Method for purifying inactivated rabies virus vaccine

Abstract

The invention belongs to the technical field of rabies vaccines, and particularly relates to a purification method of a rabies virus inactivated vaccine, which comprises the following steps: step 1, carrying out passage by using a Walvax-2 cell strain through a cell factory method, and carrying out multiplication culture on the passage production cells in a microcarrier bioreactor; 2, cleaning the cells with a treating fluid, inoculating the cells with a rabies virus fixed strain, and preparing and culturing a rabies virus solution; step 3, carrying out ultrafiltration concentration on the rabies virus stock solution; step 4, purifying the rabies virus concentrated solution; step 5, inactivating the rabies virus; and 6, hydrolyzing to obtain the rabies vaccine stock solution. By adopting the composite Capto Core 700 column chromatography, impure proteins can be effectively removed, the recovery rate is high, the quality of the rabies vaccine obtained by adopting a mode of firstly purifying and then adopting beta-propiolactone inactivation is excellent, and the effect of the finally obtained human diploid cell rabies vaccine stock solution is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of rabies vaccines, and particularly relates to a purification method for inactivated rabies virus vaccines. Background Art

[0002] Rabies is a disease that affects mammals, mainly with canids (dogs, wolves, foxes, bush dogs, and jackals), procyonids (raccoons), viverrids (mongoose), mephitids (skunks), and chiropterans (bats) as hosts or carriers. All mammals are susceptible to this disease, and almost all human rabies infections are secondary to animal bites or scratches. Inhalation of the virus, inoculation with inadequately inactivated vaccines, or transplantation of infected corneas, tissues, and organs can also occur. The main host of human rabies in most parts of the world is the dog. Although cats are not hosts of the rabies virus, they are also important virus carriers. Rabies mainly presents as an acute invasive viral encephalitis, which is transmitted through exposure to the saliva of infectious viruses or other routes. The disease can be transmitted from animal to animal or from animal to human. However, most existing processes use subcultured cells for production, and human diploid cell vaccines still have the problems of small batch sizes and poor product quality uniformity. Summary of the Invention

[0003] To solve the problems raised in the above background art, the present invention provides a purification method for inactivated rabies virus vaccines, which has the characteristic of good rabies vaccine effect.

[0004] To achieve the above object, the present invention provides the following technical solution: A purification method for inactivated rabies virus vaccines, including a purification method for inactivated rabies virus vaccines:

[0005] Step 1: Subculture with Walvax-2 cell line by the method of cell factory, and place the subcultured production cells in a microcarrier bioreactor for amplification culture;

[0006] Step 2: Wash the cells with a treatment solution, inoculate a fixed strain of rabies virus on the cells, and prepare a culture of rabies virus solution;

[0007] Step 3: Ultrafilter and concentrate the rabies virus stock solution;

[0008] Step 4: Purify the concentrated rabies virus solution;

[0009] Step 5: Inactivate the rabies virus;

[0010] Step 6: Hydrolyze to obtain the rabies vaccine stock solution.

[0011] Preferably, in Step 3, a 750KD hollow fiber is used to ultrafilter and concentrate the rabies virus stock solution.

[0012] Preferably, in step four, a composite Capto Core 700 column chromatography is used to remove impurity proteins.

[0013] Preferably, in step five, a rabies vaccine is obtained by inactivating with β-propiolactone.

[0014] Preferably, in step two, when the cells are passaged to the 15th generation, the virus titer increases from 3.0 logLD50 / ml at the P1 generation to more than 7.0 logLD50 / ml at the P15 generation.

[0015] Preferably, in step six, a rabies vaccine virus strain sensitive to human diploid cells is obtained, and the titer of the prepared rabies virus solution can reach about 7.5 logLD50 / ml.

[0016] Preferably, in step two, the treatment solution is a sterile PBS solution containing sodium butyrate and protamine sulfate solution.

[0017] Preferably, in step six, after inactivation, the heating temperature is 38 - 40°C for hydrolysis to obtain the rabies vaccine bulk.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] In the present invention, by using a composite Capto Core 700 column chromatography, impurity proteins can be effectively removed with a high recovery rate. The rabies vaccine obtained by first purifying and then inactivating with β-propiolactone has better quality, and finally, the effect of obtaining the rabies vaccine bulk from human diploid cells is good. Detailed implementation manners

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Embodiment

[0022] The present invention provides the following technical solutions: A purification method for an inactivated rabies virus vaccine, including a purification method for an inactivated rabies virus vaccine:

[0023] Step one: Using the cell factory method, passage with the Walvax-2 cell line, and place the passaged production cells in a microcarrier bioreactor for amplification culture;

[0024] Step two: Wash the cells with the treatment solution, inoculate a fixed rabies virus strain on the cells, and prepare and culture a rabies virus solution;

[0025] Step 3: Ultrafiltration and concentration of the rabies virus stock solution;

[0026] Step 4: Purification of the rabies virus concentrate;

[0027] Step 5: Inactivation of the rabies virus;

[0028] Step 6: Hydrolysis to obtain the rabies vaccine stock solution.

[0029] Specifically, in Step 3, 750KD hollow fibers are used for ultrafiltration and concentration of the rabies virus stock solution.

[0030] Specifically, in Step 4, a composite Capto Core 700 column chromatography is used to remove impurity proteins.

[0031] Specifically, in Step 5, the rabies vaccine is obtained by the inactivation method using β-propiolactone.

[0032] Specifically, in Step 2, when the cells are passaged to the 15th generation, the virus titer increases from 3.0 logLD50 / ml at the P1 generation to more than 7.0 logLD50 / ml at the P15 generation.

[0033] Specifically, in Step 6, a rabies vaccine virus strain sensitive to human diploid cells is obtained, and the titer of the prepared rabies virus solution can reach about 7.5 logLD50 / ml.

[0034] Specifically, in Step 2, the treatment solution is a sterile PBS solution containing sodium butyrate and protamine sulfate solution.

[0035] Specifically, in Step 6, after inactivation, the heating temperature is 38 - 40°C, and hydrolysis is carried out to obtain the rabies vaccine stock solution.

[0036] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A purification method for inactivated rabies virus vaccine, characterized in that: It includes a purification method for inactivated rabies virus vaccine: Step 1: Subculture with Walvax-2 cell line by the method of cell factory, and place the subcultured production cells in a microcarrier bioreactor for amplification culture; Step 2: Wash the cells with a treatment solution, inoculate a fixed strain of rabies virus on the cells, and prepare a culture of rabies virus liquid; Step 3: Ultrafilter and concentrate the crude rabies virus liquid; Step 4: Purify the concentrated rabies virus liquid; Step 5: Inactivate the rabies virus; Step 6: Hydrolyze to obtain the crude rabies vaccine liquid.

2. The purification method for inactivated rabies virus vaccine according to claim 1, characterized in that: In step 3, 750KD hollow fiber is used to ultrafilter and concentrate the crude rabies virus liquid.

3. The purification method for inactivated rabies virus vaccine according to claim 1, characterized in that: In step 4, a composite Capto Core 700 column chromatography is used to remove impurity proteins.

4. The purification method for inactivated rabies virus vaccine according to claim 1, characterized in that: In step 5, the rabies vaccine is obtained by the inactivation method with β-propiolactone.

5. The purification method for inactivated rabies virus vaccine according to claim 1, characterized in that: In step 2, when the cells are subcultured to the 15th generation, the virus titer rises from 3.0 logLD50 / ml at the P1 generation to more than 7.0 logLD50 / ml at the P15 generation.

6. The purification method for inactivated rabies virus vaccine according to claim 1, characterized in that: In step 6, a rabies vaccine virus strain sensitive to human diploid cells is obtained, and the titer of the prepared rabies virus liquid can reach about 7.5 logLD50 / ml.

7. The purification method for inactivated rabies virus vaccine according to claim 1, characterized in that: In step 2, the treatment solution is a sterile PBS solution containing sodium butyrate and protamine sulfate solution.

8. The purification method for inactivated rabies virus vaccine according to claim 1, characterized in that: In step 6, after inactivation, the heating temperature is 38 - 40 °C, and hydrolysis is carried out to obtain the crude rabies vaccine liquid.