Efficient cat herpes virus purification process

Abstract

The invention relates to the technical field of biological viruses, and particularly discloses an efficient cat herpesvirus purification process which comprises the following steps: 1) clarifying virus harvesting liquid; and 2) concentrating, washing and filtering the clarified liquid by using a hollow fiber column. And the virus content of the final sample is not obviously lost, the total protein can be reduced to be less than 80 mu g / ml, and the turbidity is as low as 20.3 NTU. As the cat herpes virus has the characteristics of temperature sensitivity, poor shear force tolerance, similar virions and impure proteins in size, incapability of removing impure proteins through a filtering method and the like, a great challenge is brought to a downstream purification process, and the method provides an efficient cat herpes disease purification process; the method greatly reduces the antigen loss, has the characteristics of low production cost, excellent purification effect and the like, avoids the use of multi-step purification steps such as a chromatographic process, and enables the purification process to be simpler and more efficient.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological viruses, and particularly relates to a high-efficiency feline herpes virus purification process. BACKGROUND

[0002] Feline herpes virus-1 (FHV-1) is the main pathogen causing infectious rhinotracheitis in cats, which can cause acute upper respiratory system diseases in cats. The virus has a wide prevalence, and was first discovered in the United States, and then in Canada, the Netherlands, the United Kingdom and other countries, and has now become a worldwide epidemic. The virus mainly attacks kittens, and the clinical symptoms include increased eye and nasal secretions, sneezing, nasal congestion, oral ulcers, elevated body temperature and a series of upper respiratory symptoms, and complications such as chronic sinusitis and keratoconjunctivitis. After the infected cats recover, the virus is latent in the trigeminal nerve, optic nerve, tonsil, submandibular lymph node, nasal turbinates and oral and nasal secretions, which can cause persistent infection and latent infection, and can also reactivate to cause secondary disease in infected cats, causing difficulties in the diagnosis and prevention of the disease. At present, the prevention of the disease is still mainly based on vaccine immunization.

[0003] In recent years, the technology of the biological product industry has been continuously improved, and the use of suspension culture process to produce antigens has been widely applied. The host cell proteins (HCP) in the culture system and the protein substances contained in the culture medium are significantly increased compared with the adherent culture system. Due to the poor temperature and shear resistance of FHV-1, the large size of the virus particles and other characteristics, in large-scale production, great challenges are caused to the downstream purification process. However, with the continuous improvement of the quality requirements of vaccine products, especially for pet vaccines, each biological product enterprise regards the reduction of the content of impurities in the vaccine as a key technical point to improve the market competitiveness of the product. Therefore, a corresponding process technical solution should be designed to solve the problem.

[0004] There is no report and patent on the purification process of feline herpes virus antigen in the published articles and patents. SUMMARY

[0005] (1) Technical problems solved

[0006] In view of the deficiencies in the prior art, the present application provides a high-efficiency feline herpes virus purification process, which solves the problem that the virus recovery rate is less than 10% caused by the common clarification process due to the large molecular weight and complex structure of FHV-1, and the difficulty of the downstream purification process is increased by 2-4 times due to the increase of impurities caused by suspension culture compared with adherent culture. The existing downstream purification process for adherent culture production of antigens cannot meet the suspension culture

[0007] The demand for large-scale production of FHV-1 and the problem that large protein complexes in the culture system cannot be solved by tangential flow technology.

[0008] (II) Technical solutions

[0009] To achieve the above object, the present application is implemented by the following technical solutions: An efficient feline herpesvirus purification process, the method steps are as follows:

[0010] S1, first, the virus harvest liquid is clarified;

[0011] The virus harvest liquid is treated by continuous flow centrifugation at 9000 rpm, and then clarified by a 0.3-30 μm depth filter;

[0012] S2, then use hollow fiber column to concentrate and wash the clarified liquid; use 500KD-750KD hollow fiber column to concentrate the clarified liquid to the target concentration, then wash the concentrated liquid with wash buffer (0.01 mol / L, pH 7.2-7.4 PBS) 20-40 times, empty the hollow fiber column pipeline, and collect the concentrated liquid;

[0013] Preferably, in step S1,

[0014] The method steps for clarifying treatment using a depth filter with a pore size of 0.3-30 μm are as follows:

[0015] S301, prepare the filter: select a depth filter with a pore size of 0.3-30 μm;

[0016] S302, pretreat the filter: place the test liquid in the upstream container, provide power by peristaltic pump, open the filter exhaust valve to exhaust air in the system, when the gas is exhausted, then close the exhaust valve, place the beaker on the balance and peel off the membrane material and rinse the membrane material according to the instructions;

[0017] S303, filter the virus harvest liquid: start timing when the first drop of liquid is collected, and record the relevant parameters such as upstream pressure, filtration volume, filtration time, turbidity, etc. at appropriate intervals, wherein large particles such as cell debris and protein complexes in the liquid are trapped by the depth filter, and the virus can pass through smoothly.

[0018] Preferably, in step S2, the method steps for concentrating the clarified liquid to the target concentration using a 500KD-750KD hollow fiber column are as follows:

[0019] S401, prepare the equipment: first, make sure that the 500KD-750KD hollow fiber column has been correctly installed and connected to the system, the hollow fiber column has a specific pore size that allows small molecules and solvents to pass through, while the virus is trapped, thereby achieving the effect of concentration;

[0020] S402, concentration operation: the clarified liquid is pumped into the hollow fiber column, and the system shear force is adjusted to be in the range of 2000 S -1 ~ 6000 S -1 The macromolecular substances will be retained in the column, while the small molecules and culture medium, etc. will flow out through the membrane pores, and the target substance will be gradually concentrated in the column to the target concentration with the flow of the liquid. The concentration multiple is 20-40, preferably 30.

[0021] Preferably, in step S2, the concentrated liquid is washed with the washing liquid for 20-40 times, and the specific method steps are as follows:

[0022] S501, preparation of washing buffer: 0.01 mol / L, pH 7.2-7.4 PBS buffer is prepared as the washing buffer, and the concentration meets the experimental requirements;

[0023] S502, washing: the virus washing buffer is slowly pumped into the concentrated liquid, and the concentrated liquid is washed for 20-40 times by controlling the flow rate of 365-1100 ml / min and the transmembrane pressure, so as to ensure that the impurities in the concentrated liquid are effectively removed;

[0024] S503, collection of concentrated liquid: when the impurities in the concentrated liquid meet the requirements and reach the predetermined washing times, the washing is stopped, the hollow fiber column pipeline is emptied, and the concentrated liquid is collected;

[0025] S504, cleaning and maintenance: the hollow fiber column is cleaned to remove the residual substances and maintain its performance.

[0026] Preferably, in step S2, the specific method steps for emptying the hollow fiber column pipeline and collecting the concentrated liquid are as follows:

[0027] S601, stop washing: first, the washing buffer (0.01 mol / L, pH 7.2-7.4 PBS) is used for washing, and the washing times reach the predetermined 20-40 times, then the washing process is stopped;

[0028] S602, collection of sample: the filter outlet is closed, and the sample in the hollow fiber column and the pipeline is emptied into the collection bottle at a speed of 8000 S -1 for 5 minutes, the corresponding valve is opened, and the sample in the hollow fiber column and the pipeline is emptied into the collection bottle;

[0029] S603, pipeline flushing: the residual sample in the pipeline is flushed into the collection bottle with an appropriate amount of washing liquid, and the color and clarity of the sample at the collection port are observed to confirm whether it has been completely flushed, so as to ensure the recovery rate;

[0030] S604, record and detect: during the collection of the concentrated liquid, the volume and time of the collection should be recorded, and the collected concentrated liquid should be preliminarily detected in quality, including determination of its toxic dose, turbidity and residual amount of impure protein;

[0031] S605, close the system and clean up: the used hollow fiber column and pipeline are properly cleaned and disinfected, the whole system is closed, and is properly stored.

[0032] (Three) beneficial effects

[0033] Compared with the prior art, the beneficial effects of the present application are:

[0034] (1) The clarification conditions of the FHV-1 suspension culture virus liquid are optimized and improved, and the optimized method can reduce the turbidity of the feed liquid by more than 95%, reduce the pressure of the subsequent hollow fiber concentration and purification system, and improve the system load;

[0035] (2) The concentration and purification conditions of the FHV-1 suspension culture virus liquid are optimized, the system flux is significantly increased, the washing volume is significantly reduced, the process time is effectively reduced, and the production cost is reduced;

[0036] (3) Avoid using chromatography, which can minimize the risk of FHV-1 in large-scale production, improve the purification efficiency, and through the previous process optimization, the virus recovery rate is improved while the impure protein is controlled below 80 μg / ml, and the turbidity is as low as 20.3 NTU, which is a more simple and efficient purification process. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The figure is a schematic diagram of the connection of the purification equipment. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. The following embodiments are used to illustrate the present application, but not to limit the scope of the present application. If the specific technology or condition is not specified in the embodiments, it is carried out according to the technology or condition described in the literature in the art, or according to the product instruction. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel. All other embodiments obtained by ordinary skilled in the art without making creative efforts belong to the scope of protection of the present application.

[0039] The main instruments and equipment in the embodiments of the present application are:

[0040] Continuous flow centrifuge: Model "GQ142G", purchased from Shanghai Junmiao Separation Technology Co., Ltd.; Hollow fiber membrane column: 500KD, 750KD hollow fiber membrane column, purchased from Sibei Pure Trade (Shanghai) Co., Ltd.; Depth filter: Model "SC-03" (pore size 0.3μm-1μm), purchased from Arios Bioscience (Shanghai) Co., Ltd.; Peristaltic pump: Model "BT100-1L", purchased from Baoding Langeng Constant Flow Pump Co., Ltd.

[0041] Purification system connection process:

[0042] Upstream: Virus harvest liquid storage tank → continuous flow centrifuge (inlet pipe diameter 10mm) → harvest liquid after centrifugation;

[0043] Midstream: Harvested liquid after centrifugation → selection of screen mesh filtration or pore size 1μm-1.5μm filter filtration or depth filter filtration, and the hollow fiber column purification (inlet pressure sensor connection) is carried out by selecting the better one;

[0044] Downstream: Hollow fiber column permeate outlet → waste liquid collection tank; Concentrated liquid outlet → filter washing buffer mixing tank → product collection bottle (sterile sampling port)

[0045] Connection requirements: All pipes are made of silica gel material (inner diameter 12.7mm), and clamps are used for sealing at the interface to avoid pressure fluctuations caused by leakage.

[0046] As shown in Figure 1 , the embodiment of the present application provides a technical scheme: an efficient feline herpesvirus purification process, the method steps are as follows:

[0047] S1, first, the virus harvest liquid is clarified;

[0048] The virus harvest liquid is treated by continuous flow centrifugation at 9000rpm, and then clarified by 400 mesh, 500 mesh or 700 mesh screen, pore size 1μm-1.5μm filter or 0.3μm-30μm depth filter;

[0049] S2, then the clarified liquid is concentrated and washed by a hollow fiber column;

[0050] The clarified liquid is concentrated to the target concentration by a 500KD-750KD hollow fiber column, and then the concentrated liquid is washed by a filter washing liquid for 20-40 times, the hollow fiber column pipeline is emptied, and the concentrated liquid is collected;

[0051] S3, finally, the residual amount of impurities in the final sample can be reduced to below 80μg / ml dose.

[0052] In step S1, the method steps for clarifying treatment using 400 mesh, 500 mesh or 700 mesh screen are as follows:

[0053] S101, preparing the screen: cutting the screen to the appropriate size and cleaning it for high-pressure steam sterilization;

[0054] S102, filtering the virus harvest liquid: placing the screen on the funnel and filtering the virus liquid by gravity, collecting the virus liquid;

[0055] The method steps for clarification using a filter with a pore size of 1 μm-1.5 μm are as follows:

[0056] S201, preparing the filter: selecting a filter with a pore size of 1 μm-1.5 μm;

[0057] S202, filtering the virus harvest liquid: loading the test liquid into the upstream container, powered by a peristaltic pump (flow rate 150 LMH), filtering the virus liquid, and collecting the virus liquid;

[0058] The method steps for clarification using a depth filter with a pore size of 0.3 μm-30 μm are as follows:

[0059] S301, preparing the filter: selecting a depth filter with a pore size of 0.3 μm-30 μm;

[0060] S302, pre-treating the filter: loading the test liquid into the upstream container, powered by a peristaltic pump, opening the filter air exhaust valve to exhaust the air in the system, and when the gas is exhausted, then closing the air exhaust valve, placing the beaker on the balance and peeling and wetting the membrane according to the instructions;

[0061] S303, filtering the virus harvest liquid: starting the timer when the first drop of liquid is collected, and finally recording the relevant parameters such as upstream pressure, filtration volume, filtration time, turbidity, etc. at appropriate intervals, wherein the impurities such as cell debris and protein complexes in the liquid are intercepted by the depth filter, and the virus can pass through smoothly.

[0062] The method steps for concentrating the clarified liquid to the target concentration using a 500 KD, 750 KD hollow fiber column in step S2 are as follows:

[0063] S401, preparing the equipment: first, it is necessary to ensure that the 500 KD, 750 KD hollow fiber column has been correctly installed and connected to the system, the hollow fiber column has a specific pore size that allows small molecules and solvents to pass through, while the virus is intercepted, thereby achieving the effect of concentration;

[0064] S402, concentration operation: pumping the clarified liquid into the hollow fiber column, adjusting the system shear force to 2000 S -1 -6000 S -1In the process, as the liquid flows, macromolecular substances will be trapped in the column, while small molecules and culture medium, etc. will flow out through the membrane pores, which will result in the target substances being gradually concentrated in the column to the target concentration.

[0065] In step S2, the concentrated solution is washed with a washing buffer for 20-40 times, and the specific method steps are as follows:

[0066] S501, preparing a washing buffer: 0.01 mol / L, pH 7.2-7.4 PBS buffer is configured as the washing buffer, and the concentration thereof is required to meet the experimental requirements;

[0067] S502, washing: the washing buffer (0.01 mol / L, pH 7.2-7.4 PBS) is slowly pumped into the concentrated solution, and the concentrated solution is washed for 20-40 times by controlling the flow rate and transmembrane pressure, so as to ensure that the impurities in the concentrated solution are effectively removed;

[0068] S503, collecting the concentrated solution: when the impurities in the concentrated solution meet the requirements and reach the predetermined number of washing times, the washing is stopped, the hollow fiber column pipeline is emptied, and the concentrated solution is collected;

[0069] S504, cleaning and maintenance: the hollow fiber column is cleaned to remove residual substances and maintain its performance.

[0070] Through preliminary experiments and combined with the research progress at home and abroad, 0.01 mol / L, pH 7.2-7.4 PBS buffer is selected as the washing buffer, because its ion concentration, ion type, pH range and other physicochemical parameters are close to human body fluids, and it is more stable for most viruses.

[0071] And in large-scale production, its configuration is simpler, the batch difference is smaller, and because

[0072] FHV-1 is difficult to sterilize, and this buffer is simple to sterilize and has a low contamination rate.

[0073] In step S2, the specific method steps for emptying the hollow fiber column pipeline and collecting the concentrated solution are as follows:

[0074] S601, stopping washing: first, the washing process is stopped after the number of washing times using the washing buffer (0.01 mol / L, pH 7.2-7.4 PBS) reaches the predetermined 20-40 times;

[0075] S602, collecting the sample: the filter outlet is closed, and the 8000S -1 speed is cycled for 5 minutes, the corresponding valve is opened, and the sample in the hollow fiber column and the pipeline is emptied into the collection bottle;

[0076] S603, flushing the pipeline: using the appropriate amount of washing filtrate to flush the residual sample in the pipeline to the collection bottle, pay attention to observe the color and clarity of the sample at the collection port, which is used to confirm whether it has been completely flushed clean to ensure the recovery rate;

[0077] S604, recording and detection: during the collection of the concentrated liquid, the volume and time of collection should be recorded, and the collected concentrated liquid should be preliminarily detected for quality, including determination of its toxic dose, turbidity and residual amount of impure protein;

[0078] S605, closing the system and cleaning: the used hollow fiber column and pipeline are properly cleaned and disinfected, the entire system is closed, and is properly stored.

[0079] Example 1: cat herpes virus purification method and its effect

[0080] S1, first, the virus harvest liquid is clarified;

[0081] The FHV-1 virus harvest liquid is treated by continuous flow centrifugation at 9000 rpm to obtain a pre-filtered feed liquid, which is clarified using a 400-mesh, 500-mesh, 700-mesh screen, a filter with a pore size of 1 μm and 1.5 μm, and a depth filter with a pore size of 0.3 μm to 30 μm;

[0082] Among them, the method steps for clarifying using a 400-mesh, 500-mesh, 700-mesh screen are as follows:

[0083] S101, prepare the screen: cut the screen to the appropriate size and clean it for high-pressure steam sterilization;

[0084] S102, filter the virus harvest liquid: place the screen on the funnel, filter the virus feed liquid by gravity, collect the virus feed liquid, and obtain samples filtered by 400-mesh, 500-mesh, and 700-mesh screens, respectively, labeled as SW-400, SW-500, and SW-700;

[0085] The method steps for clarifying using a filter with a pore size of 1 μm and 1.5 μm are as follows:

[0086] S201, prepare the filter: select a filter with a pore size of 1 μm and 1.5 μm;

[0087] S202, filter the virus harvest liquid: load the test feed liquid into the upstream container, provide power by peristaltic pump (flow rate 150 LMH), filter the virus feed liquid, collect the virus feed liquid, and label them as PP-1.0 (pore size 1 um) and PP-1.5 (pore size 1.5 um), respectively;

[0088] The method steps for clarifying using a depth filter with a pore size of 0.3 μm to 30 μm are as follows:

[0089] S301, preparing filter: selecting deep filter with pore size in the range of 0.3 μm to 30 μm;

[0090] S302, pre-treating filter: loading test liquid into upstream container, powered by peristaltic pump (flow rate 150 LMH), opening filter air exhaust valve to exhaust air in the system, when the air is exhausted, then closing the air exhaust valve, placing the beaker on the balance and peeling and washing the membrane material according to the instructions;

[0091] S303, filtering virus harvest liquid: starting timing when the first drop of liquid is collected, recording relevant parameters such as upstream pressure, filtration volume, filtration time, turbidity, etc. at appropriate intervals, wherein large particles such as cell debris and protein complexes in the liquid are intercepted by the deep filter, and the virus can pass through smoothly. The samples filtered by the corresponding type of deep filter are respectively marked as SC-81, SC-12, SC-36, SC-05, SC-03, and SC-04.

[0092] S2, then using a hollow fiber column to concentrate and wash the clarified liquid;

[0093] Concentrating the clarified liquid to the target concentration using a 500KD or 750KD hollow fiber column, then washing the concentrated liquid 20 to 40 times with a washing liquid, emptying the hollow fiber column pipeline, and collecting the concentrated liquid;

[0094] The method steps for concentrating the clarified liquid to the target concentration using a 500KD or 750KD hollow fiber column are as follows:

[0095] S401, preparing equipment: first, it is necessary to ensure that the 500KD or 750KD hollow fiber column has been correctly installed and connected to the system, the hollow fiber column has a specific pore size that allows small molecules and solvents to pass through, while the virus is intercepted, thereby achieving the effect of concentration;

[0096] S402, concentration operation: pumping the clarified liquid into the hollow fiber column, adjusting the shear force of the system to 2000 S -1 , 4000 S -1 or 6000 S -1 , as the liquid flows, macromolecular substances will be intercepted in the column, while small molecules and culture medium, etc. will flow out through the membrane pores, this process will cause the target substance to be gradually concentrated to the target concentration in the column.

[0097] Further improvement, in step S2, the concentrated liquid is washed 20 to 40 times with a virus washing buffer (0.01 mol / L, pH 7.2 to 7.4 PBS).

[0098] S501, prepare the washing buffer: prepare 0.01 mol / L, pH 7.2-7.4 PBS buffer as the washing buffer, and the concentration should meet the experimental requirements;

[0099] S502, perform the washing: slowly pump the virus preservation solution into the concentrated solution, and perform the washing for 20-40 times by controlling the flow rate at 365, 730 or 1095 ml / min and the transmembrane pressure at 5 psi, so as to ensure that the impurities in the concentrated solution are effectively removed;

[0100] S503, collect the concentrated solution: when the impurities in the concentrated solution meet the requirements and reach the predetermined washing times, stop the washing, empty the hollow fiber column pipeline, and collect the concentrated solution;

[0101] S504, clean and maintain: clean the hollow fiber column to remove the residual substances and maintain the performance.

[0102] Through the preliminary experiment, 0.01 mol / L, pH 7.2-7.4 PBS buffer is selected as the washing buffer, because the physicochemical parameters such as ion concentration, ion type and pH range are close to the body fluid of the organism, and most viruses are more stable.

[0103] And in large-scale production, its configuration is simpler, the batch difference is smaller, and because

[0104] FHV-1 is difficult to filter, and the buffer is simple to filter bacteria, and the pollution rate is low.

[0105] In step S2, the specific method steps of emptying the hollow fiber column pipeline and collecting the concentrated solution are as follows:

[0106] S601, stop the washing: first, stop the washing process when the number of times of washing using the virus preservation solution reaches the predetermined 20-40 times;

[0107] S602, collect the sample: close the filter outlet and use 8000S -1 Speed cycle for 5 minutes, open the corresponding valve, empty the sample in the hollow fiber column and the pipeline to the collection bottle;

[0108] S603, flush the pipeline: flush the residual sample in the pipeline to the collection bottle with an appropriate amount of washing solution, and observe the color and clarity of the sample at the collection port to confirm whether it has been completely flushed, so as to ensure the recovery rate;

[0109] S604, record and detect: during the collection of the concentrated solution, the volume and time of collection should be recorded, and the collected concentrated solution should be preliminarily detected for quality, including determination of the titer, turbidity and residual amount of impurities;

[0110] S605, close the system and clean up: clean and disinfect the used hollow fiber column and pipeline, close the whole system, and store properly.

[0111] Finally, the residual amount of impurities in the final sample can be reduced to below 80 μg / ml of the dose.

[0112] The results are shown in Table 1, which shows that the use of screens and PP filter cartridges (pore size 1 μm and 1.5 μm filters) does not significantly reduce the antigen titer and the clarification effect is still not ideal; the use of deep filter SC-03 has a slight loss of titer, but its clarification effect is significant. There is no significant difference in the removal rate of impurities in the feed liquid treated by the clarification process.

[0113] Table 1. Virus titer and impurity removal in the clarification process

[0114]

[0115] Example 2, Optimization of the feline herpesvirus purification method

[0116] I. Effect of hollow fiber column on virus titer and impurity removal in the concentration and purification process

[0117] According to the method of Example 1, wherein the concentration and purification conditions are: 500KD or 750KD hollow fiber column, and other conditions are as shown in Example 1.

[0118] The results are shown in Table 2, which shows that the use of 500KDa and 750KDa does not significantly reduce the virus titer and the purification recovery rate is ideal, the use of 750KDa has a better impurity removal rate and shorter purification process time, so the subsequent purification uses 750KDa hollow fiber for purification.

[0119] Table 2. Virus titer and impurity removal in the concentration and purification process using 500KD and 750KD hollow fiber columns

[0120]

[0121] II. Effect of shear force of hollow fiber system on virus titer and impurity removal in the concentration and purification process

[0122] According to the method of Example 1, wherein the system uses 2000S -1 , 4000S -1 or 6000S -1 shear force, and other conditions are as shown in Example 1.

[0123] The results are shown in Table 3, which shows that the use of different shear forces has little effect on total protein, but only 2000S -1 has the most ideal antigen recovery rate, so 2000S -1Purification was performed. Table 3. Hollow fiber system with 2000S -1 , 4000S -1 or 6000S -1 Effect of shear concentration purification process virus titer and impurity removal

[0124]

[0125] Three, hollow fiber system washing filter multiple concentration purification process virus titer and impurity removal

[0126] According to the method of example 1, wherein the system uses 20-40 times washing and filtering concentration, and other conditions are shown in example 1.

[0127] The results are shown in table 4, the results show that with the increase of washing and filtering multiple, the total protein has a downward trend, but 30 times and 40 times washing and filtering has little difference in impurity removal, so the subsequent use of 30 times washing and filtering scheme.

[0128] Table 4. Hollow fiber system with 20-40 times washing and filtering concentration purification process virus titer and impurity removal

[0129]

[0130] According to the method of example 1, wherein the system uses SC-03 and PP-1.0 for subsequent experiments, and other conditions are shown in example 1.

[0131] The results are shown in table 5, the results show that using SC-03 has no significant change in virus titer compared with PP-1.0, but the total protein of hollow fiber and the effect of turbidity depth filter after purification are better.

[0132] Table 5. Hollow fiber system with 20-40 times washing and filtering concentration purification process virus titer and impurity removal

[0133]

[0134] Through the above optimization of purification process, the total protein of virus liquid can be reduced to below 80 μg / ml, and the turbidity is as low as 20.3 NTU. The optimal conditions are selected as depth filter SC-03, hollow fiber pore size 750 KDa, shear force 2000 -1 , and washing and filtering multiple 30 times. Because feline herpes virus has poor temperature and shear resistance compared with other viruses, it cannot be sterilized and filtered, which brings great challenge to the downstream purification process. This method is a high-efficiency purification process for feline herpes virus produced by suspension culture, which has the characteristics of low production cost and excellent purification effect, greatly reduces the risk of aseptic risk of feline herpes virus which cannot be sterilized and filtered, avoids the use of chromatography process, and makes the purification process simpler and more efficient.

[0135] The foregoing merely illustrates the principles of the application and application of its more particular aspects and embodiments. This description and the examples are not intended to limit the scope of the application to these precise forms. Many variations are possible with the intended scope of the application. While some embodiments of the application have been described, these have been presented by way of example only and are not intended to limit the scope of the application. Any combination of the recited features can be used in accordance with the teaching of this application without departing from the scope of the application. Any reference signs in the claims should not be construed as limiting the scope of the claims.

[0136] Furthermore, it should be understood that although the description has been set forth in the context of certain embodiments, the description is not intended to be limited to those embodiments alone. The description is intended to be illustrative, and not restrictive. The scope of the application is defined by the appended claims, rather than the description and the examples are intended as illustrative only and are not intended to limit the scope of the application.

Claims

1. A highly efficient feline herpesvirus purification process, characterized in that, The process steps are as follows: S1, the virus harvest fluid is clarified; The virus harvest fluid is treated by continuous flow centrifugation at 9000 rpm, and then clarified by a depth filter with a pore size of 0.3-30 μm to obtain a clarified liquid; S2, the clarified liquid is concentrated and washed by a hollow fiber column; The clarified liquid obtained in step S1 is concentrated to a target concentration by a 500KD-750KD hollow fiber column, and then washed 20-40 times with a washing buffer to obtain a concentrated liquid, wherein the washing buffer is PBS with a concentration of 0.01 mol / L and a pH of 7.2-7.

4.

2. The feline herpesvirus purification process according to claim 1, characterized in that: In step S1, the virus harvest fluid is treated by continuous flow centrifugation at 9000 rpm, and then clarified by a depth filter with a pore size of 0.3-30 μm, and the steps are as follows: S301, prepare the filter: select a depth filter with a pore size of 0.3-30 μm; S302, pretreat the filter: place the virus liquid obtained in S102 in an upstream container, provide power by a peristaltic pump, open the filter exhaust valve to exhaust air in the system, close the exhaust valve after the air is exhausted, place the beaker on the balance and remove the skin and rinse the membrane material; S303, filter the virus harvest fluid: large particles such as cell debris and protein complexes in the liquid are trapped by the depth filter, and the virus can pass through smoothly.

3. The feline herpesvirus purification process according to claim 1, characterized in that: In step S2, the clarified liquid obtained in step S1 is concentrated to a target concentration by a 500KD-750KD hollow fiber column, and the steps are as follows: S401, prepare the equipment: connect the 500KD-750KD hollow fiber column with the depth filter in step S1; S402, concentration operation: the clarified liquid is pumped into the hollow fiber column, and the system shear force is adjusted to 2000 s -1 -6000 s -1 With the flow of the liquid, macromolecular substances will be intercepted in the column, while small molecules and culture medium, etc. will flow out through the membrane holes. This process will cause the target substance to be gradually concentrated in the column to the target concentration. The concentration multiple is 20-40 times.

4. The feline herpesvirus purification process according to claim 3, characterized in that: In step S2, the concentrated liquid is washed 20-40 times with a washing buffer, and the specific steps are as follows: S501, prepare the washing buffer: prepare PBS buffer with a concentration of 0.01 mol / L and a pH of 7.2-7.4 as the washing buffer; S502, perform washing: slowly pump the washing buffer into the concentrated antigen liquid, and wash the concentrated liquid 20-40 times by controlling the flow rate of 365-1100 ml / min and the transmembrane pressure to ensure that the impurities in the concentrated liquid are effectively removed; the washing buffer is PBS with a concentration of 0.01 mol / L and a pH of 7.2-7.4; S503, collect the concentrated liquid: when the impurities in the concentrated liquid meet the requirements and reach the predetermined number of washing times, stop the washing, empty the hollow fiber column pipeline, and collect the concentrated liquid; S504, clean and maintain: clean the hollow fiber column to remove residual substances and maintain its performance.

5. The feline herpesvirus purification process according to claim 4, characterized in that: In step S2, the specific steps for emptying the hollow fiber column pipeline and collecting the concentrated liquid are as follows: S601, stop washing: first, stop the washing process when the number of washing times using the washing buffer reaches the predetermined 20-40 times; the washing buffer is PBS with a concentration of 0.01 mol / L and a pH of 7.2-7.4; S602, collect sample: close the filter outlet and use 8000S -1 Speed cycle 5 minutes, open the corresponding valve, empty the sample in the hollow fiber column and the pipeline into the collection bottle; S603, flushing pipeline: use appropriate amount of washing filtrate to flush the residual sample in the pipeline to the collection bottle, pay attention to observe the color and clarity of the sample at the collection port, which is used to confirm whether it has been completely flushed clean to ensure the recovery rate; S604, record and detect: during the collection of the concentrated liquid, the volume and time of collection should be recorded, and the collected concentrated liquid should be preliminarily detected in quality, including determination of its toxic dose, turbidity and residual amount of impure protein; S605, close the system and clean up: properly clean and disinfect the used hollow fiber column, membrane bag and pipeline, close the whole system, and save.

6. The purification process of claim 1, wherein, The pore size of the deep filter is 0.3-1 μm.

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