Prediction method for virus ultraviolet inactivation effect in liquid biological product

Abstract

The invention provides a method for predicting the ultraviolet inactivation effect of viruses in a liquid biological product. The method comprises the following steps: (1) detecting the absorbance value of a to-be-detected liquid biological product at a preset wavelength; (2) constructing a relation curve of the ultraviolet dose of the BSA / virus mixed solution and the decline degree of the virus titer; (3) constructing a relation curve of the ultraviolet dose of the SMZ working solution and the SMZ degradation degree; (4) constructing a relation curve of the operation parameters and the SMZ degradability; and (5) substituting preset operation parameters into the relation curve of the operation parameters and the SMZ degradability, substituting the obtained result into the relation curve of the ultraviolet dose and the SMZ degradability, substituting the obtained result into the relation curve of the ultraviolet dose and the decline of the indication virus titer, and judging the ultraviolet inactivation effect of the virus according to a predicted value of the decline of the indication virus titer. The method is accurate in prediction result, does not need to consume liquid biological products, and can be used for various virus inactivation effects under the condition that the ultraviolet dose cannot be accurately calculated.

Description

Technical Field

[0001] This invention belongs to the field of virus inactivation effect detection and evaluation technology, specifically relating to a method for predicting the ultraviolet inactivation effect of viruses in liquid biological products. Background Technology

[0002] Ultraviolet virus inactivation technology for liquid biological products is a physical disinfection method that uses ultraviolet radiation to destroy the nucleic acid structure of viruses and render them infective. It has important application value in fields such as biopharmaceuticals and blood products.

[0003] Traditional ultraviolet irradiation methods typically employ static or simple flow treatment, where the liquid is irradiated with ultraviolet light in a fixed container. This method relies primarily on the direct irradiation of ultraviolet light; however, due to uneven or excessively thick liquid layers, irradiation dead zones can easily occur, leading to unstable virus inactivation effects and potentially damaging proteins and other effective components.

[0004] In contrast, the continuous centrifugation ultraviolet method uses physical centrifugal force to uniformly thin liquid samples to a thickness of micrometers, forming a unidirectional, stable, high-speed flowing uniform liquid film. High-energy short-wave ultraviolet irradiation ensures that every virus particle within the liquid receives sufficient ultraviolet energy, achieving highly efficient inactivation. This technology has demonstrated excellent inactivation effects on various biological products, especially for parvoviruses that are difficult to inactivate using traditional methods. A single-step treatment can reduce virus titers by 4-5 logs, meeting regulatory requirements while maintaining protein activity and purity recovery rates exceeding 90%, with minimal or almost no impact on product composition.

[0005] Although continuous centrifugation ultraviolet (CCI) is highly effective, and its operating parameters (such as sample flow rate and centrifugation conditions) are correlated with ultraviolet dose and inactivation effect, the unconventional geometry of the CCI inactivation device, which is the basis of this technology, results in turbulent internal flow and random liquid distribution within a certain range. This makes it difficult to accurately calculate the actual ultraviolet dose received by virus-containing samples using existing theoretical calculation methods. Furthermore, there is currently a lack of reliable methods for predicting the effectiveness of virus ultraviolet inactivation. Therefore, before being put into continuous production, extensive experimental trials using actual products and indicator viruses are necessary to explore operating parameters. This not only wastes products and indicator viruses but also increases the workload of equipment cleaning and validation.

[0006] Therefore, developing a method that can accurately predict the UV inactivation effect of viruses in liquid biological products has important practical significance and application value. Summary of the Invention

[0007] The problem to be solved by the present invention is to provide a method for predicting the effect of ultraviolet inactivation of viruses in liquid biological products.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: This invention provides a method for predicting the ultraviolet inactivation effect of viruses in liquid biological products, comprising: (1) Detect the absorbance value of the liquid biological product to be tested at a preset wavelength; (2) Prepare a BSA solution, which includes a first BSA solution with an absorbance value at a preset wavelength that is less than or equal to the absorbance value of the liquid biological product to be tested at a preset wavelength and / or a second BSA solution with an absorbance value at a preset wavelength that is greater than or equal to the absorbance value of the liquid biological product to be tested at a preset wavelength. Mix the BSA solution with an indicator virus to obtain a BSA / virus mixture. Detect the decrease in indicator virus titer before and after the BSA / virus mixture is irradiated with different UV doses at a preset wavelength. Construct a curve showing the relationship between the UV dose of the BSA / virus mixture and the decrease in indicator virus titer. (3) Prepare SMZ working solution, the absorbance value of which at a preset wavelength is the same as the absorbance value of the liquid biological product to be tested at a preset wavelength, detect the degree of SMZ degradation of the SMZ working solution before and after being irradiated with different UV doses at a preset wavelength, and construct the relationship curve between UV dose and SMZ degradation of the SMZ working solution. (4) The SMZ working solution was treated with ultraviolet irradiation at a preset wavelength using an ultraviolet virus inactivation device. The SMZ degradation degree of the SMZ working solution before and after ultraviolet irradiation was detected under different operating parameters, and the relationship curve between operating parameters and SMZ degradation degree was constructed. (5) Substitute the preset operating parameters of the ultraviolet virus inactivation device into the relationship curve between the operating parameters and the SMZ degradation degree to calculate the predicted value of SMZ degradation degree. Substitute the predicted value of SMZ degradation degree into the relationship curve between the ultraviolet dose of the SMZ working solution and the SMZ degradation degree to calculate the predicted value of ultraviolet dose. Substitute the predicted value of ultraviolet dose into the relationship curve between the ultraviolet dose of the BSA / virus mixed solution and the decrease in the indicator virus titer to obtain the predicted value of the decrease in the indicator virus titer. Based on the predicted value of the decrease in the indicator virus titer, determine the ultraviolet inactivation effect of the ultraviolet virus inactivation device on the liquid biological product under the preset wavelength and preset operating parameters.

[0009] In some embodiments, in step (2), the absorbance value of the BSA solution at a preset wavelength is equal to the absorbance value of the liquid biological product to be tested at a preset wavelength.

[0010] In some embodiments, in step (2), the BSA solution includes at least a first BSA solution with an absorbance value at a preset wavelength lower than that of the test liquid biological product at the preset wavelength, and a second BSA solution with an absorbance value at a preset wavelength higher than that of the test liquid biological product at the preset wavelength. The relationship curve between the UV dose of the BSA / virus mixture and the decrease in the indicated virus titer includes the relationship curve between the UV dose of the first BSA / virus mixture and the decrease in the indicated virus titer, and the relationship curve between the UV dose of the second BSA / virus mixture and the decrease in the indicated virus titer. In step (5), the predicted ultraviolet dose is substituted into the relationship curve between the ultraviolet dose of the first BSA / virus mixed solution and the indicator virus titer decrease and the relationship curve between the ultraviolet dose of the second BSA / virus mixed solution and the indicator virus titer decrease to obtain the first predicted value and the second predicted value.

[0011] More preferably, the absorbance value of the first BSA solution at a preset wavelength is 5% to 80% of the absorbance value of the liquid biological product to be tested at the preset wavelength, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%.

[0012] More preferably, the absorbance value of the second BSA solution at a preset wavelength is 120% to 180% of the absorbance value of the liquid biological product to be tested at the preset wavelength, for example, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%.

[0013] In some preferred embodiments, the absorbance value of the first BSA solution at a preset wavelength is 5% to 20% of the absorbance value of the liquid biological product to be tested at the preset wavelength.

[0014] In some preferred embodiments, the absorbance value of the second BSA solution at a preset wavelength is 150% to 180% of the absorbance value of the liquid biological product to be tested at the preset wavelength.

[0015] Furthermore, the absorbance value of the liquid biological product to be tested at a preset wavelength is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or any value between any two of the above values.

[0016] Further, in step (2), the BSA solution is placed in a first sterile container and irradiated with ultraviolet light using a parallel light ultraviolet irradiator.

[0017] Further, the SMZ working solution was placed in a second sterile container and irradiated with ultraviolet light using a parallel light ultraviolet irradiator.

[0018] In some embodiments, in steps (2) and (3), the method for detecting the ultraviolet dose is as follows: using an ultraviolet radiometer to detect the ultraviolet intensity at multiple points at the location of the liquid surface, calculating the average incident ultraviolet intensity, and calculating the ultraviolet dose according to the following formula: , —D represents the ultraviolet dose, in mJ / cm². 2 ; —— The average incident ultraviolet radiation intensity is expressed in mW / cm². 2 ; —— Transmittance is calculated using the following formula: ; —H represents the liquid level height, in cm; —t represents the duration of ultraviolet radiation, measured in seconds.

[0019] Furthermore, the average incident ultraviolet intensity ( The detection method is as follows: After turning on the ultraviolet lamp until its radiation intensity is balanced, place the photosensitive surface of the ultraviolet radiometer probe at the position of the liquid surface in the petri dish in step (2), and test n points evenly distributed within the light-receiving range of the liquid surface below the center of the ultraviolet lamp, record the incident ultraviolet intensity (Ii) at each point, and calculate the average incident ultraviolet intensity (Ii) according to the following formula. : , —— The average incident ultraviolet radiation intensity is expressed in mW / cm². 2 ; —n is the number of points uniformly selected; —I i The intensity of incident ultraviolet radiation measured at point i is given in mW / cm². 2 .

[0020] Among them, the preferred values ​​are 10≤n≤50.

[0021] In some specific embodiments, in step (2), the indicator virus titer is detected using a cell culture method, expressed as log... 10 (TCID) 50 The value indicates the viral titer. The rate of decrease in viral titer is the difference in viral titer before and after ultraviolet irradiation.

[0022] Furthermore, the specific operation of the virus titer detection method is as follows: For each test sample, the first well contains 900 μL of cell culture medium + 100 μL of the sample to be tested; this is a 10-fold dilution, written as 10. -1 The second well contains 900 μL of cell culture medium + 100 μL of a 10-fold diluted sample. This is a 100-fold dilution, written as 10. -2 Dilute sequentially to 10 -10 100 μL of each dilution was inoculated into a cell plate pre-cultured with indicator cells. Multiple replicate wells were set up for each dilution, and cell control wells (culture medium only) were also set up. The plates were incubated at 37°C and 5% CO2 for 7 days and then removed. Cell pathogenesis was observed under a microscope, and the number of positive wells for each dilution was recorded. The viral titer was calculated according to the Spearman-Karber method.

[0023] More specifically, the indicator cells used were A9 cells, and the cell culture medium used was DMEM-LOW medium containing 2% FBS.

[0024] Furthermore, the rate of decrease in viral titer = positive control test titer - experimental group test titer.

[0025] In some specific embodiments, in step (3), the concentration of the SMZ working solution is calculated according to the following formula: , —C represents the concentration of the SMZ working solution, in μg / mL; —K is the absorbance coefficient of the SMZ working solution; —The target absorbance value is the absorbance value of the liquid biological product to be tested at a preset wavelength.

[0026] More specifically, the absorbance coefficient is the absorbance value of a 1 mol / L SMZ standard solution measured at 254 nm using a cuvette with a 1 cm optical path.

[0027] In some embodiments, in steps (3) and (4), the degree of SMZ degradation is the difference in absorbance of the SMZ working solution at its absorption peak wavelength before and after the SMZ working solution is irradiated with different UV doses at a preset wavelength.

[0028] According to some embodiments of the present invention, in step (2), a standard “UV dose-drop titer” relationship curve indicating the virus MVM is plotted with the degree of decrease in viral titer as the vertical axis (Y-axis) and the corresponding UV dose as the horizontal axis (X-axis).

[0029] According to some embodiments of the present invention, in step (3), with Plot the standard "UV dose-SMZ degradation rate" relationship curve of SMZ working solution with the vertical axis (Y-axis) and the corresponding UV dose as the horizontal axis (X-axis).

[0030] According to some embodiments of the present invention, in step (4), the "operating parameters-SMZ degradation degree" relationship curve of the liquid biological product virus ultraviolet inactivation instrument is plotted with the mean value of ∆A264.5 (1cm) as the vertical axis (Y axis) and the corresponding operating flow rate as the horizontal axis (X axis).

[0031] In some specific embodiments, in step (5), the ultraviolet virus inactivation device is a continuous centrifugal ultraviolet virus inactivation device, and the operating parameter is the sample flow rate or centrifugation speed.

[0032] In some specific embodiments, the preset wavelength is 240~260nm, such as 240nm, 241nm, 242nm, 243nm, 244nm, 245nm, 246nm, 247nm, 248nm, 249nm, 250nm, 251nm, 252nm, 253nm, 254nm, 255nm, 256nm, 257nm, 258nm, 259nm, and 260nm.

[0033] In some specific embodiments, the effective active ingredient of the liquid biological product to be tested is protein.

[0034] Furthermore, the liquid biological products to be tested include, but are not limited to, vaccine preparations, toxin preparations, toxoid preparations, immune serum, blood products, immunoglobulin preparations, antigen preparations, allergen preparations, cytokine preparations, hormone preparations, enzyme preparations, fermentation broth, monoclonal antibody preparations, or in vitro immunodiagnostic products.

[0035] In some specific embodiments, the indicator virus includes, but is not limited to, 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.

[0036] Compared with the prior art, the present invention has the following advantages: This invention constructs curves showing the relationship between UV dose and the rate of decrease in indicator virus titer for a BSA / virus mixed solution, the relationship between UV dose and SMZ degradation rate for the SMZ working solution, and the relationship between operating parameters and SMZ degradation rate. The preset operating parameters of the UV virus inactivation device are substituted into the curves showing the relationship between operating parameters and SMZ degradation rate to calculate the predicted value of SMZ degradation rate. The predicted value of SMZ degradation rate is then substituted into the curve showing the relationship between UV dose and SMZ degradation rate for the SMZ working solution to calculate the predicted value of UV dose. Finally, the predicted value of UV dose is substituted into the curve showing the relationship between UV dose and the rate of decrease in indicator virus titer for the BSA / virus mixed solution to obtain the predicted value of the rate of decrease in indicator virus titer. Based on the predicted value of the rate of decrease in indicator virus titer, the effectiveness of the UV virus inactivation device on liquid biological products under preset wavelengths and preset operating parameters is determined. The prediction results of this invention are accurate, require no loss of liquid biological products, and can be used to predict the virus inactivation effect in complex instruments, irregular flow fields, or situations where accurate calculation of UV dose is not possible. It has high reference and application value when using UV for virus inactivation of liquid biological products with extremely low transmittance. Attached Figure Description

[0037] Figure 1 The standard "UV dose-decreasing titer" relationship curve for the indicator virus MVM constructed in Example 1; Figure 2 This is the standard "UV dose-SMZ degradation rate" curve for the SMZ working solution constructed in Example 1; Figure 3 The curve showing the relationship between "operating parameters - SMZ degradation degree" for the liquid biological product virus ultraviolet inactivation instrument constructed in Example 1. Detailed Implementation

[0038] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the embodiments of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0039] In this invention, the absorbance values ​​are all measured using a cuvette with a 1 cm optical path.

[0040] "A254nm(1cm)" indicates the absorbance value measured at a wavelength of 254nm using a cuvette with an optical path of 1cm.

[0041] "A264.5nm(1cm)" indicates the absorbance value measured at a wavelength of 264.5nm using a cuvette with an optical path of 1cm.

[0042] "Same" means completely identical or the difference is within ±0.1%. The absorbance value of SMZ working solution at the preset wavelength and the absorbance value of the test liquid biological product at the preset wavelength are the same, meaning that under the same detection conditions, the difference between the absorbance value of SMZ working solution at the preset wavelength and the absorbance value of the test liquid biological product at the preset wavelength is within ±0.1%.

[0043] "Molar absorptivity ε": According to Beer-Lambert's law: A = εbc, where A is absorbance, ε is molar absorptivity, b is the optical path length of the cuvette, and c is the solution concentration.

[0044] The present invention will be further described below with reference to the accompanying drawings and Embodiment 1.

[0045] Example 1: This example provides a method for predicting the UV inactivation effect of viruses in liquid biological products. The inactivation effect is determined by predicting the decrease in the indicator virus titer after UV irradiation of a liquid biological product containing an indicator virus. Taking a 10 mg / mL monoclonal antibody solution as an example, the specific process is as follows: 1. Take a 10 mg / mL monoclonal antibody solution to be tested and measure its absorbance at a wavelength of 254 nm. The absorbance of the monoclonal antibody solution A254nm(1cm) = 5.56.

[0046] 2. Establish the relationship between "UV dose and viral titer reduction rate" as an indicator of viral load. 2.1. Using BSA solution as the light-adjusting solution and MVM as the indicator virus, BSA solutions with concentrations of 1.66 mg / mL, 10 mg / mL, and 30 mg / mL were mixed with the indicator virus at a ratio of 100:1 (v / v) to obtain BSA / virus mixtures. The A254nm (1 cm) at a wavelength of 254 nm of the BSA / virus mixture was detected. The A254nm values ​​corresponding to the BSA concentrations from low to high were 0.5, 3, and 9, respectively.

[0047] 2.2. Establish the relationship between "UV dose and viral titer reduction rate" as an indicator of the virus under parallel UV irradiation: 2.2.1. A certain amount of the above BSA / virus mixture was poured into different sterile culture dishes, and the liquid level (H) of the BSA / virus mixture in the sterile culture dishes was measured. In this example, it was 0.8 cm.

[0048] 2.2.2. After turning on the UV lamp until its radiation intensity reaches equilibrium, place the sterile cultured blood containing the BSA / virus mixture directly under the UV lamp for UV irradiation to inactivate the virus, and record the irradiation time. Collect samples after different irradiation times and label them as the experimental group, with the samples before UV irradiation serving as the control group. Perform serial dilutions on both the experimental and control groups, and detect the virus titer using cell culture methods. The specific procedures are as follows: For each test sample, the first well contains 900 μL of cell culture medium + 100 μL of the sample to be tested; this is a 10-fold dilution, written as 10. -1 The second well contains 900 μL of cell culture medium + 100 μL of a 10-fold diluted sample. This is a 100-fold dilution, written as 10. -2 Dilute sequentially to 10 -10 100 μL of each dilution was seeded into a 96-well cell plate pre-cultured with indicator cells (A9 cells in this example). Ten replicate wells were set up for each dilution, and cell control wells (containing only culture medium) were also included. The plates were incubated at 37°C with 5% CO2 and observed for 7 days. Cellular pathogenesis was then observed under a microscope, and the number of positive wells for each dilution was recorded. The viral titer was calculated using the Spearman-Karber method, expressed as the logarithm of the half-maximal cell infection dose (log). 10 (TCID) 50 The value represents the viral titer, and the rate of decrease in viral titer = positive control test titer - experimental group test titer). The rate of decrease in viral titer for each BSA / virus mixture is recorded. In this example, the cell culture medium used is DMEM-LOW medium containing 2% FBS.

[0049] 2.2.3. After turning on the UV lamp until its radiation intensity is balanced, place the photosensitive surface of the UV radiometer probe at the position of the liquid surface in the petri dish in step (2), and evenly distribute 30 points within the light-receiving range of the liquid surface below the center of the UV lamp to test, record the incident UV intensity (Ii) at each point, and calculate the average incident UV intensity (Ii) according to the following formula. : , —— The average incident ultraviolet radiation intensity is expressed in mW / cm². 2 ; —n is the number of points to be uniformly selected. In this embodiment, n=30; —I i The intensity of incident ultraviolet radiation measured at point i is given in mW / cm². 2 .

[0050] The embodiment obtained =0.3151mW / cm2 .

[0051] Calculate the UV dose (D) received by the BSA / virus mixture using the following formula: , —D represents the ultraviolet dose, in mJ / cm². 2 ; —I avg The average ultraviolet radiation intensity is expressed in mW / cm². 2 ; ——T 254 Transmittance is calculated using the following formula: ; —H is the liquid level (depth of the liquid in the petri dish), in cm. In this example, H=0.8cm; —t represents the duration of ultraviolet radiation, measured in seconds.

[0052] In this embodiment, the UV dose received by BSA / virus mixed solutions with different BSA concentrations and the decrease in indicator virus titer are summarized in Table 1. Based on the data in Table 1, a standard "UV dose-titer decrease" relationship curve for the indicator virus MVM is plotted with the decrease in indicator virus titer as the ordinate (Y-axis) and the corresponding UV dose as the abscissa (X-axis). Figure 1 .

[0053] Table 1

[0054] 3. Establish a standard "UV dose-SMZ degradation rate" relationship for SMZ working solution: 3.1. Preparation of SMZ working solution: Calculate the required concentration of SMZ working solution using the following formula: , —C represents the concentration of the SMZ working solution, in μg / mL; —K is the absorbance coefficient (or molar absorbance coefficient ε) of the SMZ working solution, that is, the absorbance value A254nm (1cm) of a 1 mol / L SMZ standard solution at 254nm. It is a characteristic constant of SMZ, but it may fluctuate between 18±2 due to the influence of the detection instrument or detection environment. In this example, it is 18.55. —In this embodiment, the target A254 value is 5.56.

[0055] In this embodiment, the required concentration of SMZ working solution is 103.2 μg / mL, and an SMZ working solution with a concentration of 103.2 μg / mL is prepared. The absorption peak wavelength of the SMZ working solution is scanned spectrally, and its absorbance at the absorption peak wavelength is detected. In this embodiment, the absorption peak of the SMZ working solution is located at 264.5 nm, and A264.5nm (1cm) = 6.51.

[0056] 3.2. Establish the standard "UV dose-degradation rate" relationship for SMZ working solution under parallel UV irradiation: Referring to section 2.2.1 above, replace the BSA / virus mixture in the culture dish with an equal volume of SMZ working solution, i.e., pour SMZ working solution into the sterile culture dish and control the thickness H of the SMZ working solution in the sterile culture dish to be 0.8 cm.

[0057] Referring to section 2.2.2 above, after turning on the UV lamp until its radiation intensity reaches equilibrium, place the sterile culture blood containing SMZ working solution directly under the UV lamp for UV irradiation to inactivate the virus (at the same location as the sterile culture blood containing BSA / virus mixture in section 2.2.2). Record the UV irradiation time and the difference in absorbance of the SMZ working solution at 264.5 nm before and after irradiation for different times. As a measure of the degradation rate of the SMZ working fluid, .

[0058] Referring to section 2.2.3 above, calculate the UV dose (D) received by the SMZ working solution according to the formula: , —D represents the ultraviolet dose, in mJ / cm². 2 ; —I avg The average ultraviolet radiation intensity is expressed in mW / cm². 2 ; ——T 254 Transmittance is calculated using the following formula: ; —H is the liquid level (depth of the liquid in the petri dish), in cm. In this example, H=0.8cm; —t represents the duration of ultraviolet radiation, measured in seconds; —— The results were obtained using the detection method described in section 2.2.3 above. Slight fluctuations occurred in each test, but these are negligible. The results of this test... =0.3221mW / cm 2 .

[0059] In this embodiment, the UV dose received by the SMZ working solution and the corresponding... Statistics are presented in Table 2. Based on the data in Table 2, using... Plot the standard "UV dose-SMZ degradation rate" relationship curve for SMZ working solution using the ordinate (Y-axis) and the corresponding UV dose as the abscissa (X-axis). See [link to curve]. Figure 2 .

[0060] Table 2

[0061] 4. Establish the relationship between "operating parameters - SMZ degradation degree" for the UV inactivation instrument for liquid biological products. A continuous centrifugal ultraviolet virus inactivation device (Suzhou Liangchen, LCGC-PB3 continuous centrifugal ultraviolet virus inactivation device. See patent CN118557759B) was used. The SMZ working solution prepared in section 3.1 was used instead of the test monoclonal antibody solution for the instrument operation under ultraviolet light irradiation. The operation steps are as follows: (1) Turn on the ultraviolet lamp (emission wavelength is 254nm) and balance the intensity; (2) Turn on the continuous centrifugal ultraviolet virus inactivation device and set the parameters (centrifugation speed = 300 rpm, temperature control = 5 ± 5℃). (3) The SMZ working solution was introduced into the instrument at different flow rates to simulate the test solution. Samples were taken before and after UV irradiation, and the absorbance of the SMZ working solution at 264.5 nm before and after UV irradiation was measured. The degradation rate of the SMZ working solution at different flow rates was calculated. .

[0062] In this embodiment, the operating parameters (flow rate) of the continuous centrifugal ultraviolet virus inactivation device and the corresponding... Statistics are presented in Table 3. Based on the data in Table 3, using... The mean value is plotted on the ordinate (Y-axis), and the corresponding operating flow rate is plotted on the abscissa (X-axis). The relationship curve between the operating parameters and the SMZ degradation rate of the UV inactivation instrument for liquid biological products is shown below. Figure 3 .

[0063] Table 3

[0064] 5. Predicting the UV inactivation effect of viruses in liquid biological products By utilizing the degradation rate of SMZ working solution running on a continuous centrifugal ultraviolet virus inactivation device, and comparing it with the pre-established standard "UV dose-degradation rate" of SMZ working solution and the standard "UV dose-virus titer reduction rate" of virus, the predicted results of "operating parameter-virus titer reduction rate" for samples directly running on the continuous centrifugal ultraviolet virus inactivation device were obtained, as shown in Table 4: Table 4

[0065] In this embodiment, the relationship between the viral standard "UV dose-viral titer reduction rate" has three relationship curves. The predicted viral titer reduction (logs) is a range value, with the lower limit being the predicted value substituted into the relationship curve corresponding to MVM (A254=9), and the upper limit being the predicted value substituted into the relationship curve corresponding to MVM (A254=0.5). Figure 1 In practical applications, the mean value of the three viral titer decreases (logs) obtained by substituting the UV dose D into the relationship between the indicator virus standard "UV dose - viral titer decrease" is ±0.5. In this embodiment, the detection limit is 0.5 logs, and the result of predicting the undetected viral titer is recorded as ">Positive control titer - detection limit". In this embodiment, the positive control titer is the indicator virus MVM titer in the BSA / virus mixture before UV irradiation, specifically 5.88 logs.

[0066] 6. Verification of prediction results: Take 10 mg / mL monoclonal antibody solution and add MVM virus at a ratio of 100:1. Perform continuous ultraviolet virus inactivation on the LCGC-PB3 continuous centrifugation ultraviolet virus inactivation device according to the operating method and parameters described in 3.1. Take samples before and after ultraviolet irradiation and perform serial dilution. Detect the actual virus titer using cell culture method. Refer to the virus titer cell culture method detection steps described in 1.2.2. The comparison with the predicted virus titer decrease is shown in Table 5.

[0067] Table 5

[0068] As shown in Table 5, the actual viral titer decreases all fall within the corresponding predicted range, indicating reliable prediction results. This embodiment is applicable to liquid biological products with an A254nm (1cm) value of 0.5~9. In other embodiments, the concentration of the SMZ working solution can be adjusted according to the A254nm (1cm) value of the liquid biological product to be tested, so that the A254nm (1cm) value of the SMZ working solution matches that of the test solution. If necessary, the concentration of the light-adjusting solution can be adjusted to ensure that the A254nm (1cm) value of the liquid biological product to be tested falls within the A254nm (1cm) range of the light-adjusting solution.

[0069] The method for predicting the UV inactivation effect of viruses in liquid biological products of the present invention can be used to predict the virus inactivation effect under conditions of complex instruments, irregular flow fields, or when the UV dose cannot be accurately calculated. It has high reference and application value for using UV inactivation schemes for liquid biological products (with extremely low transmittance, usually transmittance T < 0.1%, transmittance T ≈ 50% when A254nm (1cm) = 0.3; transmittance T = 0.1% when A254nm (1cm) = 3; transmittance T = 0.0000001% when A254nm (1cm) = 9).

[0070] 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 predicting the effect of ultraviolet inactivation of viruses in a liquid biological product, characterized in that, It comprises: (1) detecting the absorbance value of the liquid biological product to be tested at a preset wavelength; (2) preparing a BSA solution, which includes a first BSA solution with an absorbance value at a preset wavelength less than or equal to the absorbance value of the liquid biological product to be tested at a preset wavelength, and / or a second BSA solution with an absorbance value at a preset wavelength greater than or equal to the absorbance value of the liquid biological product to be tested at a preset wavelength, mixing the BSA solution with the indicator virus to obtain a BSA / virus mixed solution, detecting the decline of the indicator virus titer before and after the BSA / virus mixed solution is irradiated with different ultraviolet doses at a preset wavelength, and constructing a relationship curve of the ultraviolet dose of the BSA / virus mixed solution and the decline of the indicator virus titer; Preparing a SMZ working solution with an absorbance value at a preset wavelength identical to the absorbance value of the liquid biological product to be tested at a preset wavelength, detecting the SMZ degradation degree before and after the SMZ working solution is irradiated with different ultraviolet doses at a preset wavelength, and constructing a relationship curve of the ultraviolet dose of the SMZ working solution and the SMZ degradation degree; (4) using an ultraviolet virus inactivation device to irradiate the SMZ working solution at a preset wavelength, detecting the SMZ degradation degree before and after the SMZ working solution is irradiated with different operating parameters, and constructing a relationship curve of the operating parameters and the SMZ degradation degree; (5) bringing the preset operating parameters of the ultraviolet virus inactivation device into the relationship curve of the operating parameters and the SMZ degradation degree to calculate the predicted value of the SMZ degradation degree, bringing the predicted value of the SMZ degradation degree into the relationship curve of the ultraviolet dose of the SMZ working solution and the SMZ degradation degree to calculate the predicted value of the ultraviolet dose, substituting the predicted value of the ultraviolet dose into the relationship curve of the ultraviolet dose of the BSA / virus mixed solution and the decline of the indicator virus titer to obtain the predicted value of the decline of the indicator virus titer, and judging the ultraviolet virus inactivation effect of the ultraviolet virus inactivation device on the liquid biological product at a preset wavelength and preset operating parameters according to the predicted value of the decline of the indicator virus titer.

2. The method for predicting the effect of ultraviolet inactivation of viruses in a liquid biological product according to claim 1, characterized in that, In step (2), the BSA solution at least includes a first BSA solution with an absorbance value at a preset wavelength less than the absorbance value of the liquid biological product to be tested at a preset wavelength, and a second BSA solution with an absorbance value at a preset wavelength greater than the absorbance value of the liquid biological product to be tested at a preset wavelength, and the relationship curve of the ultraviolet dose of the BSA / virus mixed solution and the decline of the indicator virus titer includes a relationship curve of the ultraviolet dose of the first BSA / virus mixed solution and the decline of the indicator virus titer, and a relationship curve of the ultraviolet dose of the second BSA / virus mixed solution and the decline of the indicator virus titer, In step (5), the predicted value of the ultraviolet dose is substituted into the relationship curve of the ultraviolet dose of the first BSA / virus mixed solution and the decline of the indicator virus titer and the relationship curve of the ultraviolet dose of the second BSA / virus mixed solution and the decline of the indicator virus titer to obtain a first predicted value and a second predicted value.

3. The method for predicting the effect of ultraviolet inactivation of viruses in a liquid biological product according to claim 1, characterized by, In step (2), the absorbance value of the BSA solution at the preset wavelength is equal to the absorbance value of the liquid biological product to be measured at the preset wavelength.

4. The method for predicting the effect of ultraviolet inactivation of viruses in a liquid biological product according to claim 2, characterized by, The first BSA solution has an absorbance value at the preset wavelength of 5% to 80% of the absorbance value of the liquid biological product to be measured at the preset wavelength, and / or, The second BSA solution has an absorbance value at the preset wavelength of 120% to 180% of the absorbance value of the liquid biological product to be measured at the preset wavelength.

5. The method for predicting the effect of ultraviolet inactivation of viruses in a liquid biological according to claim 4, characterized in that, The absorbance value of the liquid biological product to be measured at the preset wavelength is 1 to 10.

6. The method of predicting the effect of UV inactivation of viruses in a liquid biological product according to claim 1, characterized in that, In step (2), the BSA solution is placed in a first sterile container and subjected to ultraviolet irradiation using a parallel light ultraviolet irradiator. In step (3), the SMZ working solution is placed in a second sterile container and subjected to ultraviolet irradiation using a parallel light ultraviolet irradiator.

7. The method for predicting the effect of UV inactivation of viruses in a liquid biological according to claim 6, characterized in that, In steps (2) and (3), the detection method of the ultraviolet dose is as follows: the ultraviolet intensity of multiple points at the position of the liquid surface is detected using an ultraviolet irradiator, the average incident ultraviolet intensity is calculated, and the ultraviolet dose is calculated according to the following formula: ， - D is the UV dose in mJ / cm 2 ; — Average incident UV intensity in mW / cm2 2 ; — For the light transmittance, the calculation formula is ; H is the height of the liquid surface, in cm; t is the time of ultraviolet irradiation, in sec.

8. The method of claim 1, wherein the method is characterized by: In step (2), the virus titer was measured by cell culture method, and the virus titer was expressed as log 10 (TCID 50 ).

9. The method of claim 1, wherein the method is characterized by, In step (3), the concentration of the SMZ working solution is calculated according to the following formula: ， C is the concentration of the SMZ working solution, in μg / mL; K is the absorbance coefficient of the SMZ working solution; The target absorbance value is the absorbance value of the liquid biological product to be measured at the preset wavelength.

10. The method of predicting the effect of UV inactivation of viruses in a liquid biological product according to claim 9, characterized in that, The absorbance coefficient is the absorbance value of a 1 mol / L SMZ standard solution at 254 nm, measured using a 1 cm optical path cuvette.

11. The method of predicting the effect of UV inactivation of viruses in a liquid biological product according to claim 1, characterized in that, In steps (3) and (4), the SMZ degradation degree is the difference in absorbance of the SMZ working solution at the absorption peak wavelength before and after the SMZ working solution is irradiated with different ultraviolet doses at the preset wavelength.

12. The method of claim 1, wherein the method is characterized by: In step (5), the ultraviolet virus inactivation device is a continuous centrifugal ultraviolet virus inactivation device, and the operating parameter is the sample flow rate or the centrifugal speed.

13. The method of claim 1, wherein the method is characterized by: The preset wavelength is 240 to 260 nm.

14. The method of claim 1, wherein the method is characterized by: The effective active ingredient of the liquid biological product to be measured is protein.

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