Virus inactivation method of liquid biological product

Abstract

The invention provides a virus inactivation method of a liquid biological product, which adopts a virus inactivation device to carry out virus inactivation on the liquid biological product, and the virus inactivation device comprises a box body, a console, an overflowing container arranged in the box body and an ultraviolet lamp group arranged in the box body, a liquid biological product is continuously introduced into the liquid inlet buffer bin through the peristaltic pump, upwards overflows through the liquid inlet buffer bin, enters the temporary liquid storage bin after passing through the plurality of quartz round tubes, and is finally discharged from the second flow guide silicone tube; the liquid biological product is irradiated by the first ultraviolet lamp and the second ultraviolet lamp to inactivate viruses in the process of flowing in the quartz round tubes. According to the invention, the virus inactivation effect can be improved while the biological activity of the liquid biological product is ensured, and continuous and stable virus inactivation of the liquid biological product can be realized. The virus inactivation device is easy to clean and maintain and long in service life, and the virus inactivation method based on the virus inactivation device is more economical, simpler and more convenient and suitable for large-scale treatment of liquid biological products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of virus inactivation, and particularly relates to a virus inactivation method for liquid biological products. BACKGROUND

[0002] Due to the risk of virus contamination in the preparation process of biological products from eukaryotic / prokaryotic cells, tissues, living insects or animals, in order to ensure product safety, manufacturers need to prove that their process equipment can provide sufficient virus removal / inactivation capacity, and enterprises are required to take necessary measures to remove / inactivate viruses for different products.

[0003] The mechanism of virus inactivation includes: 1. Virus capsid / envelope lipid destruction: the envelope contains lipid substances, so viruses with envelopes can be quickly destroyed by lipid solvents, such as diethyl ether, chloroform or sodium deoxycholate, which can inactivate lipid envelope viruses. Physical effects such as osmotic pressure changes, freeze-thaw, heat and drying can cause envelope destruction. 2. Virus protein denaturation: chemical agents that can denature proteins can also denature viral proteins, such as phenol, formaldehyde, hypochlorite, acid and alkali. Heating-induced denaturation is also an effective inactivation method. In general, viruses are weakly resistant to heat, and 60℃ for a few minutes can significantly reduce their infectivity. 3. Damage to viral nucleic acids: ionizing radiation such as X-rays and gamma rays can cut nucleic acids and damage viral gene function. In addition, dyes such as acridine orange or neutral red can bind to viral nucleic acids, and exposure to light can cause nucleic acid degradation and viral inactivation.

[0004] Methods for virus removal include: 1. Virus removal filtration: also known as nanomembrane filtration, using a filter membrane with a pore size of about 20 nm to filter the biological product solution, using the principle of sieving, in nanomembrane filtration, viruses or other pathogens larger than the pore size of the filter membrane are trapped on the membrane, and smaller biological products can pass through the filter membrane and remain in the solution; 2. Chromatography: using the differences in affinity or project action of various components with the stationary phase to separate various components; 3. Fractionated precipitation method: ethanol precipitation method, fractionated precipitation under low temperature environment to remove viruses, and also has the effect of inactivating viruses. Octanoic acid precipitation method, under acidic conditions, octanoic acid can bind to most proteins and precipitate, achieving the purpose of virus removal; 4. S / D method: using an organic solvent / detergent mixture (S / D) to destroy the lipids of lipid envelope viruses; 5. Wet heat / dry heat method: a method of physically inactivating viruses using temperature and humidity; 6. Low pH method: inactivating viruses by lowering the pH value and increasing the temperature.

[0005] The above method will cause damage to protein biological products to some extent, compared with short-time ultraviolet irradiation. Ultraviolet virus inactivation belongs to a physical method, has broad spectrum, and can basically kill all microorganisms. Virus inactivation is fast, only a few seconds are needed, no chemicals need to be added, the sample is not polluted, and the product quality is not affected. The effect of ultraviolet rays on nucleic acid causes abnormal chemical bonds to be formed between adjacent pyrimidine molecules of viral DNA or RNA, thereby hindering the replication of DNA or RNA, so as to achieve virus inactivation. For this purpose, the patent CN220714425U provides a device capable of continuous virus inactivation, but in actual use, it is found that the consistency of the flow rate of the liquid biological product in the device is not good enough, leading to uneven disinfection, and the liquid biological product receives a large resistance when flowing in the device, and there are still problems of protein adhesion and residue after long-time use, and the virus inactivation effect needs to be further improved. In addition, the device also has problems of large cleaning difficulty, small adjustable space of the ultraviolet lamp, and difficult operation.

[0006] Therefore, it is necessary to further improve the device and provide a liquid biological product virus inactivation method capable of improving the virus inactivation effect while ensuring the biological activity of the liquid biological product, continuously and stably inactivating viruses, and being more economical and simple. SUMMARY

[0007] The problem to be solved by the present application is to provide a liquid biological product virus inactivation method capable of improving the virus inactivation effect while ensuring the biological activity of the liquid biological product, continuously and stably inactivating viruses, and being more economical and simple.

[0008] To solve the above technical problems, the present application adopts the following technical solutions:

[0009] The present application provides a liquid biological product virus inactivation method, the effective component of the liquid biological product is protein, a virus inactivation device is used to inactivate viruses of the liquid biological product, the virus inactivation device comprises a box body, a control console, a flow container arranged in the box body, and an ultraviolet lamp group arranged in the box body,

[0010] The inner wall of the box body is provided with a reflective coating,

[0011] The overflow container comprises a first support fixedly arranged in the box, a plurality of cylindrical quartz round tubes arranged on the first support and extending in a direction perpendicular to the horizontal plane, a liquid inlet buffer bin arranged on the first support below the plurality of quartz round tubes and in communication with the lower end openings of the plurality of quartz round tubes, a first flow guide silica gel pipe in communication with the liquid inlet buffer bin, a temporary liquid storage bin arranged above the plurality of quartz round tubes and in communication with the upper end openings of the plurality of quartz round tubes, and a second flow guide silica gel pipe in communication with the temporary liquid storage bin, the plurality of quartz round tubes are arranged in sequence and in close alignment on the same plane, an inner coating is arranged on the inner side wall of each quartz round tube, the material of the inner coating is polyethylene glycol, and a peristaltic pump is arranged on the first flow guide silica gel pipe,

[0012] The ultraviolet lamp group comprises a first ultraviolet lamp group and a second ultraviolet lamp group arranged on opposite sides of the overflow container, the first ultraviolet lamp group comprises a second support slidably arranged in the box and a first ultraviolet lamp arranged on the second support, and the second ultraviolet lamp group comprises a third support slidably arranged in the box and a second ultraviolet lamp arranged on the third support, the first ultraviolet lamp and the second ultraviolet lamp are cylindrical and extend in a direction perpendicular to the horizontal plane, the lengths of the first ultraviolet lamp and the second ultraviolet lamp are greater than or equal to the length of the quartz round tube, the plane on which the first ultraviolet lamp and the second ultraviolet lamp are arranged and the plane on which the plurality of quartz round tubes are arranged are perpendicular to each other and intersect at the central axis of the plurality of quartz round tubes, and the control console is used to control the movement of the second support and the third support to adjust the distance between the first ultraviolet lamp, the second ultraviolet lamp and the plane on which the plurality of quartz round tubes are arranged,

[0013] The liquid biological product is continuously introduced into the liquid inlet buffer bin through the peristaltic pump, flows upwards through the plurality of quartz round tubes from the liquid inlet buffer bin and then enters the temporary liquid storage bin, and finally is discharged from the second flow guide silica gel pipe, and the liquid biological product is irradiated by the first ultraviolet lamp and the second ultraviolet lamp during the flowing process in the plurality of quartz round tubes to inactivate viruses. According to some embodiments of the present application, the lengths of the first ultraviolet lamp and the second ultraviolet lamp are equal to the length of the quartz round tube and are arranged in alignment.

[0014] In the embodiments of the present application, the thickness of the inner coating is 1-50 nm, and is further preferably 10-30 nm.

[0015] In the embodiments of the present application, the liquid inlet buffer bin is in close connection with the lower end openings of the plurality of quartz round tubes, and the connection is sealed by a silica gel pad around the connection. The temporary liquid storage bin is in close connection with the upper end openings of the plurality of quartz round tubes, and the connection is sealed by a silica gel pad around the connection.

[0016] In the embodiment of the present application, the material of each quartz round tube is high-purity JGS1 / JGS2.

[0017] In the embodiment of the present application, an outer coating layer is arranged on the outer sidewall of each quartz round tube, and the material of the outer coating layer is amorphous fluorine-containing polymer.

[0018] In the embodiment of the present application, the thickness of the outer coating layer is 1-50 nm, and is further preferably 10-30 nm.

[0019] In the embodiment of the present application, the inner diameter of each quartz round tube is 0.1-1 cm, the tube wall thickness of each quartz round tube is 0.05-0.5 cm, the thickness of the inner coating layer of each quartz round tube is 1-50 nm, and the length of each quartz round tube is 5-20 cm.

[0020] In the embodiment of the present application, the outer diameter of the first ultraviolet lamp and the second ultraviolet lamp is 1-3 cm respectively, the length of the first ultraviolet lamp and the second ultraviolet lamp is 5-20 cm respectively, and the distance from the first ultraviolet lamp and the second ultraviolet lamp to the plane where the plurality of quartz round tubes are located is 0.5-5 cm respectively.

[0021] In the embodiment of the present application, the power of the first ultraviolet lamp and the second ultraviolet lamp is 5-50 W respectively, and the irradiation wavelength of the first ultraviolet lamp and the second ultraviolet lamp is 250-260 nm.

[0022] In the embodiment of the present application, the number of quartz round tubes is 5-15.

[0023] In actual application, in the embodiment of the present application, the total protein content of the liquid biological product is 0.01-50 mg / mL, and the flow rate of the liquid biological product is controlled by the peristaltic pump to be 0.1-15 mL / s. In actual operation, appropriate quartz round tubes and ultraviolet lamps can be selected according to the required degree of virus inactivation, and the distance between the ultraviolet lamp and the quartz round tube and the flow rate of the liquid biological product can be adjusted at any time.

[0024] In the embodiment of the present application, an ultraviolet lamp tube adjustment reference scale is engraved on the bottom surface of the box body corresponding to the positions of the second support and the third support, so as to facilitate recording the distance between the ultraviolet lamp tube and the quartz round tube after adjusting the position of the ultraviolet lamp each time.

[0025] The viruses that can be inactivated by the present application include, but are not limited to, one or more of the following: Reoviridae, Rhabdoviridae, Orthomyxoviridae, Filoviridae, Coronaviridae, Bunyaviridae, Phenuiviridae, Flaviviridae, Paramyxoviridae, Togaviridae, Arenaviridae, Picornaviridae, Caliciviridae, Retroviridae, Poxviridae, Herpesviridae, Iridoviridae, Papovaviridae, and Parvoviridae.

[0026] According to some embodiments, the virus includes one or more of encephalomyocarditis virus, porcine parvovirus, murine parvovirus, and heterophile mouse virus.

[0027] The liquid biological product to which the present application is applicable includes, but is not limited to, vaccine preparation, toxin preparation, toxoid preparation, immune serum, blood product, immunoglobulin preparation, antigen preparation, allergen preparation, cytokine preparation, hormone preparation, enzyme preparation, fermentation broth, monoclonal antibody preparation, or in vitro immunodiagnostic preparation.

[0028] According to some embodiments of the present application, the liquid biological product is a thrombin solution.

[0029] Specifically, the flow rate of the liquid biological product is controlled by the peristaltic pump to be 0.1-1 mL / s, the liquid biological product flows in the plurality of quartz round tubes and is irradiated by the first and second ultraviolet lamps for 5-20 s, and the liquid biological product receives an ultraviolet dose of 30-40 mJ / cm 2 .

[0030] Further, the plurality of quartz round tubes are 7-11 in number, each quartz tube has an inner diameter of 2-4 mm and a length of 80-90 mm.

[0031] In the embodiment of the present application, the ultraviolet inactivation device further comprises a thermometer, a hygrometer, an ultraviolet detector, and a temperature control dehumidifier arranged in the cabinet and connected with the control console, the control console is provided with a display screen capable of displaying the temperature, humidity, and power of the ultraviolet lamp group in the cabinet, and an ultraviolet lamp switch, a thermometer switch, a hygrometer switch, an ultraviolet detector switch, and a temperature adjusting knob for adjusting the temperature control dehumidifier. The cabinet is further provided with a leakage sensor, and the control console is provided with a leakage sensor alarm lamp.

[0032] In the embodiment of the present application, the cabinet is provided with an openable movable panel which can be used to check the internal structure of the device, adjust or replace the ultraviolet lamp tube, and the like, and is also convenient for continuously feeding the liquid biological product to be inactivated into the first flow guide silica gel tube through an external pipeline, and convenient for connecting the second flow guide silica gel tube to guide the liquid biological product after inactivation of the virus to the next process or a collection bin through another external pipeline.

[0033] The application adopts a new virus inactivation device to inactivate viruses in liquid biological products. The main body container for flowing is a quartz round tube. The liquid biological products have small resistance in the quartz round tube and are not easy to be blocked. The liquid biological products have uniform flow rate in the quartz round tube, and the disinfection is more uniform. The quartz round tube is convenient to clean after being disassembled, and manpower and material resources are saved. The tube wall of the quartz round tube has a transmittance of 92% or more at 254 nm wavelength ultraviolet light, and the ultraviolet light loss is small. The inner wall of the quartz round tube is coated with a transparent high molecular material (low toxicity PEG) with protein adhesion resistance. The transparent high molecular material (low toxicity PEG) with protein adhesion resistance not only does not affect the disinfection effect of ultraviolet light on the liquid biological products, but also enhances the smoothness of the inner wall of the quartz round tube and the flowability of the liquid biological products in the quartz round tube. The transparent high molecular material (low toxicity PEG) with protein adhesion resistance avoids the adhesion of proteins in the liquid biological products to the inner wall of the quartz round tube and the denaturation and coagulation of the proteins on the inner wall of the quartz round tube after being irradiated by ultraviolet light. The transparent high molecular material (low toxicity PEG) with protein adhesion resistance maintains the ultraviolet transmittance stable for a long time, improves the service life of the device, is more economical and environmentally friendly, and also avoids the decrease of the quality of the liquid biological products caused by the adhesion of proteins in the liquid biological products to the inner wall of the quartz round tube. The outer wall of the quartz round tube is coated with a high molecular material coating (Teflon represents an amorphous fluoropolymer) that increases the strength of the tube wall. The high molecular material coating has the advantages of high ultraviolet transmittance and low optical refractive index, does not affect the disinfection effect of ultraviolet light on the biological materials, and has the characteristics of heat resistance, low temperature resistance, stable chemical properties, corrosion resistance and low friction coefficient. To some extent, the high molecular material coating can increase the service life of the quartz round tube, reduce the replacement frequency of consumables, and thus reduce the use cost. The virus inactivation device adopted by the application is also provided with a liquid inlet buffer bin. Even if the liquid inlet speed is fast, the speed of the liquid biological products pushing into the quartz round tube after passing through the liquid inlet buffer bin can be ensured to be consistent, so as to avoid the problems of inconsistent flow rate of biological materials in the quartz round tube, accumulation, pollution and feeding difficulty caused by the adhesion of proteins to the inner wall. In addition, the first ultraviolet lamp and the second ultraviolet lamp in the virus inactivation device adopted by the application are fixed by movable supports, which is convenient and fast to operate. The liquid biological products flowing out of the virus inactivation device of the application can be directly used for downstream production. The main components in the virus inactivation device adopted by the application can be reused, can be disassembled and cleaned, or can be self-cleaned with disinfectant. The method is simple, saves resources and energy, and is suitable for large-scale industrial production.

[0034] Compared with the prior art, the application has the following advantages:

[0035] The application adopts a new virus inactivation device with a new structure to inactivate viruses in liquid biological products. While ensuring the biological activity of the liquid biological products, the virus inactivation effect is improved, and the liquid biological products can be continuously and stably inactivated. The virus inactivation device adopted by the application is easy to clean and maintain, has a long service life, and the virus inactivation method based thereon is more economical and simple, and is suitable for industrialized large-scale production of liquid biological products. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Schematic diagram of the overall structure of the virus inactivation device used in the embodiment (partially in perspective view);

[0037] Figure 2 Schematic diagram of the structure of the flow-through container of the virus inactivation device of Figure 1

[0038] Figure 3 Schematic diagram of the sectional structure of the partial components of the virus inactivation device of Figure 1

[0039] Figure 4 Schematic diagram of the top view structure of the partial components of the virus inactivation device of Figure 1

[0040] Figure 5 Schematic diagram of the partial front view structure inside the box of the virus inactivation device of Figure 1 In the above drawings, 1, box; 2, flow-through container; 21, first support; 22, quartz round tube; 23, liquid inlet buffer bin; 24, first flow guide silica gel tube; 25, temporary liquid storage bin; 26, second flow guide silica gel tube; 31, first ultraviolet lamp group; 311, second support; 312, first ultraviolet lamp; 32, second ultraviolet lamp group; 321, third support; 322, second ultraviolet lamp; 4, control console; 41, ultraviolet lamp switch; 42, thermometer switch; 43, hygrometer switch; 44, ultraviolet detector switch; 45, leakage sensor alarm lamp; 46, temperature adjusting knob; 5, thermometer; 6, hygrometer; 7, ultraviolet detector, 8, temperature control dehumidifier;

[0041]

[0042] The number of quartz round tubes 22 in the embodiment is only schematic and does not represent that the virus inactivation device used in the embodiment is only five quartz round tubes 22, nor does it represent that in other embodiments it can only be five quartz round tubes 22. Figure 2 DETAILED DESCRIPTION The following further describes the present application in conjunction with the embodiments shown in the drawings.

[0043] In the following, only certain exemplary embodiments are described simply. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the embodiments of the present application. Therefore, the drawings and the description are considered to be exemplary in nature rather than limiting.

[0044] In the description of the present application, it should be noted that the positional descriptions such as "upper", "lower", etc. are all according to the drawings.

[0045] Figure 1 ​​​​"inner," "outer" are defined as positions relative to the center of a device or component, with "inner" being closer to the center of the device or component and "outer" being farther from the center of the device or component. These terms are used only to facilitate the description of the application and are not intended to limit or restrict the scope of the application as described.

[0046] The accompanying drawings provided by the present application are only schematic illustrations of the structure of the virus inactivation device, which are convenient for readers to understand, and do not represent a limitation on the structure of the virus inactivation device for biological products.

[0047] In the embodiments of the present application, unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connecting", "fixing" and the like should be understood in a broad sense, for example, can be fixedly connected, or can be detachably connected, or can be integrated. For those skilled in the art, the specific meanings of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0048] In order to simplify the disclosure of the embodiments of the present application, the components and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the embodiments of the present application. The specifications and models of the components can be adjusted according to actual needs.

[0049] The virus inactivation device used in the following embodiments is shown in Figures 1 to 5 It includes a box body 1, a control console, a flow container 2 arranged in the box body 1, a UV lamp group arranged in the box body 1, a thermometer 5, a hygrometer 6, a UV detector 7, a temperature and humidity controller 8 and a leakage sensor.

[0050] Specifically, the inner wall of the box body 1 is provided with a reflective coating for reflecting the ultraviolet light irradiated onto the box body 1 to the flow container 2 to reduce the loss. The box body 1 is provided with an openable movable panel which can be used to check the internal structure of the device, adjust or replace the ultraviolet lamp tube, etc., and also facilitates the introduction of liquid biological products to be inactivated into the flow container 2, and facilitates the introduction of liquid biological products after virus inactivation into the next process or collection bin.

[0051] Specifically, the overflow container 2 includes a first support 21 fixedly installed inside the housing 1, a plurality of cylindrical quartz tubes 22 installed on the first support 21 and extending in a direction perpendicular to the horizontal plane, an inlet buffer chamber 23 installed on the first support 21 below the plurality of quartz tubes 22 and connected to the lower opening of the plurality of quartz tubes 22, a first flow guiding silicone tube 24 connected to the inlet buffer chamber 23, a temporary liquid storage chamber 25 installed above the plurality of quartz tubes 22 and connected to the upper opening of the plurality of quartz tubes 22, and a second flow guiding silicone tube 26 connected to the temporary liquid storage chamber 25. The plurality of quartz tubes 22 are arranged in close alignment on the same plane. Each quartz tube 22 has an inner coating on its inner wall. The thickness of the inner coating is 1 to 50 nm and the material of the inner coating is polyethylene glycol. A peristaltic pump is installed on the first flow guiding silicone tube 24. The inlet buffer chamber 23 is tightly connected to the lower openings of multiple quartz tubes 22, and the connection is sealed with a silicone gasket. The temporary storage chamber 25 is tightly connected to the upper openings of multiple quartz tubes 22, and the connection is sealed with a silicone gasket. Each quartz tube 22 is made of high-purity JGS1 / JGS2 material. Each quartz tube 22 has an outer coating on its outer wall, made of amorphous fluoropolymer, with a thickness of 1–50 nm. Each quartz tube 22 has an inner diameter of 0.1–1 cm, a wall thickness of 0.05–0.5 cm, an inner coating thickness of 1–50 nm, and a length of 5–20 cm. The number of quartz tubes 22 is 5–15; the specific number can be selected according to the specific application.

[0052] Specifically, the ultraviolet lamp assembly includes a first ultraviolet lamp assembly 31 and a second ultraviolet lamp assembly 32 respectively disposed on opposite sides of the flow container 2. The first ultraviolet lamp assembly 31 includes a second bracket 311 slidably disposed within the housing 1 and a first ultraviolet lamp 312 disposed on the second bracket 311. The second ultraviolet lamp assembly 32 includes a third bracket 321 slidably disposed within the housing 1 and a second ultraviolet lamp 322 disposed on the third bracket 321. The first ultraviolet lamp 312 and the second ultraviolet lamp 322 are respectively cylindrical and extend along... Extending perpendicularly to the horizontal plane, the lengths of the first UV lamp 312 and the second UV lamp 322 are greater than or equal to the length of the quartz tube 22. The plane containing the first UV lamp 312 and the second UV lamp 322 intersects perpendicularly with the plane containing the multiple quartz tubes 22, and the line of intersection is the central axis of the multiple quartz tubes 22. The control console 4 is used to control the movement of the second support 311 and the third support 321 to adjust the distance between the first UV lamp 312 and the second UV lamp 322 and the plane containing the multiple quartz tubes 22. The outer diameters of the first UV lamp 312 and the second UV lamp 322 are 1–3 cm, the lengths of the first UV lamp 312 and the second UV lamp 322 are 5–20 cm, and the distances from the first UV lamp 312 and the second UV lamp 322 to the plane containing the multiple quartz tubes 22 are 0.5–5 cm. The power of the first ultraviolet lamp 312 and the second ultraviolet lamp 322 are 5 to 50W respectively, and the irradiation wavelength of the first ultraviolet lamp 312 and the second ultraviolet lamp 322 is 250 to 260nm.

[0053] Specifically, the control panel 4 is equipped with a display screen that can display the temperature, humidity and power of the ultraviolet lamp group inside the cabinet 1, as well as an ultraviolet lamp switch 41, a thermometer switch 42, a hygrometer switch 43, an ultraviolet detector switch 44, a temperature adjustment knob 45 for adjusting the temperature control dehumidifier, and a leak sensor alarm light 46.

[0054] In the following examples and comparative examples, virus titer detection was performed using a cell culture method: The samples to be tested were serially diluted using cell culture medium containing 2% FBS. For each test sample, the first well contained 900 μl of cell culture medium + 100 μl of the test sample (10-fold dilution, denoted as 10⁻¹); the second well contained 900 μl of cell culture medium + 100 μl of the 10-fold diluted test sample (100-fold dilution, denoted as 10⁻²); and so on, with each dilution being 10-fold. 100 μl of each dilution was seeded onto a 96-well cell plate pre-seeded with indicator cells (ST cells). The 96-well cell plates were incubated at 37°C in a 5% CO₂ incubator. After 5 days, the plates were removed and the lesions (CPE) were observed under a microscope. Normal cells served as a blank control. The test results were recorded, and the virus titer (median cellular infection dose, TCID50) was calculated using the Spearman-Karber method.

[0055] The FⅡa potency calculation method refers to the relevant thrombin potency standard detection method in the 2020 edition of the Chinese Pharmacopoeia - Part II - Main Text - Thrombin Lyophilized Powder, and uses an automated coagulation analyzer for potency determination.

[0056] Example 1

[0057] The sample used in this embodiment was a pre-prepared thrombin (FIIa) solution with a total protein content of 10 mg / mL, an initial FIIa titer of 10 IU / mL, and an initial PPV viral titer of 7.55 logs.

[0058] In this embodiment, the virus inactivation device uses 11 quartz tubes 22, each with an inner diameter of 2 mm and a length of 87 mm. The thickness of both the inner and outer coatings is 20 nm. The first ultraviolet lamp 312 and the second ultraviolet lamp 322 both have an inner diameter of 15 mm, a length of 87 mm, a power of 10 W, and an irradiation wavelength of 254 nm. The distance from the first ultraviolet lamp 312 and the second ultraviolet lamp 322 to the plane containing all the quartz tubes 22 is 3.5 cm.

[0059] The virus inactivation method is as follows: Turn on the first UV lamp 312 and the second UV lamp 322. After equilibration for 10 minutes, turn on the peristaltic pump to start sample injection, controlling the injection flow rate at 0.3 ml / s. The sample overflows upwards through the inlet buffer chamber 23, passes through the quartz tube 22, and enters the temporary storage chamber 25. Finally, it is discharged from the second guide silicone tube 26. During the flow of the sample in the quartz tube 22, it is irradiated by the first UV lamp 312 and the second UV lamp 322 to inactivate the virus. The duration of sample flow through the quartz tube 22 and receiving UV irradiation is approximately 10 seconds. The maximum UV intensity measured at various points on the UV lamp tube plane is approximately 8000 uW / cm². 2 The average value is approximately 3500 uW / cm 2 The sample received a UV dose of 35 mJ / cm². 2 .

[0060] Virus titer results showed that after inactivation treatment, the morphology of the cells was normal under light microscopy, and no cytopathic effect (CPE) was observed. The PPV viral titer decreased by 4.38 logs, and the FIIa titer of the thrombin solution after inactivation treatment was 84.7% of the initial FIIa titer.

[0061] Example 2

[0062] The sample to be processed in this embodiment is the same as in Embodiment 1, and the virus device used is basically the same as in Embodiment 1, except that the number of quartz tubes 22 is 7 and the inner diameter of each quartz tube 22 is 4mm. The virus inactivation operation is the same as in Embodiment 1.

[0063] The viral titer results of this embodiment show that after inactivation treatment, the indicator cells were morphologically normal and no CPE was observed under a light microscope. The PPV viral titer decreased by 4.12 logs, and the FIIa titer of the thrombin solution after inactivation treatment was 85.6% of the initial FIIa titer.

[0064] Example 3

[0065] The sample used in this embodiment was a pre-prepared thrombin (FIIa) solution with a total protein content of 10 mg / mL, an initial FIIa titer of 10 IU / mL, and an initial EMCV viral titer of 8.26 logs. The virus inactivation device and operating method used in this embodiment were exactly the same as in Example 1.

[0066] The viral titer results of this embodiment show that after inactivation treatment, the indicator cells were morphologically normal and no CPE was observed under a light microscope. The EMCV viral titer decreased by 4.22 logs, and the FIIa titer of the thrombin solution after inactivation treatment was 83.5% of the initial FIIa titer.

[0067] Example 4

[0068] The sample to be processed used in this embodiment is the same as in Embodiment 3. The virus inactivation device and operating method used in this embodiment are exactly the same as in Embodiment 2.

[0069] The viral titer results of this embodiment show that after inactivation treatment, the indicator cells were morphologically normal and no CPE was observed under a light microscope. The EMCV viral titer decreased by 4.10 logs, and the FIIa titer of the thrombin solution after inactivation treatment was 86.6% of the initial FIIa titer.

[0070] Comparative Example 1

[0071] The samples used in this comparative example are the same as those in Example 3. The virus inactivation device used in this comparative example is the ultraviolet inactivation device CN220714425U. The short inner diameter of the inactivation tube is 1 cm, the long inner diameter of the inactivation tube is 5 mm, the length of the inactivation tube is 92 mm, the vertical distance between the inactivation tube and the ultraviolet lamp tube is 3 cm, the power is 10 W, and the irradiation wavelength is 254 nm.

[0072] The virus inactivation method is as follows: turn on the ultraviolet lamp, equilibrate for 10 minutes, then turn on the peristaltic pump to start the sample injection, control the injection flow rate to 0.5 ml / s, and the sample flows through the quartz tube 22 and is irradiated by ultraviolet light for about 50 seconds.

[0073] The viral titer results of this comparative example show that, after inactivation treatment, as observed under a light microscope, at a dilution of 10... -2CPE was observed in 4 out of 8 replicate wells, with an EMCV virus titer decrease of 3.38 logs. The FIIa titer of the inactivated thrombin solution was 78.0% of the initial FIIa titer. This method cannot effectively inactivate the virus under the above conditions. In addition, the protein denatures and adheres to the tube wall after irradiation, resulting in a low activity recovery rate.

[0074] Comparative Example 2

[0075] The samples used in this comparative example were the same as those in Example 3. This comparative example used the method described in the national standard (HJ 2522-2012) for parallel light ultraviolet radiation irradiation. 49 ml of the sample solution was placed in a 90 mm petri dish, with the irradiation area and the dish bottom area totaling 49 cm². 2 The thickness of the liquid layer parallel to the ultraviolet light (light path length) is 1 cm. However, due to the action of the magnetic stir bar, the bottom liquid can still receive ultraviolet light irradiation. The distance between the ultraviolet lamp (20W straight type) and the petri dish is about 15 cm, and the ultraviolet intensity is 2000 uW / cm. 2 The irradiation time was 15 seconds, and the average ultraviolet dose was approximately 30 mJ / cm². 2 .

[0076] The viral titer results of this comparative example show that, after inactivation treatment, as observed under a light microscope, at a dilution of 10... -4 CPE was observed in 3 out of 8 replicate wells, with an EMCV virus titer decrease of 1.50 logs. The FIIa titer of the inactivated thrombin solution was 77.0% of the initial FIIa titer. Because this method involves non-continuous influent, it cannot meet the needs of large-scale production.

[0077] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A method for inactivating viruses in a liquid biological product, characterized in that, The active ingredient of the liquid biological product is protein. A virus inactivation device is used to inactivate the virus in the liquid biological product. The virus inactivation device includes a housing, a control panel, a flow container housed within the housing, and an ultraviolet lamp assembly housed within the housing. The inner wall of the box is coated with a reflective material. The flow container includes a first support fixedly mounted inside the housing; multiple cylindrical quartz tubes extending perpendicular to the horizontal plane and mounted on the first support; an inlet buffer chamber mounted on the first support below the multiple quartz tubes and connected to the lower openings of the multiple quartz tubes; a first flow-guiding silicone tube connected to the inlet buffer chamber; a temporary storage chamber above the multiple quartz tubes and connected to the upper openings of the multiple quartz tubes; and a second flow-guiding silicone tube connected to the temporary storage chamber. The multiple quartz tubes are arranged sequentially and closely aligned on the same plane. Each quartz tube has an inner coating material of polyethylene glycol on its inner wall. The first flow-guiding silicone tube is connected to a peristaltic pump. The ultraviolet lamp assembly includes a first ultraviolet lamp assembly and a second ultraviolet lamp assembly disposed on opposite sides of the flow container. The first ultraviolet lamp assembly includes a second bracket slidably disposed within the housing and a first ultraviolet lamp disposed on the second bracket. The second ultraviolet lamp assembly includes a third bracket slidably disposed within the housing and a second ultraviolet lamp disposed on the third bracket. The first and second ultraviolet lamps are respectively cylindrical and extend in a direction perpendicular to the horizontal plane. The lengths of the first and second ultraviolet lamps are greater than or equal to the lengths of the quartz tubes. The plane containing the first and second ultraviolet lamps intersects perpendicularly with the plane containing the plurality of quartz tubes, and the line of intersection is the central axis of the plurality of quartz tubes. The control console is used to control the movement of the second and third brackets to adjust the distance between the first and second ultraviolet lamps and the plane containing the plurality of quartz tubes. The liquid biological product is continuously fed into the inlet buffer chamber by the peristaltic pump. After overflowing upwards through the inlet buffer chamber and passing through the multiple quartz tubes, it enters the temporary storage chamber and is finally discharged from the second guide silicone tube. During the flow of the liquid biological product in the multiple quartz tubes, it is irradiated by the first ultraviolet lamp and the second ultraviolet lamp to inactivate the virus.

2. The method for virus inactivation of liquid biological products according to claim 1, characterized in that, Each quartz tube has an outer coating on its outer wall. The outer coating is made of an amorphous fluoropolymer and has a thickness of 1–50 nm.

3. The method for virus inactivation of liquid biological products according to claim 1, characterized in that, The inner diameter of each quartz tube is 0.1–1 cm, the wall thickness is 0.05–0.5 cm, the thickness of the inner coating is 1–50 nm, and the length is 5–20 cm. The outer diameters of the first and second ultraviolet lamps are 1–3 cm, the lengths of the first and second ultraviolet lamps are 5–20 cm, the distances from the first and second ultraviolet lamps to the plane containing the plurality of quartz tubes are 0.5–5 cm, the power of the first and second ultraviolet lamps is 5–50 W, and the irradiation wavelengths of the first and second ultraviolet lamps are 250–260 nm.

4. The method for virus inactivation of liquid biological products according to claim 1, characterized in that, The ultraviolet inactivation device also includes a thermometer, a hygrometer, an ultraviolet detector, and a temperature-controlled dehumidifier, which are installed inside the chamber and connected to the control panel. The control panel is equipped with a display screen that can display the temperature, humidity, and power of the ultraviolet lamp group inside the chamber, as well as buttons for controlling the thermometer, hygrometer, and ultraviolet detector, and a knob for adjusting the temperature of the temperature-controlled dehumidifier.

5. The method for virus inactivation of liquid biological products according to claim 4, characterized in that, The number of the plurality of quartz tubes is 5 to 15.

6. The method for virus inactivation of liquid biological products according to claim 4, characterized in that, The total protein content of the liquid biological product is 0.01–50 mg / mL, and the flow rate of the liquid biological product is controlled by the peristaltic pump to be 0.1–15 mL / s.

7. The method for virus inactivation of liquid biological products according to claim 6, characterized in that, The viruses mentioned include one or more of the following families: Reoviridae, Rhabdoviridae, Orthomyxoviridae, Filoviridae, Coronaviridae, Bunyaviridae, Bonaviviridae, Flaviviridae, Paramyxoviridae, Clonorviridae, Arenaviridae, Microribonucleoviridae, Caliciviridae, Retroviridae, Poxviridae, Herpesviridae, Iridoviridae, Papillomaviridae, and Parvoviridae.

8. The method for virus inactivation of liquid biological products according to claim 7, characterized in that, The liquid biological products are vaccine preparations, toxin preparations, toxoid preparations, immune serum, blood products, immunoglobulin preparations, antigen preparations, allergen preparations, cytokine preparations, hormone preparations, enzyme products, fermentation broth, monoclonal antibody preparations, or in vitro immunodiagnostic products.

9. The method for virus inactivation of liquid biological products according to claim 8, characterized in that, The flow rate of the liquid biological product is controlled to be 0.1–1 mL / s by the peristaltic pump. The duration of irradiation of the liquid biological product within the plurality of quartz tubes by the first and second ultraviolet lamps is 5–20 s, and the ultraviolet dose received by the liquid biological product is 30–40 mJ / cm². 2 .

10. The method for virus inactivation of liquid biological products according to claim 9, characterized in that, The number of the plurality of quartz tubes is 7 to 11, and the inner diameter of each quartz tube is 2 to 4 mm and the length is 80 to 90 mm.

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