides highly pure rabies virus prepared by the method of the invention and a vaccine composition containing the rabies virus.

The determination shows that the rabies virus prepared by the invention has high purity, the removal rate of the hybrid protein is far more than 90 percent, so the residual quantity of the hybrid protein is far less than 10 percent, and better less than or equal to 5 percent.

The rabies virus (inactivated) prepared by the invention can be used as immunogen for stimulating animals to generate immune response aiming at the rabies virus, thereby protecting animals (human beings) from being infected by the rabies virus.

One preferred composition is a prophylactic vaccine composition. The vaccine composition of the present invention may be a monovalent or multivalent vaccine composition.

These vaccines comprise the highly purified rabies virus prepared according to the present invention, and are usually combined with "pharmaceutically acceptable carriers" including any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive viral particles. Such vectors are well known to those of ordinary skill in the art. In addition, these carriers may act as immunostimulants ("adjuvants").

Preferred adjuvants that enhance the effect of the composition include, but are not limited to: (1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) ISA720 adjuvant; (3) freund's adjuvant, etc.

The vaccine compositions of the invention (including rabies virus, pharmaceutically acceptable carriers and/or adjuvants) typically contain diluents such as water, saline, glycerol, ethanol and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may be present in such vehicles.

More specifically, vaccines, including immunogenic compositions, comprise an immunologically effective amount of rabies virus, as well as the other desired components described above. An "immunologically effective amount" refers to an amount that is effective for treatment or prevention in a single dose or in a continuous dose administered to an individual. The amount will depend on the physiological condition of the animal (e.g., human), the ability of the immune system to synthesize antibodies, the degree of protection desired, and other relevant factors.

In the present invention, the vaccine composition or immunogenic composition can be prepared into an injectable agent, such as a liquid solution or emulsion; it can also be made into solid form suitable for preparing solution or suspension, or liquid excipient before injection. The formulation may also be emulsified or encapsulated in liposomes, enhancing the adjuvant effect in the pharmaceutically acceptable carrier described above.

The conventional approach is to administer the immunogenic composition by injection from the parenteral (subcutaneous or intramuscular) route. Other formulations suitable for other modes of administration include oral and transdermal applications, and the like. The therapeutic dose may be a single dose regimen or a multiple dose regimen. The vaccine may be administered in combination with other immunomodulators.

The main advantages of the invention include:

1. the invention unexpectedly develops a virus density gradient centrifugal purification device for the first time, which can simply, conveniently and timely remove air bubbles in a continuous flow density gradient centrifugal rotor in the process of purifying the virus by using a sucrose density gradient, particularly can remove the air bubbles in the sucrose density gradient, thereby avoiding the vibration of the rotor and the distribution of the sucrose density gradient in the rotor caused by the air bubbles and further preparing a high-quality virus vaccine.

2. The virus density gradient centrifugal purification device has the advantages of simple structure and convenient operation, and is suitable for industrial production.

3. The density gradient centrifugation has small shearing force to the virus, and is easy to collect complete virus particles.

4. The rabies virus density gradient centrifugal purification method has high antigen recovery rate and protein removal rate of ovalbumin, host protein, pancreatin, bovine serum, antibiotics and total protein content.

5. The rabies virus density gradient centrifugal purification method is carried out by using the virus density gradient centrifugal purification device to prepare the high-quality vaccine, can be directly purified without concentration step or concentrated by a smaller multiple before density gradient centrifugation, and has stronger operability compared with the traditional molecular sieve.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1

Preparation of rabies Virus harvesting solution

Materials: SPF chick embryo (11 days old)

The method comprises the following steps: a) embryo taking and embryo processing (removing head and viscera, and cutting into small tissue blocks); b) digesting the chick embryo tissue blocks; c) dispersing and collecting chicken embryo fibroblasts; d) counting cells and infecting rabies viruses according to the number of the cells; e) culturing at 33-35 deg.C for 1 day; f) changing the culture solution, and culturing at 33-35 deg.C for 4 days; g) collecting the harvest liquid, adding a fresh maintenance liquid, and continuously culturing for 2 days; h) a second harvest was performed.

Example 2

The present embodiment provides a virus density gradient centrifugation and purification apparatus, a schematic structural diagram of which is shown in fig. 1, the virus density gradient centrifugation and purification apparatus includes a loop pipeline and a continuous flow density gradient centrifuge, the loop pipeline is a diamond pipeline, and the continuous flow density gradient centrifuge includes a continuous flow density gradient centrifuge rotor 1;

the first pipe orifice is communicated with the upper end of the continuous flow density gradient centrifugal rotor through a first pipe 10, the second pipe orifice is communicated with the lower end of the continuous flow density gradient centrifugal rotor through a second pipe 11, the third pipe orifice is connected with a first bottle body 13 through a third pipe 12, the first bottle body is a sample bottle, the fourth pipe orifice is connected with a second bottle body 15 through a fourth pipe 14, and the second bottle body is a receiving bottle;

a liquid conveying device 18 is arranged on the third pipeline, and the liquid conveying device is a bidirectional conveying peristaltic pump;

the continuous flow density gradient centrifuge is a manufacturer alpha Weishiman model eKII ultracentrifuge, wherein the model of a continuous flow density gradient centrifuge rotor is K3, the continuous flow density gradient centrifuge rotor is provided with an inner cavity for containing liquid materials, and the volume of the inner cavity is 3.2L.

Example 3

The virus was purified using the virus density gradient centrifugation purification apparatus of example 2, with the following specific steps:

1. providing a viral harvest

279kg of rabies virus harvest was prepared according to the method of example 1, centrifuged by a Hitachi continuous flow centrifuge, the supernatant was collected and concentrated by ultrafiltration 10-fold to obtain 28kg of rabies virus harvest.

2. Preparation of sucrose Density gradient

2.1, when the continuous flow density gradient centrifuge rotor 1 is at 0rpm, adding a PBS buffer solution (pH7.4, concentration of 0.01mM) into the first bottle 13, closing the first valve 6 and the third valve 8, opening the delivery device 18 (peristaltic pump), and allowing the PBS to flow out to the second bottle 15 through the third tube 12, the tube between the third tube opening 3 and the second tube opening 4, the second tube 11, the continuous flow density gradient centrifuge rotor 1, the first tube 10, the tube between the first tube opening 2 and the fourth tube opening 5, and the fourth tube 14 in sequence, so that the PBS is filled in the continuous flow density gradient centrifuge rotor 1 and the whole tube system. Since the third tube 12 passes through the outside air before the PBS solution is added to the first bottle 13, the third tube 12 can take in air (bubbles) when the PBS is conveyed by the conveying device 18 (peristaltic pump), so that the entire tubing system and the upper and lower ends of the continuous flow density gradient centrifuge rotor 1 contain bubbles.

2.2, opening the first valve 6 and the third valve 8, closing the fifth valve 16 and the sixth valve 17, and opening the delivery device 18 (peristaltic pump), wherein the PBS solution in the first bottle 13 flows through the third tube 12, and then flows out of the fourth tube 14 to the second bottle 15 through the third tube 3, the second tube 4 to the fourth tube 5, the third tube 3, and the first tube 2 to the fourth tube 5. Since the tube mouth of the third tube 12 is already in the first bottle 13PBS, in this step 2.2, the PBS delivered by the delivery device 18 (peristaltic pump) is free of air bubbles, and therefore this step 2.2 is able to remove air bubbles in the third tube 12, the ring line and the fourth tube 14.

2.3, opening a fifth valve 16 and a sixth valve 17, closing the first valve 6 and the third valve 8, opening a conveying device 18 (peristaltic pump), and allowing the PBS to flow out of the second bottle 15 through a third pipeline 12, a pipeline between the third pipe orifice 3 and the second pipe orifice 4, a second pipeline 11, a continuous flow density gradient centrifugal rotor 1, a first pipeline 10, a pipeline between the first pipe orifice 2 and the fourth pipe orifice 5, and a fourth pipeline 14 in sequence. During the PBS flow, PBS flows in from the lower end of the continuous flow density gradient centrifuge rotor 1 and flows out from the upper end of the continuous flow density gradient centrifuge rotor 1, so that the PBS buffer without air bubbles can discharge the air bubbles at the upper end of the continuous flow density gradient centrifuge rotor 1.

2.4, opening the first valve 6 and the third valve 8, closing the second valve 7 and the fourth valve 9, opening the conveying device 18 (peristaltic pump), and allowing the PBS to sequentially flow through the third pipeline 12, the pipeline between the third pipe orifice 3 and the first pipe orifice 2, the first pipeline 10, the continuous flow density gradient centrifugal rotor 1, the second pipeline 11, the pipeline between the second pipe orifice 4 and the fourth pipe orifice 5, and the fourth pipeline 14 to flow out of the second bottle body 15. During the flow of the PBS, the PBS flows in from the upper end of the continuous flow density gradient centrifugal rotor 1 and flows out from the lower end of the continuous flow density gradient centrifugal rotor 1, so that the PBS without air bubbles can discharge the air bubbles at the lower end of the continuous flow density gradient centrifugal rotor 1.

Steps 2.1-2.4 allow the continuous flow density gradient centrifuge rotor 1 and the entire pipe system to be free of air bubbles.

2.5, opening the second valve 7 and the fourth valve 9, replacing the PBS solution in the first bottle 13 with a 60% (wt%) sucrose solution, closing the fifth valve 16 and the sixth valve 17, starting the delivery device 18 (peristaltic pump), and allowing the sucrose solution in the first bottle 13 to sequentially pass through the third pipe 12, then respectively pass through the third pipe orifice 3, the pipe between the second pipe orifice 4 and the fourth pipe orifice 5, and the pipe between the third pipe orifice 3 and the first pipe orifice 2 and the fourth pipe orifice 5, and then flow out of the fourth pipe 14 to the second bottle 15. Since the third tube 12 passes through the outside air during the process of replacing the PBS solution in the first bottle 13 with the sucrose solution, the third tube 12 can take in air (bubbles) at the beginning of the sucrose solution delivered by the delivery device 18 (peristaltic pump), however, in this step 2.5, the sucrose solution flows out to the second bottle 15 only through the third tube 12, the circular tube and the fourth tube 14, and therefore, the sucrose solution delivered by the delivery device 18 (peristaltic pump) does not contain bubbles during the continuous flow of the sucrose solution, and therefore, the sucrose solution in the third tube 12, the circular tube and the fourth tube 14 does not contain bubbles as the sucrose solution continuously flows.

2.6, opening a fifth valve 16 and a sixth valve 17, closing the first valve 6 and the third valve 8, and opening a conveying device 18 (a peristaltic pump), wherein the bubble-free 60 percent (wt percent) sucrose solution enters the continuous flow density gradient centrifugal rotor 1 through a third pipeline 12, a pipeline between the third pipe orifice 3 and the second pipe orifice 4 and a second pipeline 11 in sequence, so that the bubble-free sucrose solution enters the continuous flow density gradient centrifugal rotor 1 and is contacted with the PBS buffer solution in the rotor 1, the flow rate of the 60 percent (wt percent) sucrose solution entering the continuous flow density gradient centrifugal rotor is 100ml/min and 1600ml (1/2 which is the cavity volume of the continuous flow density gradient centrifugal rotor), wherein the PBS solution in the rotor 1 passes through the first pipeline 10, the pipeline between the first pipe orifice 2 and the fourth pipe orifice 5 during the process that the sucrose solution enters the continuous flow density gradient centrifugal rotor 1, And a fourth pipe 14 which flows out to the second bottle 15.

2.7 opening the first valve 6 and the third valve 8 and closing the sixth valve 17, the continuous flow density gradient centrifuge rotor 1 is rotated and centrifuged at 5000rpm, so that the PBS and the sucrose solution in the continuous flow density gradient centrifuge rotor 1 form a longitudinal density gradient, and the closer to the outer wall of the continuous flow density gradient centrifuge rotor 1, the higher the concentration of sucrose. During the centrifugal rotation of the continuous flow density gradient centrifuge rotor 1, gas dissolved in the PBS and sucrose solutions is generated during the rotation of the continuous flow density gradient centrifuge rotor 1, and generated bubbles appear at the upper and lower ends of the continuous flow density gradient centrifuge rotor 1.

2.8, keeping the continuous flow density gradient centrifugal rotor 1 rotating at 5000rpm for centrifugation, replacing 60% (wt%) of the sucrose solution in the first bottle 13 with a PBS buffer solution (pH7.4, concentration of 0.01mM), closing the fifth valve 16 and the sixth valve 17, opening the conveying device 18 (peristaltic pump), and allowing the PBS solution in the first bottle 13 to sequentially pass through the third pipe 12, and then to flow out from the fourth pipe 14 to the second bottle 15 through the third pipe 3, the pipe between the second pipe 4 and the fourth pipe 5, the pipe between the third pipe 3 and the first pipe 2 and the fourth pipe 5. Since the third tube 12 passes through the outside air during the process of replacing the sucrose solution in the first bottle 13 with the PBS solution, the third tube 12 can take in air (bubbles) at the beginning of the PBS buffer solution conveyed by the conveying device 18 (peristaltic pump), however, in this step 2.8, the PBS solution flows out to the second bottle 15 only through the third tube 12, the circular tube and the fourth tube 14, and therefore, the PBS solution conveyed by the conveying device 18 (peristaltic pump) does not contain bubbles during the continuous flow of the PBS solution, and therefore, the PBS solution in the third tube 12, the circular tube and the fourth tube 14 does not contain bubbles as the PBS solution continuously flows.

2.9 sequential removal of air bubbles at the lower and upper ends of the continuous flow Density gradient centrifuge rotor 1

during the centrifugal process of keeping the continuous flow density gradient centrifugal rotor 1 rotating at 5000rpm, the fifth valve 16 and the sixth valve 17 are opened, the second valve 7 and the fourth valve 9 are closed, the conveying device 18 (a peristaltic pump, PBS flows out to the second bottle 15 through the third pipe 12, the pipe between the third pipe orifice 3 and the first pipe orifice 2, the first pipe 10, the continuous flow density gradient centrifugal rotor 1, the second pipe 11, the pipe between the second pipe orifice 4 and the fourth pipe orifice 5 and the fourth pipe 14 in sequence, in the PBS flow process, PBS flows in from the upper end of the continuous flow density gradient centrifugal rotor 1 at the flow rate of 250ml/min, liquid in the rotor 1 is pushed from top to bottom, and flows out from the lower end of the density gradient centrifugal rotor 1, so that bubbles at the lower end of the density gradient centrifugal rotor 1 are pushed out and removed.

during the rotation and centrifugation process of the continuous flow density gradient centrifuge rotor 1 at 5000rpm, the second valve 7 and the fourth valve 9 are opened, the first valve 6 and the third valve 8 are closed, the conveying device 18 (peristaltic pump) is started, and the PBS flows out to the second bottle body 15 through the third pipeline 12, the pipeline between the third nozzle 3 and the second nozzle 4, the second pipeline 11, the continuous flow density gradient centrifuge rotor 1, the first pipeline 10, the pipeline between the first nozzle 2 and the fourth nozzle 5 and the fourth pipeline 14 in sequence at the flow rate of 250 ml/min. In the flowing process of the PBS, the PBS flows in from the lower end of the continuous flow density gradient centrifugal rotor 1 at the flow rate of 250ml/min, liquid in the rotor 1 is pushed from bottom to top and flows out from the upper end of the density gradient centrifugal rotor 1, and thus air bubbles at the upper end of the density gradient centrifugal rotor 1 are pushed out and removed;

therefore, in step 2.9, the bubbles generated in the rotation process of the continuous flow density gradient centrifugal rotor 1 can be removed in time, so that the adverse effect that the bubbles can cause the vibration of the continuous flow density gradient centrifugal rotor to influence the density gradient inside the rotor (such as the distribution of sucrose density gradient, and further influence the final purification effect) is avoided.

3. Sample introduction:

3.1 the centrifugal speed of the continuous flow density gradient centrifugal rotor 1 is slowly and uniformly increased from 5000rpm to 35000rpm within 30min, and meanwhile, in the process of increasing the rotating speed of the rotor 1, PBS is fed into the continuous flow density gradient centrifugal rotor 1 according to the step 2.9.2 at the flow rate of 150 ml/min. Keeping the continuous flow density gradient centrifugal rotor 1 rotating at 35000rpm for centrifugation, replacing the PBS solution in the first bottle 13 with the virus harvest solution, opening the first valve 6 and the third valve 8, closing the fifth valve 16 and the sixth valve 17, starting the conveying device 18 (peristaltic pump), after the virus harvest solution in the first bottle 13 sequentially passes through the third pipeline 12, passing through the third pipeline 3, the pipeline between the second pipeline 4 and the fourth pipeline 5, the pipeline between the third pipeline 3 and the first pipeline 2 and the fourth pipeline 5, respectively, and flowing out of the fourth pipeline 14 to the second bottle 15. Since the third tube 12 passes through the outside air during the process of replacing the PBS solution in the first bottle 13 with the virus harvest solution, the third tube 12 can take in air (bubbles) at the beginning of the virus harvest solution conveyed by the conveying device 18 (peristaltic pump), however, in this step 3.1, the virus harvest solution flows out to the second bottle 15 only through the third tube 12, the circular tube and the fourth tube 14, and therefore, the virus harvest solution conveyed by the conveying device 18 (peristaltic pump) does not contain bubbles during the continuous flow of the virus harvest solution, and therefore, the virus harvest solution in the third tube 12, the circular tube and the fourth tube 14 does not contain bubbles as the virus harvest solution continuously flows.

3.2 keeping the continuous flow density gradient centrifuge rotor 1 rotating at 35000rpm for centrifugation, opening the fifth valve 16 and the sixth valve 17, closing the first valve 6 and the third valve 8, starting the conveying device 18 (peristaltic pump), allowing the virus harvest to enter the continuous flow density gradient centrifuge rotor 1 from the lower end of the continuous flow density gradient centrifuge rotor 1 through the third pipeline 12, the pipeline between the third nozzle 3 and the second nozzle 4 and the second pipeline 11 in sequence, wherein the sample injection speed of the virus harvest is 150ml/min, and at this time, the first bottle 13 still has residual excess virus harvest.

3.3 keeping the continuous flow density gradient centrifuge rotor 1 rotating at 35000rpm for centrifugation, opening the first valve 6 and the third valve 8, closing the fifth valve 16 and the sixth valve 17, replacing the virus harvest solution in the first bottle 13 with PBS buffer solution (pH7.4, concentration 0.01mM), starting the conveying device 18 (peristaltic pump), after the PBS buffer solution in the first bottle 13 passes through the third tube 12 in sequence, passing through the third tube 3, the tube between the second tube 4 and the fourth tube 5, the tube between the third tube 3 and the first tube 2 and the fourth tube 5, respectively, and flowing out from the fourth tube 14 to the second bottle 15. Since the third tube 12 passes through the outside air during the process of replacing the virus harvest in the first bottle 13 with the PBS buffer, the third tube 12 can take in air (air bubbles) at the beginning of the PBS buffer transported by the transport device 18 (peristaltic pump), however, in this step 3.3, the PBS buffer flows out to the second bottle 15 only through the third tube 12, the circular tube and the fourth tube 14, and therefore, the PBS buffer transported by the transport device 18 (peristaltic pump) does not contain air bubbles during the continuous flow of the PBS buffer, and therefore, the PBS buffer in the third tube 12, the circular tube and the fourth tube 14 does not contain air bubbles as the PBS buffer continuously flows.

3.4 opening the fifth valve 16 and the sixth valve 17, closing the first valve 6 and the third valve 8, keeping the continuous flow density gradient centrifuge rotor 1 rotating at 35000rpm for centrifugation, and simultaneously, during the centrifugation process of the continuous flow density gradient centrifuge rotor 1, opening the conveying device 18 (peristaltic pump), the PBS buffer solution sequentially passes through the third pipeline 12, the pipeline between the third nozzle 3 and the second nozzle 4 and the second pipeline 11, enters the continuous flow density gradient centrifuge rotor 1 from the lower end of the continuous flow density gradient centrifuge rotor 1, the feed speed of the PBS buffer solution is 150ml/min, the feed of the PBS buffer solution is started, after the continuous flow density gradient centrifuge rotor 1 rotating at 35000rpm for centrifugation for 60min, the virus harvest solution is distributed in different longitudinal sucrose gradient layers due to different molecular weights in the continuous flow density gradient centrifuge rotor 1, and the substances with smaller molecular weights pass through the first pipeline 10, The pipe between the first nozzle 2 and the fourth nozzle 5, and the fourth pipe 14, flow out to the second bottle 15.

4. Collecting a sample:

the sixth valve 17 is closed and the continuous flow density gradient centrifuge rotor is slowed down to 0 revolutions at 35000rpm/50min, and the longitudinal density gradient in the continuous flow density gradient centrifuge rotor 1 will form a transverse density gradient under the action of gravity. The empty first bottle 13 is placed, the sixth valve 17 is opened, the conveying device 18 (peristaltic pump) is started to carry out reverse conveying, air enters the continuous flow density gradient centrifugal rotor 1 through the fourth pipeline 14, the pipeline between the fourth pipe orifice 5 and the first pipe orifice 2 and the first pipeline 10, therefore, the gradient sample in the continuous flow density gradient centrifuge rotor 1 will flow into the first bottle 13 through the second pipe 11, the pipe between the second nozzle 4 and the third nozzle 3, and the third pipe 12, and pass through the first bottle 13 which is continuously replaced with new one (each first bottle is collected at 50 ml/bottle), thereby obtaining different gradient samples in the continuous flow density gradient centrifugation rotor 1, detecting proteins by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), as shown in FIG. 2, the results of the western blot analysis of the proteins separated by SDS-PAGE are shown in FIG. 3.

As can be seen from fig. 2 and 3: the main antigen (G) protein of the rabies virus is distributed intensively and has clear bands and good purification effect.

In step 3.2, when the virus harvest liquid starts to be injected into the continuous flow density gradient centrifugal rotor and the injection is finished, the effluent liquid (namely the flow-through liquid) of the upper end outlet of the continuous flow density gradient centrifugal rotor is collected from the fourth pipeline (14) port, and the content of the antigen (rabies virus) in the flow-through liquid is carried out. The test results are given in table 1 below.

Table 1 content of antigen (rabies virus) in flow-through (n ═ 3)

As can be seen from Table 1, no antigen (rabies virus) was eluted from the upper outlet of the continuous flow density gradient centrifuge rotor at the beginning of the virus harvest and at the end of the virus harvest.

Example 4 rabies Virus harvest was purified by injection at a flow rate of 200ml/min

This example is similar to example 3 except that in step 3.2 the rabies virus harvest in the first vial is fed into the continuous flow density gradient centrifuge rotor (1) at a flow rate of 200ml/min and the results of the SDS-PAGE and western blot tests are shown in fig. 4 and 5, respectively.

As can be seen from fig. 4 and 5: the distribution of the major antigen (G) protein of rabies virus is concentrated and the band is clear. The purification effect is good, and the difference with the sampling speed of 150ml/min is small.

Example 5 detection of rabies virus-enriched gradient centrifugation layer (i.e. viral gradient liquid)

In this example, virus purification was performed in parallel on 3 batches as described in example 3, and the harvest collected in step 4 was examined by uv absorbance to determine a gradient centrifugation layer rich in rabies virus for uniformity and reproducibility both batch to batch and intra-batch.

(A) Detection of gradient centrifugation layer (i.e. virus gradient) rich in rabies virus in 3 parallel samples (i.e. 3 batches of virus harvest prepared in parallel according to step 1 of example 3, respectively), profiles of the 3 batches of samples were subjected to profile stacking as shown in fig. 6, showing a high degree of coincidence between the curves.

(B) The same batch of samples was divided into 3 equal portions (i.e., 1 batch of viral harvest was prepared according to step 1 of example 3, and divided into 3 parallel portions), and the profiles of the 3 samples were subjected to profile superposition as shown in FIG. 7, showing a high degree of coincidence between the curves.

As can be seen from FIGS. 6 and 7, the purification of the virus using the method of example 3 ensures excellent uniformity and reproducibility between and within batches.

Example 6 isolation of rabies Virus harvests at different fold concentrations

1. Providing virus harvests of different concentration factors

Rabies virus harvest was prepared according to the method of example 1, clarified and centrifuged by a Hitachi continuous flow centrifuge and 154L of supernatant was collected. Taking 14L of the samples as sample 1; another 140L of the supernatant was concentrated by ultrafiltration 10-fold to 14L as sample 2.

2.

Samples

1 and 2 were purified separately as described in example 3.

The results of detection of SDS-PAGE and western blot are shown in FIG. 8.

As can be seen from FIG. 8, RABV enrichment is very obvious after 10-fold concentration and purification of the virus harvest, and the sample loading time is saved by 90% compared with that when the virus harvest is not concentrated. Thus, a harvest that is not concentrated or a harvest that is less concentrated can be used. However, for time saving reasons, the rabies virus harvest can be concentrated prior to density gradient centrifugation.

Example 7 purification Effect test

Antigen recovery rate is 100% total antigen after purification/total antigen before purification;

ovalbumin removal rate ═ 1-total ovalbumin content after purification/total ovalbumin content before purification × 100%;

host protein removal rate ═ 1-total host protein content after purification/total host protein content before purification × 100%;

bovine serum albumin removal rate 1-total bovine serum albumin content after purification/total bovine serum albumin content before purification 100%;

total protein removal rate ═ 1-total protein content after purification/total protein content before purification × 100%; antibiotic removal rate ═ 1-total antibiotic content after purification/total antibiotic content before purification × 100%;

the concentration was measured in different fold ratios (i.e., ratio of sample volumes before and after density gradient centrifugation) as described in example 3, and the results are shown in tables 2 and 3 below.

TABLE 2

TABLE 3

The results show that: the concentration multiple of the sample after density gradient centrifugation is high, the antigen recovery rate of the sample is up to about 80%, the removal rates of ovalbumin, host protein, antibiotics, pancreatin and bovine serum are all up to more than 99%, and the removal rate of total protein is also up to more than 98%.

Comparative example

Comparative example 1

1. Providing a viral harvest

Preparing rabies virus harvest liquid according to the method of example 1, clarifying and centrifuging the rabies virus harvest liquid by a Hitachi continuous flow centrifuge, collecting supernatant, performing ultrafiltration concentration for 10 times to obtain concentrated samples A, B, C, D, E, F, H, I and J, and performing ultrafiltration concentration for 60 times to obtain a concentrated sample G.

Samples A, B and C were purified by molecular sieve (agarose gel exclusion) and samples D, E and F were purified by continuous flow sucrose density gradient centrifugation as in example 3. Sampling before and after purification, respectively, and calculating the impurity removal rate results are shown in table 4 below.

Table 4 impurity removal results

As can be seen from Table 4, the removal rate of ovalbumin and the removal rate of antibiotics both reach 99.9 percent in two modes, and the purification method of trypsin removal rate density gradient centrifugation is superior to the molecular sieve chromatography.

Molecular sieve purification concentration is 60 times, continuous flow density gradient centrifugation only needs 10 times of concentration, and the loss rate of the antigen concentrated by 60 times is higher than that concentrated by 10 times. The results are given in Table 5 below.

TABLE 5 results of antigen recovery

The recovery of antigen decreases with increasing concentration factor. The molecular sieve chromatography technology requires that virus liquid is concentrated by 60 times to have purification operability, and sucrose density gradient centrifugation only needs 10 times or even no concentration to carry out purification. Sucrose density gradient centrifugation is more operable than molecular sieve purification.

Comparative example 2

In the same manner as in example 3, except that in step 3.2, the rabies virus harvest in the first vial was fed into the continuous flow density gradient centrifuge rotor (1) at a flow rate of 250ml/min, the results of the SDS-PAGE and western blot tests are shown in FIG. 9 and FIG. 10, respectively.

As can be seen from fig. 9 and 10, at the loading rate of 250ml/min, the bands of non-rabies virus structural proteins in the lanes are significantly reduced, and the purity of the virus is significantly higher than other samples purified at lower loading rates, since under the loading rate, the complete virus particles have no time to enter the sucrose gradient layer. It is expected that as the loading rate continues to increase, more and more virus particles are lost with the sample effluent, particulate matter larger in size than rabies virus particles enters the sucrose layer, and the purification effect will be significantly reduced.

Comparative example 3

In the same manner as in example 3, step 3.2, the rabies virus harvest in the first vial was fed into the continuous flow density gradient centrifuge rotor (1) at a flow rate of 50ml/min, and the results of the SDS-PAGE and western blot tests are shown in FIGS. 11 and 12.

As can be seen from FIGS. 11 and 12, the sample was flowing through the rotor chamber for a longer period of time with the largest yield of major antigen (G-protein) but incomplete removal of the hetero-proteins (especially 34-36 tubes) when the sample was injected at a flow rate of 50ml/min,

comparative example 4

This example is similar to the procedure of example 3, except that 60% (wt%) sucrose solution was changed to 30% + 60% (wt%) sucrose solution, specifically, 0.6L 30% (wt%) sucrose solution was added to the continuous flow density gradient centrifuge rotor, and 1L 60% (wt%) sucrose solution was added, and the purification effect was examined by SDS-PAGE and WB methods.

The continuous sucrose densities formed by the two different sucrose solutions after purification is complete are shown in fig. 13, where the repeat experiments for sucrose density for the 3 batch 60% (wt%) sucrose solution protocol in example 3 are shown in fig. 14; density gradient centrifugation in 30% + 60% (wt%) sucrose concentration protocol SDS-PAGE and western blot detection of purified samples is shown in FIGS. 15 and 16

As can be seen from fig. 13, the 30% + 60% (wt%) sucrose solution formed a sucrose concentration gradient with a smaller distance span than the 60% (wt%) sucrose solution. The concentration width of 60% (wt%) cane sugar is wide, the collection range is small, the product is easy to enrich, the collection range is large, and the separation effect is good.

As can be seen from fig. 14, the 60% (wt%) sucrose solution formed a sucrose gradient with excellent stability and reproducibility.

As can be seen from tables 15-16, the 30% + 60% (wt%) sucrose solution protocol was relatively closer in distance between protein peaks of different molecular weights after purification than the 60% (wt%) sucrose solution, and the host protein content after the major peak of the virus was significantly greater than the 60% (wt%) sucrose solution protocol. The western blot results show that the 60% (wt%) sucrose solution protocol has a more concentrated band of G protein and a greater amount, indicating that the 60% (wt%) sucrose solution protocol forms a sucrose gradient that is more favorable for the enrichment of rabies virus particles.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A virus density gradient centrifugation purification device, characterized in that the device comprises a loop-shaped pipeline and a continuous flow density gradient centrifuge, wherein the continuous flow density gradient centrifuge comprises a continuous flow density gradient centrifuge rotor (1);

a first pipe orifice (2), a third pipe orifice (3), a second pipe orifice (4) and a fourth pipe orifice (5) are sequentially arranged on the annular pipe, a first valve (6) is arranged on the pipe between the first pipe orifice and the third pipe orifice, a second valve (7) is arranged on the pipe between the third pipe orifice and the second pipe orifice, a third valve (8) is arranged on the pipe between the second pipe orifice and the fourth pipe orifice, and a fourth valve (9) is arranged on the pipe between the fourth pipe orifice and the first pipe orifice;

the first pipe orifice is connected with the upper end of the continuous flow density gradient centrifugal rotor through a first pipeline (10), the second pipe orifice is connected with the lower end of the continuous flow density gradient centrifugal rotor through a second pipeline (11), the third pipe orifice is connected with a first bottle body (13) through a third pipeline (12), and the fourth pipe orifice is connected with a second bottle body (15) through a fourth pipeline (14);

a fifth valve (16) is arranged on the first pipeline, and a sixth valve (17) is arranged on the second pipeline;

and a liquid conveying device (18) is arranged on the third pipeline.

2. The apparatus of claim 1 wherein said annular conduit is a diamond shaped conduit.

In another preferred example, the first nozzle (2), the third nozzle (3), the second nozzle (4) and the fourth nozzle (5) are respectively positioned at four diamond corners of the diamond pipeline.

3. The device of claim 1, wherein the liquid delivery device is a bi-directional delivery device.

4. Use of a virus density gradient centrifugation purification device according to claim 1 for purification of rabies virus containing material by density gradient centrifugation.

5. A rabies virus density gradient centrifugal purification method is characterized by comprising the following steps:

wherein F2 is 90-240 ml/min;

6. The method of claim 5, wherein V1/V0 is 0.4-0.6.

7. The method of claim 5, wherein the method is performed using the virus density gradient centrifugation purification device of claim 1.

8. The method of claim 7, wherein said method comprises the steps of:

1. the rabies virus-containing material;

2. preparing a sucrose density gradient;

2.1, when the continuous flow density gradient centrifugal rotor (1) is at 0rpm, adding a buffer solution into a first bottle body (13), closing a first valve (6) and a third valve (8), starting a conveying device (18), and enabling the buffer solution to flow out to a second bottle body (15) through a third pipeline (12), a pipeline between a third pipe orifice (3) and a second pipe orifice (4), a second pipeline (11), the continuous flow density gradient centrifugal rotor (1), a first pipeline (10), a pipeline between a first pipe orifice (2) and a fourth pipe orifice (5), and a fourth pipeline (14) in sequence to enable the buffer solution to fill the continuous flow density gradient centrifugal rotor (1) and the whole pipeline system;

2.2, opening the first valve (6) and the third valve (8), closing the fifth valve (16) and the sixth valve (17), starting the conveying device (18), and allowing the buffer solution in the first bottle body (13) to flow out of the fourth pipeline (14) to the second bottle body (15) through the third pipeline (3), the pipeline between the second pipeline (4) and the fourth pipeline (5), the third pipeline (3) and the pipeline between the first pipeline (2) and the fourth pipeline (5) after passing through the third pipeline (12);

2.3, opening a fifth valve (16) and a sixth valve (17), closing a first valve (6) and a third valve (8), starting a conveying device (18), and allowing the buffer solution to flow out to a second bottle body (15) through a third pipeline (12), a pipeline between a third pipe orifice (3) and a second pipe orifice (4), a second pipeline (11), a continuous flow density gradient centrifugal rotor (1), a first pipeline (10), a pipeline between the first pipe orifice (2) and a fourth pipe orifice (5), and a fourth pipeline (14) in sequence;

2.4, opening the first valve (6) and the third valve (8), closing the second valve (7) and the fourth valve (9), starting the conveying device (18) (peristaltic pump), and allowing the buffer solution to flow out of the second bottle body (15) through the third pipeline (12), the pipeline between the third pipe orifice (3) and the first pipe orifice (2), the first pipeline (10), the continuous flow density gradient centrifugal rotor (1), the second pipeline (11), the pipeline between the second pipe orifice (4) and the fourth pipe orifice (5), and the fourth pipeline (14) in sequence;

2.5, opening a second valve (7) and a fourth valve (9), replacing the slow solution in the first bottle body (13) with 55-65% (wt%) of a sucrose solution, closing a fifth valve (16) and a sixth valve (17), starting a conveying device (18), and enabling the sucrose solution in the first bottle body (13) to sequentially pass through a third pipeline (12), and then respectively pass through a third pipe orifice (3), a pipeline between the second pipe orifice (4) and the fourth pipe orifice (5), a third pipe orifice (3), and a pipeline between the first pipe orifice (2) and the fourth pipe orifice (5), and then flow out of the fourth pipeline (14) to the second bottle body (15);

2.6, opening a fifth valve (16) and a sixth valve (17), closing the first valve (6) and the third valve (8), opening a conveying device (18), and allowing a sucrose solution to enter the continuous flow density gradient centrifugal rotor (1) through a third pipeline (12), a pipeline between a third pipe orifice (3) and a second pipe orifice (4) and a second pipeline (11) in sequence, so that the sucrose solution enters the continuous flow density gradient centrifugal rotor (1) and is in contact with a buffer solution in the rotor (1), wherein the volume of the sucrose solution entering the continuous flow density gradient centrifugal rotor is V1;

2.7, opening the first valve (6) and the third valve (8), closing the sixth valve (17), and performing rotation centrifugation on the continuous flow density gradient centrifugal rotor (1) at the speed of T1 to enable the buffer solution and the sucrose solution in the continuous flow density gradient centrifugal rotor (1) to form a longitudinal density gradient, wherein during the centrifugal rotation of the continuous flow density gradient centrifugal rotor (1), gas dissolved in the buffer solution and the sucrose solution is generated during the rotation of the continuous flow density gradient centrifugal rotor (1), and generated gas bubbles appear at the upper end and the lower end of the continuous flow density gradient centrifugal rotor (1);

2.8, keeping the continuous flow density gradient centrifugal rotor (1) rotating for centrifugation at T1, replacing 55-65% (wt%) of sucrose solution in the first bottle body (13) with buffer solution, closing the fifth valve (16) and the sixth valve (17), opening the conveying device (18), enabling the buffer solution in the first bottle body (13) to sequentially pass through the third pipeline (12), and then respectively pass through the third pipe orifice (3), the pipeline between the second pipe orifice (4) and the fourth pipe orifice (5), the third pipe orifice (3) and the pipeline between the first pipe orifice (2) and the fourth pipe orifice (5), and flow out of the fourth pipeline (14) to the second bottle body (15);

2.9 removing air bubbles at the lower end and the upper end of the continuous flow density gradient centrifugal rotor (1) in sequence;

2.9.1 removal of bubbles from the lower end of a continuous flow density gradient centrifuge rotor (1) by the following specific steps:

keeping the continuous flow density gradient centrifugal rotor (1) rotating and centrifuging at T1, opening a fifth valve (16) and a sixth valve (17), closing a second valve (7) and a fourth valve (9), starting a conveying device (18), and allowing a buffer solution to flow out to a second bottle body (15) through a third pipeline (12), a pipeline between a third pipe orifice (3) and a first pipe orifice (2), a first pipeline (10), the continuous flow density gradient centrifugal rotor (1), a second pipeline (11), a pipeline between a second pipe orifice (4) and a fourth pipe orifice (5), and a fourth pipeline (14) in sequence, so that bubbles at the lower end of the density gradient centrifugal rotor (1) are pushed out and removed;

2.9.2 removal of air bubbles at the upper end of a continuous flow density gradient centrifuge rotor (1) by the following specific steps:

keeping the continuous flow density gradient centrifugal rotor (1) in the rotating centrifugal process at T1, opening a second valve (7) and a fourth valve (9), closing a first valve (6) and a third valve (8), starting a conveying device (18), and allowing a buffer solution to flow out to a second bottle body (15) through a third pipeline (12), a pipeline between a third pipe orifice (3) and a second pipe orifice (4), a second pipeline (11), the continuous flow density gradient centrifugal rotor (1), a first pipeline (10), a pipeline between a first pipe orifice (2) and a fourth pipe orifice (5), and a fourth pipeline (14) in sequence, so that bubbles at the upper end of the density gradient centrifugal rotor (1) are pushed out and removed;

3. sample introduction:

3.1, the centrifugal speed of the continuous flow density gradient centrifugal rotor (1) is increased from T1 to T2, the continuous flow density gradient centrifugal rotor (1) is kept to rotate and centrifuge with T2, the buffer solution in the first bottle body (13) is replaced by the material containing the rabies virus, the first valve (6) and the third valve (8) are opened, the fifth valve (16) and the sixth valve (17) are closed, the conveying device (18) is started, the material containing the rabies virus in the first bottle body (13) sequentially passes through the third pipeline (12), and then respectively passes through the third pipeline (3), the pipeline between the second pipeline (4) and the fourth pipeline (5), the pipeline between the third pipeline (3) and the first pipeline (2) and the fourth pipeline (5), and flows out of the fourth pipeline (14) to the second bottle body (15);

3.2 keeping the continuous flow density gradient centrifugal rotor (1) rotating for centrifugation at T2, opening a fifth valve (16) and a sixth valve (17), closing a first valve (6) and a third valve (8), starting a conveying device (18), enabling the rabies virus-containing material to enter the continuous flow density gradient centrifugal rotor (1) from the lower end of the continuous flow density gradient centrifugal rotor (1) through a third pipeline (12), a pipeline between a third pipe orifice (3) and a second pipe orifice (4) and a second pipeline (11) in sequence, and enabling the rabies virus-containing material to have a sample injection speed of F1;

3.3 keeping the continuous flow density gradient centrifugal rotor (1) rotating for centrifugation at T2, opening the first valve (6) and the third valve (8), closing the fifth valve (16) and the sixth valve (17), replacing the rabies virus-containing material in the first bottle (13) with a buffer solution, starting the conveying device (18), and allowing the buffer solution in the first bottle (13) to sequentially pass through the third pipeline (12), and then respectively pass through the third pipe orifice (3), the pipeline between the second pipe orifice (4) and the fourth pipe orifice (5), the pipeline between the third pipe orifice (3) and the first pipe orifice (2) and the fourth pipe orifice (5), and then flow out of the fourth pipeline (14) to the second bottle (15);

3.4, opening a fifth valve (16) and a sixth valve (17), closing a first valve (6) and a third valve (8), keeping the continuous flow density gradient centrifugal rotor (1) to rotate and centrifuge at T2, and simultaneously, opening a conveying device (18) during the centrifugation process of the continuous flow density gradient centrifugal rotor (1), wherein a buffer solution sequentially passes through a third pipeline (12), a pipeline between a third pipe orifice (3) and a second pipe orifice (4) and a second pipeline (11) and enters the continuous flow density gradient centrifugal rotor (1) from the lower end of the continuous flow density gradient centrifugal rotor (1), the liquid inlet speed of the buffer solution is F2, the continuous flow density gradient centrifugal rotor (1) rotates and centrifuges at T2, and the rabies virus-containing materials are distributed in different longitudinal sucrose gradient layers in the continuous flow density gradient centrifugal rotor (1) due to different molecular weights;

4. collecting a sample:

and closing the sixth valve (17), slowing down the continuous flow density gradient centrifugal rotor 1 to 0 revolution, forming a transverse density gradient by the longitudinal density gradient in the continuous flow density gradient centrifugal rotor 1, and collecting the gradient centrifugal layer rich in the rabies virus so as to obtain the purified rabies virus.

9. A purified rabies virus, prepared by the method of claim 5.

10. Use of the purified rabies virus of claim 9 for the preparation of a vaccine against rabies virus.