A method for inactivating viruses in liquid biological products

Abstract

The present invention provides a method for inactivating viruses in liquid biological products. The method uses a virus inactivation device to inactivate viruses in liquid biological products. The virus inactivation device includes a feeding part, a centrifugation part, and a discharging part. The centrifugation part is provided with an ultraviolet lamp. The liquid biological products are continuously fed into the centrifugation part through the feeding part. Under the action of centrifugation, the liquid biological products are thinned to a liquid layer thickness of 0.01 cm to 0.2 cm, and at the same time, the viruses are inactivated by ultraviolet irradiation through the ultraviolet lamp. After the viruses are inactivated, the liquid biological products are continuously discharged from the centrifugation part to the discharging part under the action of centrifugation, and are discharged from the virus inactivation device through the discharging part. The retention time of the liquid biological products in the centrifugation part is controlled to be 5 to 60 s, the irradiation intensity of the ultraviolet lamp is 500 to 10,000 μW / cm<supgt;2< / supgt;, and the irradiation wavelength of the ultraviolet lamp is 254 to 280 nm. The present invention is a continuous method for inactivating viruses in liquid biological products.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus inactivation, and particularly relates to a method for inactivating viruses in liquid biological products. Background Art

[0002] In the biotech industry, with the increasing variety of biological products and the expanding population using them, problems such as the risk of animal-derived virus infection of humans and potential iatrogenic infections have become increasingly prominent. The "Measures for the Administration of Drug Registration" requires that biological products need to add data on the verification of virus inactivation processes. The verification study of virus inactivation / removal should adopt a method that simulates the production process, and a virus inactivation / removal verification study plan related to and reasonable for the actual production process should be designed as much as possible.

[0003] Traditional virus inactivation processes include low pH method, organic solvent / detergent (S / D) method, nanofiltration method, chromatography method, pasteurization method, etc. These methods all have disadvantages to varying degrees, such as high cost, good effect only on lipid-enveloped viruses, genotoxicity, and the optimal inactivation conditions changing with the composition of biological products. Compared with the above methods, ultraviolet disinfection belongs to a pure physical disinfection method, which has the advantages of being simple and convenient, broad-spectrum and highly efficient, without secondary pollution, easy to manage and automate. The principle of ultraviolet disinfection is to use the effect of ultraviolet light on nucleic acids to cause abnormal chemical bonds to form between adjacent pyrimidine molecules of virus DNA or RNA, thereby hindering the replication of DNA or RNA and achieving the inactivation of viruses.

[0004] However, ultraviolet disinfection is only widely used in the disinfection of tap water, space, etc., and its application in the field of biological products is not very common. According to the traditional method, when directly irradiating static biological products with ultraviolet light, since biological products (such as proteins) themselves have an absorption coefficient and will absorb part of the ultraviolet light, the virus inactivation effect of only the surface biological products is better. As the amount of biological products increases, the thickness of the liquid layer receiving ultraviolet irradiation becomes larger, and the virus inactivation effect becomes worse. Even if the stirring operation is increased, it will not bring a particularly large improvement. In addition, the larger the liquid layer thickness, the longer the ultraviolet irradiation time, and the effective substances in the upper-layer biological products will be consumed due to excessive ultraviolet irradiation. Moreover, the above traditional methods are not suitable for the virus inactivation of large-scale biological products in the production process. When developing a virus inactivation method suitable for large-scale biological products in the production process, not only the virus inactivation effect and the degree of loss of biological product activity need to be considered, but also the inactivation efficiency and energy consumption need to be concerned to achieve the purpose of obtaining a better inactivation effect at a lower cost. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a continuous method for inactivating viruses in liquid biological products.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for inactivating viruses in liquid biological products. The liquid biological products do not use nucleic acids as the active ingredient. A virus inactivation device is used to inactivate the viruses in the liquid biological products. The virus inactivation device includes a feeding part, a centrifugal part, and a discharging part. The centrifugal part is provided with an ultraviolet lamp. The liquid biological products are continuously fed into the centrifugal part through the feeding part. Under the centrifugal action, the liquid biological products are thinned to a liquid layer thickness of 0.01 cm to 0.2 cm (for example, 0.01 cm, 0.02 cm, 0.03 cm, 0.04 cm, 0.05 cm, 0.06 cm, 0.07 cm, 0.08 cm, 0.09 cm, 0.1 cm, 0.11 cm, 0.12 cm, 0.13 cm, 0.14 cm, 0.15 cm, 0.16 cm, 0.17 cm, 0.18 cm, 0.19 cm, 0.2 cm). At the same time, the viruses are inactivated by ultraviolet irradiation through the ultraviolet lamp. The liquid biological products after virus inactivation are continuously discharged from the centrifugal part to the discharging part under the centrifugal action and are discharged from the virus inactivation device through the discharging part. The retention time of the liquid biological products in the centrifugal part is controlled to be 5 to 60 s (for example, 5 s, 10 s, 15 s, 20 s, 25 s, 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, 60 s), and the irradiation intensity of the ultraviolet lamp is 500 to 10,000 μW / cm 2 (for example, 500 μW / cm 2 、1000 μW / cm 2 、

[0008] 1500 μW / cm 2 、2000 μW / cm 2 、2500 μW / cm 2 、3000 μW / cm 2 、3500 μW / cm 2 、4000 μW / cm 2 、

[0009] 4500 μW / cm 2 、5000 μW / cm 2 、5500 μW / cm 2 、6000 μW / cm 2 、6500 μW / cm 2 、7000 μW / cm 2 、

[0010] 7500 μW / cm 2 、8000 μW / cm 2 、8500 μW / cm 2 、9000 μW / cm2 , 9500 μW / cm 2 , 10000 μW / cm 2 ), and the irradiation wavelength of the ultraviolet lamp is 254 - 280 nm.

[0011] Preferably, the thickness of the liquid layer is controlled to be 0.02 cm - 0.08 cm, the retention time of the liquid biological product in the centrifugation part is controlled to be 5 - 60 s, and the irradiation intensity of the ultraviolet lamp is 2000 - 2500 μW / cm 2 , and the irradiation wavelength of the ultraviolet lamp is 254 - 280 nm.

[0012] More preferably, the thickness of the liquid layer is controlled to be 0.02 cm - 0.06 cm, the retention time of the liquid biological product in the centrifugation part is controlled to be 10 - 30 s, and the irradiation intensity of the ultraviolet lamp is 2000 - 2500 μW / cm 2 , and the irradiation wavelength of the ultraviolet lamp is 254 - 265 nm.

[0013] According to some embodiments, the discharging part includes a first container, the first container includes a body having a hollow cavity with an open upper end and a baffle cover detachably provided on the body, and a discharging port communicating with the outside is provided at the bottom of the body. The centrifugation part includes a second container, a driving assembly, and an ultraviolet lamp. The second container is arranged inside the first container. The second container is a frustum-shaped hollow structure with an open upper end, and the inner diameter of the second container gradually increases from bottom to top. The inner radius of the bottom of the second container is 10 - 200 cm. The included angle between the inner side wall and the inner bottom surface of the second container is 90° - 135° and does not include 90°. There is a gap between the upper end of the second container and the baffle cover. The driving assembly includes a rotating shaft passing through the bottom of the first container and connected to the second container and capable of driving the second container to rotate around its own axis, and a motor connected to the rotating shaft and used to drive the rotating shaft to rotate. The rotating shaft and the second container are coaxial. The ultraviolet lamp is inserted on the baffle cover and is suspended in the second container. The length direction of the ultraviolet lamp is parallel to the axis direction of the second container, and there is a gap between the ultraviolet lamp and the inner wall of the second container. The feeding part includes a feeding pipe and a peristaltic pump. One end of the feeding pipe passes through the baffle cover and extends into the second container, and the other end of the feeding pipe is communicated with the peristaltic pump to continuously feed the liquid biological product into the second container. Under the centrifugal action, the liquid biological product is thinned on the side wall of the second container, and at the same time, it advances upward from the bottom of the second container and is thrown out from the upper edge of the second container into the first container under the centrifugal action.

[0014] According to some embodiments, the feed pipe includes a first part connected to a peristaltic pump and a funnel-shaped second part connected to the first part. The upper end of the second part is open, and the vertical projection of the lower end opening of the second part on the bottom surface of the second container is close to the center of the bottom surface of the second container.

[0015] According to some embodiments, the bottom of the first container is an inclined plane, and the discharge port is arranged at the lower end of the inclined plane.

[0016] According to some embodiments, the virus inactivation device further includes a console, and a rotation speed adjustment button is provided on the console.

[0017] According to some embodiments, the virus inactivation device further includes a detection device, a collection device, or a collection / detection integrated device connected to the discharge port.

[0018] According to some embodiments, the inactivation method includes the steps of selecting a centrifugation speed and selecting a feed flow rate, specifically as follows:

[0019] Select a centrifugation speed within the range of 200 to 20000 rpm;

[0020] Obtain the surface area of the inner wall of the second container, measure the mass of the liquid bioproduct in the second container when the feed amount and the discharge amount of the second container are equal under the selected centrifugation speed. The measurement method is: under the selected centrifugation speed, introduce the liquid bioproduct into the second container at a constant feed flow rate. When the second container rotates to a constant weight, stop feeding and close the upper opening of the second container to stop discharging at the same time, and measure the mass of the liquid bioproduct in the second container.

[0021] Calculate the liquid layer thickness according to the calculation formula of the liquid layer thickness: liquid layer thickness = mass of the liquid bioproduct in the second container ÷ surface area of the inner wall of the second container. Test the liquid layer thickness at different feed flow rates, and construct the functional relationship between the feed flow rate and the liquid layer thickness.

[0022] Calculate the retention time according to the calculation formula of the retention time: retention time = mass of the liquid bioproduct in the second container ÷ feed flow rate. Test the retention time at different feed flow rates, and construct the functional relationship between the feed flow rate and the retention time.

[0023] According to the functional relationship between the feed flow rate and the liquid layer thickness and the functional relationship between the feed flow rate and the retention time, select a feed flow rate that can make both the liquid layer thickness and the retention time within the controlled range.

[0024] Further, when the inner radius of the bottom of the second container is 10 to 20 cm, select a centrifugation speed within the range of 500 to 1000 rpm.

[0025] Furthermore, when the inner radius of the bottom of the second container is 10 - 15 cm, the centrifugation speed is selected within the range of 600 - 800 rpm.

[0026] According to some specific embodiments, the ratio of the inner radius of the bottom to the inner radius of the top of the second container is 1:(1.05 - 1.2), such as 1:1.05, 1:1.06, 1:1.07, 1:1.08, 1:1.09, 1:1.10, 1:1.11, 1:1.12, 1:1.13, 1:1.14, 1:1.15, 1:1.16, 1:1.17, 1:1.18, 1:1.19, 1:1.20.

[0027] According to some specific embodiments, the power of the ultraviolet lamp is 15 - 30 W, such as 15 W, 16 W, 17 W, 18 W, 19 W, 20 W, 21 W, 22 W, 23 W, 24 W, 25 W, 26 W, 27 W, 28 W, 29 W, 30 W.

[0028] According to some specific and preferred embodiments, the inner radius of the bottom of the second container is 10 - 11.5 cm, the inner radius of the top is 11.8 - 13 cm, and the height is 18 - 20 cm.

[0029] Further preferably, the feeding speed of the liquid biological product is 1 - 5 mL / s, and the rotation speed of the centrifugation is 600 - 800 rpm.

[0030] In the embodiment of the present invention, the active ingredient of the liquid biological product is protein.

[0031] Preferably, the total protein content of the liquid biological product is 0.01 - 50 mg / mL.

[0032] Preferably, the viruses include encephalomyocarditis virus, porcine parvovirus, murine parvovirus, and heterophilic murine virus.

[0033] In the embodiment of the present invention, the liquid biological product is a vaccine preparation, a toxin preparation, a toxoid preparation, an immune serum, a blood product, an immunoglobulin preparation, an antigen preparation, an allergen product, a cytokine preparation, a hormone preparation, an enzyme product, a fermentation broth, a monoclonal antibody preparation, or an in vitro immunodiagnostic product.

[0034] The present invention has the following advantages compared with the prior art:

[0035] The present invention provides a continuous virus inactivation method for liquid biological products. By centrifugation, the feed liquid is thinned to 0.01 cm - 0.2 cm, and at the same time, under the centrifugation, the liquid biological product is controlled at an irradiation intensity of 1500 - 3500 μW / cm2 Irradiate under ultraviolet light for 5 to 60 s, and show unexpectedly better effects than the prior art in terms of balancing the virus inactivation effect and the activity loss of liquid biological products. According to the existing experimental results, for the virus inactivation of liquid biological products with proteins as the active substances, the virus titer reduction amount (log10) ≥ 4 logs and the protein titer retention degree > 80% can be achieved within a relatively short ultraviolet irradiation time, which is applicable to large-scale industrial processing and has lower energy consumption compared with the prior art. Description of the Drawings

[0036] Figure 1 It is a schematic structural diagram of the virus inactivation device used in Examples 1 to 8 and Comparative Examples 4 and 5;

[0037] Figure 2 It is a function relationship diagram of the feed flow rate and the liquid layer thickness;

[0038] Figure 3 It is a function relationship diagram of the feed flow rate and the retention time;

[0039] Figure 4 It is a microscopic observation diagram after inoculating the treated FⅡa solution in Example 1 with ST cells and culturing;

[0040] Figure 5 It is a microscopic observation diagram after inoculating the FⅡa solution to be treated in Example 1 with ST cells and culturing;

[0041] Figure 6 It is a microscopic observation diagram after inoculating the treated FⅡa solution in Example 2 with ST cells and culturing;

[0042] Figure 7 It is a microscopic observation diagram after inoculating the FⅡa solution to be treated in Example 2 with ST cells and culturing;

[0043] Figure 8 It is a microscopic observation diagram after inoculating the treated FⅡa solution in Example 3 with ST cells and culturing;

[0044] Figure 9 It is a microscopic observation diagram after inoculating the FⅡa solution to be treated in Example 3 with ST cells and culturing;

[0045] Figure 10 It is a microscopic observation diagram after inoculating the treated FⅡa solution in Example 4 with ST cells and culturing;

[0046] Figure 11 It is a microscopic observation diagram after inoculating the FⅡa solution to be treated in Example 4 with ST cells and culturing;

[0047] Figure 12Microscopic observation image after inoculating the processed FⅡa solution in Example 5 into ST cells and culturing;

[0048] Figure 13 Microscopic observation image after inoculating the FⅡa solution to be processed in Example 5 into ST cells and culturing;

[0049] Figure 14 Microscopic observation image after inoculating the processed FⅡa solution in Example 6 into ST cells and culturing;

[0050] Figure 15 Microscopic observation image after inoculating the FⅡa solution to be processed in Example 6 into ST cells and culturing;

[0051] Figure 16 Microscopic observation image after inoculating the processed FⅡa solution in Example 7 into ST cells and culturing;

[0052] Figure 17 Microscopic observation image after inoculating the FⅡa solution to be processed in Example 7 into ST cells and culturing;

[0053] Figure 18 Microscopic observation image after inoculating the processed FⅡa solution in Example 8 into ST cells and culturing;

[0054] Figure 19 Microscopic observation image after inoculating the FⅡa solution to be processed in Example 8 into ST cells and culturing;

[0055] Figure 20 Microscopic observation image after inoculating the processed FⅡa solution in Comparative Example 1 into ST cells and culturing;

[0056] Figure 21 Microscopic observation image after inoculating the processed FⅡa solution in Comparative Example 2 into ST cells and culturing;

[0057] Figure 22 Microscopic observation image after inoculating the processed FⅡa solution in Comparative Example 2 into ST cells and culturing;

[0058] Figure 23 Microscopic observation image after inoculating the processed FⅡa solution in Comparative Example 3 into ST cells and culturing;

[0059] Figure 1 Among them, 11, the first container; 111, the main body; 112, the baffle cover; 113, the discharge port; 21, the second container; 22, the driving component; 23, the ultraviolet lamp; 311, the first part; 312, the second part; 32, the peristaltic pump; 4, the console; 5, the integrated collection / detection device. Detailed implementation manners

[0060] In order to develop a virus inactivation method applicable to large-scale biological products in the production process, the inventors of the present application have conducted a large number of studies on the virus inactivation method in combination with existing devices capable of thinning biological products. For example, in order to be able to thin biological products to receive ultraviolet irradiation, Patent CN108524969A discloses a centrifugal device that uses centrifugal force to thin biological products and irradiate them with ultraviolet light, but it cannot achieve continuous inactivation and is not suitable for the inactivation of large-scale biological products in the production process. Patent CN218132575U and Patent CN218132575U also use centrifugal force to thin biological products and irradiate them with ultraviolet light, while achieving continuous inactivation. However, after a large number of virus sterilization experiments on biological products using the devices of Patent CN218132575U and Patent CN218132575U, particularly satisfactory results have not been obtained. Therefore, the inventors of the present application have further adjusted the virus inactivation device and optimized it in combination with the adjustment of virus inactivation conditions, improving the virus inactivation efficiency while enhancing the virus inactivation effect.

[0061] Specifically, the technical solution adopted in the present invention is as follows: A virus inactivation device is used to inactivate the liquid biological product. The virus inactivation device includes a feeding part, a centrifugal part, and a discharging part. The centrifugal part is provided with an ultraviolet lamp. The liquid biological product is continuously introduced into the centrifugal part through the feeding part. Under the centrifugal force, the liquid biological product is thinned to a liquid layer thickness of 0.01 cm to 0.2 cm, and at the same time, it is irradiated with ultraviolet light through the ultraviolet lamp to inactivate the virus. The liquid biological product after virus inactivation is continuously discharged from the centrifugal part to the discharging part under the centrifugal force and discharged from the virus inactivation device through the discharging part. The retention time of the liquid biological product in the centrifugal part is controlled to be 5 to 60 s, and the irradiation intensity of the ultraviolet lamp is 500 to 10,000 μW / cm 2 , and the irradiation wavelength of the ultraviolet lamp is 254 to 280 nm.

[0062] The virus inactivation device adopted includes a feeding part, a centrifugal part, a discharging part, a control console and an integrated collection / detection device. The discharging part includes a first container, and the first container includes a body having a hollow cavity with an open upper end and a baffle cover detachably arranged on the body. A discharging port communicating with the outside is provided at the bottom of the body. The centrifugal part includes a second container, a driving assembly and an ultraviolet lamp. The second container is arranged inside the first container. The second container is a frustum-shaped hollow structure with an open upper end. The inner diameter of the second container gradually increases from bottom to top. The inner bottom radius of the second container is 10 - 200 cm. The included angle between the inner side wall and the inner bottom surface of the second container is 90° - 135° and does not include 90°. There is a gap between the upper end of the second container and the baffle cover. The driving assembly includes a rotating shaft passing through the bottom of the first container and connected to the second container and capable of driving the second container to rotate around its own axis, and a motor connected to the rotating shaft and used to drive the rotating shaft to rotate. The rotating shaft is coaxial with the second container. The ultraviolet lamp is inserted into the baffle cover and suspended in the second container. The length direction of the ultraviolet lamp is parallel to the axis direction of the second container. There is a gap between the ultraviolet lamp and the inner wall of the second container. The feeding part includes a feeding pipe and a peristaltic pump. One end of the feeding pipe passes through the baffle cover and extends into the second container. The other end of the feeding pipe is communicated with the peristaltic pump, and the liquid biological product is continuously introduced into the second container. Under the centrifugal action, the liquid biological product is thinned on the side wall of the second container, and at the same time, it advances upward from the bottom of the second container and is thrown out from the upper edge of the second container into the first container under the centrifugal action. The feeding pipe includes a first part connected to the peristaltic pump and a second part connected to the first part and in a funnel shape. The upper end of the second part is an open end, and the vertical projection of the lower end opening of the second part on the bottom surface of the second container is close to the center of the bottom surface of the second container. The bottom of the first container is an inclined surface, and the discharging port is arranged at the lower end of the inclined surface. A speed regulation button is provided on the control console.

[0063] Before the formal continuous virus sterilization using the virus inactivation device of the present invention, the centrifugation speed and the feed flow rate are first selected as follows: The centrifugation speed is selected within the range of 200 to 20,000 rpm; the surface area of the inner wall of the second container is obtained, and the mass of the liquid biological product in the second container when the feed amount and the discharge amount in the second container are equal under the selected centrifugation speed condition is measured. The measurement method is: under the selected centrifugation speed condition, the liquid biological product is introduced into the second container at a constant feed flow rate. When the second container rotates and its weight remains constant, the feed is stopped and the upper opening of the second container is closed to stop the discharge at the same time. The mass of the liquid biological product in the second container is measured. The liquid layer thickness is calculated according to the calculation formula of the liquid layer thickness: liquid layer thickness = mass of the liquid biological product in the second container ÷ surface area of the inner wall of the second container. The liquid layer thickness under different feed flow rates is tested, and the functional relationship between the feed flow rate and the liquid layer thickness is constructed. The retention time is calculated according to the calculation formula of the retention time: retention time = mass of the liquid biological product in the second container ÷ feed flow rate. The retention time under different feed flow rates is tested, and the functional relationship between the feed flow rate and the retention time is constructed. According to the functional relationship between the feed flow rate and the liquid layer thickness and the functional relationship between the feed flow rate and the retention time, the feed flow rate that can make both the liquid layer thickness and the retention time within the controlled range is selected.

[0064] After a large number of experimental attempts and verifications, when the inner radius of the bottom of the second container is 10 - 20 cm, the centrifugation speed is preferably selected within the range of 500 - 1000 rpm. When the inner radius of the bottom of the second container is 10 - 15 cm, the centrifugation speed is preferably selected within the range of 600 - 800 rpm.

[0065] Some specific embodiments of the present invention show that the virus inactivation method of the present invention has unexpectedly better effects at least for protein products, and can achieve an inactivation effect with a virus titer reduction amount (log10) ≥ 4 logs and a protein potency retention degree > 80% within a relatively short ultraviolet irradiation time. It is suitable for large-scale industrial processing and has lower energy consumption compared with the prior art.

[0066] The present invention will be further described below in conjunction with embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in this industry. The technical features involved in each implementation manner of the present invention can be combined with each other as long as they do not conflict with each other.

[0067] In the following Examples 1 - 8 and Comparative Examples 4 and 5, the virus inactivation device used is as Figure 1 shown, which includes a feed part, a centrifugation part, a discharge part, a control console, and a collection / detection integrated device 5.

[0068] Specifically, the discharging part includes a first container 11. The first container 11 includes a main body 111 with a hollow cavity having an open upper end and a baffle cover 112 detachably arranged on the main body 111. A discharging port 113 communicating with the outside is provided at the bottom of the main body 111. The centrifuging part includes a second container 21, a driving assembly 22 and an ultraviolet lamp 23. The second container 21 is arranged inside the first container 11. The second container 21 is a frustum-shaped hollow structure with an open upper end. The inner diameter of the second container 21 gradually increases from bottom to top. The inner radius of the bottom of the second container 21 is 11 cm, the inner radius of the top of the second container 21 is 12 cm, the height of the second container 21 (the vertical height from the inner bottom surface to the top surface of the second container) is 19 cm, and the slant height is 19.03 cm. There is a gap between the upper end of the second container 21 and the baffle cover 112. The driving assembly 22 includes a rotating shaft passing through the bottom of the first container 11 and connected to the second container 21 and capable of driving the second container 21 to rotate around its own axis, and a motor connected to the rotating shaft and used to drive the rotating shaft to rotate. The rotating shaft is coaxial with the second container 21. The ultraviolet lamp 23 is inserted on the baffle cover 112 and suspended in the second container 21. The length direction of the ultraviolet lamp 22 is parallel to the axis direction of the second container 21. There is a gap between the ultraviolet lamp 22 and the inner wall of the second container 21. The distance between the ultraviolet lamp 22 and the axis of the second container 21 is 6 cm, and the irradiation intensity of the ultraviolet lamp 22 is 3500 μW / cm 2 . The feeding part includes a feeding pipe and a peristaltic pump 32. One end of the feeding pipe passes through the baffle cover 112 and extends into the second container 21, and the other end of the feeding pipe is communicated with the peristaltic pump 32 to continuously feed liquid biological products into the second container 21. Under the centrifugal action, the liquid biological products are thinned on the side wall of the second container 21, and at the same time, they are pushed upward from the bottom of the second container 21 and are thrown out from the upper edge of the second container 21 into the first container 11 under the centrifugal action.

[0069] The feeding pipe includes a first part 311 connected to the peristaltic pump 32 and a funnel-shaped second part 312 connected to the first part 311. The upper end of the second part 312 is open, and the vertical projection of the lower end opening of the second part 312 on the bottom surface of the second container 21 is close to the center of the bottom surface of the second container 21. The bottom of the first container 11 is an inclined plane, and the discharging port 113 is arranged at the lower end of the inclined plane. The console 4 is provided with a speed adjustment button.

[0070] After a large number of experimental attempts and verifications, when using this inactivation device, it is preferably to select the centrifugal speed within the range of 600 - 800 rpm. According to the method described above, the functional relationships between the feeding flow rate and the liquid layer thickness and between the feeding flow rate and the retention time are respectively constructed. For example, when the centrifugal speed is selected as 600 rpm and 800 rpm, the functional relationship between the feeding flow rate and the liquid layer thickness can be seen in Figure 2, the functional relationship between the feed flow rate and the retention time is shown in Figure 3 .

[0071] In the following examples and comparative examples, raw materials or reagents not specifically described are commercially available products commonly used in the art; experimental or testing methods not specifically described are experimental conditions or detection instruments commonly used in the art.

[0072] Example 1

[0073] This example provides a method for inactivating Porcine parvovirus (PPV) in a thrombin (FⅡa) solution, which is as follows:

[0074] (1) Take the FⅡa solution to be treated (self-made), with a total protein content of 10 mg / mL, an initial FⅡa titer of 10 IU / mL, and a PPV virus titer of 7.25 logs.

[0075] (2) Turn on the ultraviolet lamp and balance for 10 min; turn on the centrifuge until the rotation speed is balanced at 800 rpm; according to Figure 2 and Figure 3 select the feed flow rate according to the functional relationship under the condition of 800 rpm. In this example, 4 mL / s is selected, and the liquid layer thickness is 0.053 cm (according to Figure 2 the functional relationship under the condition of 800 rpm), and the ultraviolet irradiation duration is 11.9 s (according to Figure 3 the functional relationship under the condition of 800 rpm).

[0076] (3) Turn on the peristaltic pump, and feed the FⅡa solution to be treated into the centrifugal part at a feed flow rate of 4 mL / s. Under the action of centrifugal force, a thin liquid layer is formed on the inner side wall of the second container and moves upward. The FⅡa solution reaching the top is thrown out from the edge of the inner cylinder into the first container and finally flows out from the discharge port.

[0077] Take the treated FⅡa solution and the FⅡa solution to be treated and inoculate them into ST cells respectively. After culturing for 5 days, observe the cytopathic effect of ST cells under a microscope, and use the Karber method (8 wells / dilution) to calculate the virus titer according to the measurement results, expressed as the 50% tissue culture infective dose (TCID 50 ).

[0078] Figure 4 It shows that the ST cells in the treated FⅡa solution are normal and in good condition, with normal morphology, and no cytopathic effect is observed. Figure 5 It shows that obvious cytopathic changes occur in the ST cells in the FⅡa solution to be treated. The virus titer results show that the decrease in the PPV virus titer is 5.38 logs. After detection, the FⅡa titer of the inactivated FⅡa solution is 84.7% of the initial FⅡa titer.

[0079] Example 2

[0080] This example provides a method for inactivating encephalomyocarditis virus (EMCV) in a thrombin (FⅡa) solution, which is as follows:

[0081] (4) Take the FⅡa solution to be processed (self-made), with a total protein content of 10 mg / mL, an initial titer of FⅡa of 10 IU / mL, and an EMCV virus titer of 8.06 logs.

[0082] (5) Turn on the ultraviolet lamp and balance for 10 min; turn on the centrifuge until the rotational speed is balanced at 800 rpm; select the feed flow rate according to the functional relationship under the 800 rpm condition in Figure 2 and Figure 3 . In this example, 2.5 mL / s is selected, and the liquid layer thickness is 0.039 cm (according to the functional relationship under the 800 rpm condition in Figure 2 ), and the ultraviolet irradiation duration is 17.5 s (according to the functional relationship under the 800 rpm condition in Figure 3 ).

[0083] (6) Turn on the peristaltic pump and feed the FⅡa solution to be processed into the centrifugal part at a feed flow rate of 4 mL / s. Under the action of centrifugal force, a thin liquid layer is formed on the inner side wall of the second container and moves upward. The FⅡa solution reaching the top is thrown out from the edge of the inner cylinder into the first container and finally flows out from the discharge port.

[0084] Take the processed FⅡa solution and the FⅡa solution to be processed and inoculate them into ST cells respectively. After culturing for 5 days, observe the cytopathic effect of ST cells under a microscope, and use the Karber method (8 wells / dilution) to calculate the virus titer according to the measurement results, expressed as the 50% tissue culture infective dose (TCID 50 ).

[0085] Figure 6 It shows that the ST cells in the processed FⅡa solution are normal and in good condition, with normal morphology, and no cytopathic effect is observed. Figure 7 It shows that obvious cytopathic changes occur in the ST cells in the FⅡa solution to be processed. The virus titer results show that the decrease in the EMCV virus titer is 4.38 logs. After detection, the FⅡa titer of the inactivated FⅡa solution is 80.9% of the initial FⅡa titer.

[0086] Example 3

[0087] This embodiment provides another implementation of the method for inactivating Porcine parvovirus (PPV) in thrombin (FⅡa) solution. It is basically the same as Example 1, except that the centrifugation speed is selected as 600 rpm. In this embodiment, the selected feed flow rate is 4 mL / s, and the liquid layer thickness is 0.047 cm (according to Figure 2 the functional relationship under the condition of 600 rpm), and the ultraviolet irradiation duration is 13.9 s (according to Figure 3 the functional relationship under the condition of 600 rpm).

[0088] Figure 8 It shows that the ST cells in the treated FⅡa solution are normal and in good condition, with normal morphology, and no cytopathic effect is observed. Figure 9 It shows that obvious cytopathic changes occur in the ST cells in the FⅡa solution to be treated. The virus titer results show that the decrease in the PPV virus titer is 4.62 logs. It will be detected that the FⅡa titer of the inactivated FⅡa solution is 85.5% of the initial FⅡa titer.

[0089] Example 4

[0090] This embodiment provides another implementation of the method for inactivating Encephalomyocarditisvirus (EMCV) in thrombin (FⅡa) solution. It is basically the same as Example 2, except that the centrifugation speed is selected as 600 rpm. In this embodiment, the selected feed flow rate is 2.5 mL / s, and the liquid layer thickness is 0.064 cm (according to Figure 2 the functional relationship under the condition of 600 rpm), and the ultraviolet irradiation duration is 10.6 s (according to Figure 3 the functional relationship under the condition of 600 rpm).

[0091] Figure 10 It shows that the ST cells in the treated FⅡa solution are normal and in good condition, with normal morphology, and no cytopathic effect is observed. Figure 11 It shows that obvious cytopathic changes occur in the ST cells in the FⅡa solution to be treated. The virus titer results show that the decrease in the PPV virus titer is 4.13 logs. It will be detected that the FⅡa titer of the inactivated FⅡa solution is 83.6% of the initial FⅡa titer.

[0092] Example 5

[0093] This embodiment provides a method for inactivating Porcine parvovirus (PPV) in lysozyme (LZM) solution, which is as follows:

[0094] (7) Take the LZM solution to be processed (self-made), with a total protein content of 10 mg / mL, an initial LZM titer of 400 U / mL, and a PPV virus titer of 7.25 logs.

[0095] (8) Turn on the ultraviolet lamp and equilibrate for 10 min; turn on the centrifuge until the rotational speed is balanced at 800 rpm; select the feed flow rate according to the functional relationship under the condition of 800 rpm in Figure 2 and Figure 3 . In this example, select 4 mL / s, and the liquid layer thickness is 0.053 cm (according to the functional relationship under the condition of 800 rpm in Figure 2 ), and the ultraviolet irradiation duration is 11.9 s (according to the functional relationship under the condition of 800 rpm in Figure 3 ).

[0096] (9) Turn on the peristaltic pump and feed the LZM solution to be processed into the centrifugal part at a feed flow rate of 4 mL / s. Under the action of centrifugal force, a thin liquid layer is formed on the inner side wall of the second container and moves upward. The LZM solution reaching the top is thrown out from the edge of the inner cylinder into the first container and finally flows out from the discharge port.

[0097] Take the processed LZM solution and the LZM solution to be processed and inoculate ST cells respectively. After culturing for 5 days, observe the cytopathic effect of ST cells under a microscope, and use the Karber method (8 wells / dilution) to calculate the virus titer according to the measurement results, expressed as the 50% tissue culture infective dose (TCID 50 ).

[0098] Figure 12 It shows that the ST cells in the processed LZM solution are normal and in good condition, with normal morphology, and no cytopathic effect is observed. Figure 13 It shows that obvious cytopathic changes occur in the ST cells in the LZM solution to be processed. The virus titer results show that the decrease in the PPV virus titer is 5.26 logs. After detection, the LZM titer of the inactivated LZM solution is 93.2% of the initial LZM titer.

[0099] Example 6

[0100] This example provides a method for inactivating encephalomyocarditis virus (EMCV) in a lysozyme (LZM) solution, which is as follows:

[0101] (10) Take the LZM solution to be processed (self-made), with a total protein content of 10 mg / mL, an initial LZM titer of 400 U / mL, and an EMCV virus titer of 8.06 logs.

[0102] (11) Turn on the ultraviolet lamp and balance for 10 min; turn on the centrifuge until the rotational speed is balanced at 800 rpm; select the feed flow rate according to the functional relationship under the condition of 800 rpm in Figure 2 and Figure 3 . In this embodiment, the selected feed flow rate is 2.5 mL / s, and the liquid layer thickness is 0.039 cm (according to the functional relationship under the condition of 800 rpm in Figure 2 ), and the ultraviolet irradiation duration is 17.5 s (according to the functional relationship under the condition of 800 rpm in Figure 3 ).

[0103] (12) Turn on the peristaltic pump and feed the LZM solution to be treated into the centrifugal part at a feed flow rate of 4 mL / s. Under the action of centrifugal force, a thin liquid layer is formed on the inner side wall of the second container and moves upward. The LZM solution reaching the top is thrown out from the edge of the inner cylinder into the first container and finally flows out from the discharge port.

[0104] Take the treated LZM solution and the LZM solution to be treated and inoculate ST cells respectively. After culturing for 5 days, observe the cytopathic effect of ST cells under a microscope. Use the Karber method (8 wells / dilution) to calculate the virus titer according to the measurement results, and express it in the median cell infective dose (TCID 50 ).

[0105] Figure 14 It shows that the ST cells in the treated LZM solution are normal and in good condition, with normal morphology, and no cytopathic effect is observed. Figure 15 It shows that obvious cytopathic changes occur in the ST cells in the LZM solution to be treated. The virus titer results show that the decrease in the EMCV virus titer is 4.25 logs. The LZM titer of the LZM solution after detection and inactivation treatment is 91.6% of the initial LZM titer.

[0106] Example 7

[0107] This embodiment provides another implementation manner of the inactivation method of Porcine parvovirus (PPV) in lysozyme (LZM). It is basically the same as Example 5, except that the selected centrifugal rotational speed is 600 rpm. In this embodiment, the selected feed flow rate is 4 mL / s, and the liquid layer thickness is 0.047 cm (according to the functional relationship under the condition of 600 rpm in Figure 2 ), and the ultraviolet irradiation duration is 13.9 s (according to the functional relationship under the condition of 600 rpm in Figure 3 ).

[0108] Figure 16 It shows that the ST cells in the treated LZM solution are normal and in good condition, with normal morphology, and no cytopathic effect is observed.

[0109] Figure 17 It is shown that obvious lesions appear in ST cells in the LZM solution to be processed. The virus titer results show that the decrease in the PPV virus titer is 4.88 logs. It will be detected that the LZM titer of the inactivated LZM solution is 95.4% of the initial LZM titer.

[0110] Example 8

[0111] This example provides another implementation manner of the inactivation method of encephalomyocarditis virus (EMCV) in lysozyme (LZM). It is basically the same as Example 6, except that the centrifugal speed selected is 600 rpm. The feed flow rate selected in this example is 2.5 mL / s, and the liquid layer thickness is 0.064 cm (according to Figure 2 the functional relationship under the condition of 600 rpm in Figure 3 ), and the ultraviolet irradiation duration is 10.6 s (according to

[0112] Figure 18 It is shown that the ST cells in the processed LZM solution are normal and in good condition, with normal morphology, and no cytopathic effect is observed.

[0113] Figure 19 It is shown that obvious lesions appear in ST cells in the LZM solution to be processed. The virus titer results show that the decrease in the PPV virus titer is 4.08 logs. It will be detected that the LZM titer of the inactivated LZM solution is 92.5% of the initial LZM titer.

[0114] Comparative Example 1

[0115] This comparative example provides another inactivation method of encephalomyocarditis virus (EMCV) in thrombin (FⅡa) solution. This comparative example is basically the same as Example 1, except that the centrifugal device in Patent CN218132575U is used for virus inactivation. The bottom radius of the body of this centrifugal device is 13 cm, the top radius is 17 cm, the height is 20 cm, and the slant height is 20.39 cm. When the rotational speed is 800 rpm and the feed flow rate is 2.5 ml / s, the functional relationship between the feed flow rate and the liquid layer thickness and the functional relationship between the feed flow rate and the retention time of this comparative example are constructed according to the method of the present invention. The liquid layer thickness of the liquid inlet speed of this comparative example is 0.04 cm, and the retention time in the body is 16.9 s. It is measured that only when the FⅡa solution reaches the top layer can it receive a relatively strong average ultraviolet intensity, about 5000 μW / cm 2 , and the actual time of receiving ultraviolet irradiation is significantly lower than the retention time in the body. Figure 20It was shown that obvious lesions appeared in ST cells in the processed FⅡa solution. The virus titer results showed that the decrease in the PPV virus titer was 0.39 logs, and there was almost no inactivation effect.

[0116] This comparative example is only an example of using the centrifugal device of CN218132575U for virus inactivation. During the R & D stage, the operating parameters of this centrifugal device (including feed flow rate, centrifugal speed) were systematically adjusted, and it was impossible to obtain a technical effect equivalent to that of Comparative Example 1 within a retention time of 5 - 60 s. Compared with Example 1, the virus inactivation efficiency of this comparative example is low, the energy consumption is greater, and the cost is higher.

[0117] Comparative Example 2

[0118] This comparative example provides another method for inactivating encephalomyocarditis virus (EMCV) in thrombin (FⅡa) solution. The FⅡa solution to be processed in this comparative example is the same as that in Example 1, except that the plate centrifuge of patent CN219943241U is used for virus inactivation. When the platform radius of this centrifuge is 20 cm and the rotation speed is 300 rpm, when the feed flow rate is 0.5 ml / s, the liquid layer thickness is 0.1 cm, and the ultraviolet irradiation time is 1.2 s, and the ultraviolet intensity reaches 10000 μW / cm 2 , the virus titer results showed that the decrease in the PPV virus titer was 0.77 logs, and there was almost no inactivation effect. Figure 21 It was shown that obvious lesions appeared in ST cells in the processed FⅡa solution.

[0119] This comparative example is only an example of using the plate centrifuge of CN219943241U for virus inactivation. During the R & D stage, the operating parameters of this plate centrifuge (including feed flow rate, centrifugal speed) were systematically adjusted, and a virus inactivation effect equivalent to that of Example 1 could not be obtained.

[0120] Comparative Example 3

[0121] This comparative example provides another method for inactivating encephalomyocarditis virus (EMCV) in thrombin (FⅡa) solution. The FⅡa solution to be processed in this comparative example is the same as that in Example 1. In this comparative example, the method described in the national standard (HJ 2522 - 2012) parallel light ultraviolet radiation instrument is used for ultraviolet irradiation. This method is non - continuous liquid feeding and cannot meet the requirements of large - scale production. It is only used as a comparison reference for virus inactivation effect and protein activity retention. 49 mL of FⅡa solution is placed in a 90 - mm petri dish, and the irradiation area and the bottom area of the dish are 49 cm 2, the liquid layer thickness is 1 cm. However, due to the action of the magnetic stirrer, the bottom liquid can still receive ultraviolet light irradiation, and the ultraviolet irradiation intensity is 2000 μW / cm 2 , and the irradiation time is 15 s. Figure 22 It shows that obvious lesions appear in ST cells in the processed FⅡa solution. The virus titer results show that the decrease in the PPV virus titer is 1.80 logs. It will be detected that the FⅡa titer of the FⅡa solution after inactivation treatment is 77.5% of the initial FⅡa titer.

[0122] Comparative Example 4

[0123] This example provides another implementation of the method for inactivating Porcine parvovirus (PPV) in thrombin (FⅡa) solution. It is basically the same as Example 1, except that the selected feed flow rate in this example is 20 mL / s, the liquid layer thickness is 0.198 cm, and the ultraviolet irradiation duration is 3.16 s. The virus titer results show that the decrease in the PPV virus titer is 2.05 logs, which fails to meet the conventional requirement in the art that the virus reduction amount (log10) ≥ 4 logs, indicating that the ability of this step to remove / inactivate the virus is lower than the effective standard.

[0124] Comparative Example 5

[0125] This example provides another implementation of the method for inactivating Porcine parvovirus (PPV) in thrombin (FⅡa) solution. It is basically the same as Example 1, except that the selected feed flow rate in this example is 0.2 mL / s, the liquid layer thickness is 0.019 cm, and the ultraviolet irradiation duration is 140.2 s. The virus titer results show that the decrease in the PPV virus titer is 5.38 logs, and the FⅡa titer of the FⅡa solution after inactivation treatment is 69.4% of the initial FⅡa titer. For the same treatment volume, the treatment duration of this example is significantly longer than that of Example 1.

[0126] The above has described the present invention in detail. The purpose is to enable those skilled in this field to understand the content of the present invention and implement it. However, it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for inactivating viruses in a liquid biological product, wherein the liquid biological product does not contain nucleic acid as an effective ingredient, characterized in that: The liquid biological product is inactivated by a virus inactivation device, wherein the virus inactivation device comprises a feed part, a centrifugal part and a discharge part. The discharge part comprises a first container, the first container comprises a main body with a hollow cavity opened at the upper end and a baffle cover detachably arranged on the main body, and a discharge port communicating with the outside is arranged at the bottom of the main body. The centrifugal part includes a second container, a driving assembly and an ultraviolet lamp. The second container is arranged in the first container. The second container is a truncated cone-shaped hollow structure with an open upper end. The inner diameter of the second container gradually increases from bottom to top. The inner radius of the bottom of the second container is 10-200 cm. The angle between the inner side wall and the inner bottom surface of the second container is 90°-135° and does not include 90°. There is a gap between the upper end of the second container and the baffle cover. The driving assembly includes a rotating shaft that passes through the bottom of the first container and is connected to the second container and can drive the second container to rotate around its own axis, and a motor connected to the rotating shaft and used to drive the rotating shaft to rotate. The rotating shaft is coaxial with the second container. The ultraviolet lamp is inserted on the baffle cover and suspended in the second container. The length direction of the ultraviolet lamp is parallel to the axis direction of the second container. There is a gap between the ultraviolet lamp and the inner wall of the second container. The feed part includes a feed pipe and a peristaltic pump, one end of the feed pipe passes through the baffle cover and extends into the second container, and the other end of the feed pipe is connected to the peristaltic pump. The liquid biological product is continuously introduced into the second container through the feed part. Under the action of centrifugation, the liquid biological product is diluted on the side wall of the second container to a liquid layer thickness of 0.01 cm to 0.2 cm, and is simultaneously pushed upward from the bottom of the second container, and is thrown out from the upper edge of the second container into the first container under the action of centrifugation, and is discharged from the virus inactivation device through the discharge port. The liquid biological product is irradiated with ultraviolet light by the ultraviolet lamp to inactivate the virus, and the retention time of the liquid biological product in the second container is controlled to be 5 to 60 seconds, and the irradiation intensity of the ultraviolet lamp is 500 to 10000 μW / cm 2 The irradiation wavelength of the ultraviolet lamp is 254~280nm.

2. The method for inactivating viruses in liquid biological products according to claim 1, characterized in that: The thickness of the liquid layer is controlled to be 0.02 cm to 0.08 cm, the retention time of the liquid biological product in the second container is controlled to be 5 to 60 s, and the irradiation intensity of the ultraviolet lamp is controlled to be 2000 to 2500 μW / cm 2 .

3. The method for inactivating viruses in liquid biological products according to claim 1, characterized in that: The inactivation method comprises the steps of selecting a centrifugal speed and selecting a feed flow rate, which are specifically as follows: Select the centrifugal speed within the range of 200~20000rpm; Obtain the surface area of ​​the inner wall of the second container, and measure the mass of the liquid biological product in the second container when the feed amount and the discharge amount of the second container are equal under the selected centrifugal speed condition. The measuring method is: under the selected centrifugal speed condition, the liquid biological product is introduced into the second container at a constant feed flow rate, and when the second container rotates until the weight remains constant, the feeding is stopped and the upper end opening of the second container is closed to stop the discharge, and the mass of the liquid biological product in the second container is measured. The thickness of the liquid layer was calculated according to the formula for calculating the thickness of the liquid layer: Liquid layer thickness = mass of the liquid biological product in the second container ÷ surface area of ​​the inner wall of the second container. The thickness of the liquid layer under different feed flow rates was tested, and a functional relationship between the feed flow rate and the liquid layer thickness was established. The retention time was calculated according to the retention time calculation formula: retention time = mass of the liquid biological product in the second container ÷ feed flow rate. The retention time under different feed flow rates was tested to construct a functional relationship between feed flow rate and retention time. According to the functional relationship between the feed flow rate and the liquid layer thickness and the functional relationship between the feed flow rate and the retention time, a feed flow rate is selected that can make the liquid layer thickness and the retention time fall within the range to be controlled.

4. The method for inactivating viruses in liquid biological products according to claim 1, characterized in that: The ratio of the bottom inner radius to the top inner radius of the second container is 1:(1.05-1.2).

5. The method for inactivating viruses in liquid biological products according to claim 1, characterized in that: The inner radius of the bottom of the second container is 10-11.5 cm, the inner radius of the top is 11.8-13 cm, the height is 18-20 cm, and the power of the ultraviolet lamp is 15-30W.

6. The method for inactivating viruses in liquid biological products according to claim 5, characterized in that: The feed rate of the liquid biological product is 1-5 mL / s, and the rotation speed of the centrifugation is 600-800 rpm.

7. The virus inactivation method according to claim 1 or 2, characterized in that: The active ingredient of the liquid biological product is protein, and / or the total protein content of the liquid biological product is 0.01-50 mg / mL, and / or the virus includes encephalomyocarditis virus, porcine parvovirus, mouse parvovirus, and xenotropic mouse virus.

8. The method for inactivating viruses in liquid biological products according to claim 1 or 2, characterized in that: The liquid biological product is a vaccine preparation, a toxin preparation, a toxoid preparation, an immune serum, a blood product, an immunoglobulin preparation, an antigen preparation, an allergen preparation, a cytokine preparation, a hormone preparation, an enzyme preparation, a fermentation broth, a monoclonal antibody preparation or an in vitro immunodiagnostic product.

9. The virus inactivation method according to claim 1, characterized in that: The feeding tube includes a first part connected to the peristaltic pump and a second part connected to the first part in a funnel shape, the upper end of the second part is open, and the vertical projection of the lower end opening of the second part on the bottom surface of the second container is close to the center of the bottom surface of the second container; The bottom of the first container is an inclined surface, and the discharge port is arranged at the lower end of the inclined surface.

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