Detection method for membrane package integrity of ultrafiltration membrane and application of detection method

Abstract

The invention provides an ultrafiltration membrane package integrity detection method and application thereof, and relates to the technical field of virus preparation.The method comprises the steps that clarified virus liquid is filtered through a to-be-detected ultrafiltration membrane package, if the turbidity value of permeate liquid is larger than or equal to 4 NTU, the membrane package is incomplete, and the condition that the ultrafiltration membrane package is incomplete can be determined by detecting the turbidity value of the permeate liquid; the detection can be completed only by detecting the turbidity value, only 3-5 seconds are needed, production does not need to be stopped during detection, the method can be used for detecting a single membrane package or multiple membrane packages and systems, and the detection method is simple, rapid and effective. The monitoring of the incomplete condition in the use process of the ultrafiltration membrane is realized, and the virus purification and concentration effect is ensured. And the detection method has no influence on the physicochemical properties and safety of the virus liquid, has no influence on the membrane package structure of the ultrafiltration membrane, and improves the detection efficiency. The technical problem that in the prior art, the integrity of the membrane bag of the ultrafiltration membrane cannot be detected in the using process is solved.

Description

Technical Field

[0001] This invention relates to the field of virus preparation technology, and in particular to a method for detecting the integrity of an ultrafiltration membrane envelope and its application. Background Technology

[0002] Ultrafiltration is a part of membrane separation technology. Membrane separation technology refers to the use of semi-permeable membranes to separate, classify, purify, and enrich two- or multi-component mixtures of gas or liquid through osmosis at room temperature, driven by external energy or chemical potential difference. Membrane separation technology has many advantages: its separation process does not involve heating or cooling, the medium does not experience temperature changes, and no phase transition occurs; it does not require the use of chemicals or additives, maintaining biological stability; it can remove small molecules and bacteria while concentrating the liquid; the equipment used is compact, easy to operate, and requires little floor space; it can operate continuously at room temperature, can be directly scaled up, and can be specifically formulated with membranes.

[0003] Ultrafiltration is a sieve-based separation process that uses pressure or concentration as the driving force to separate solutions or to separate large-molecule solutes from small-molecule solutes. Due to the asymmetric microporous structure of ultrafiltration membranes, friction channels and turbulence-promoting structures are employed to reduce membrane fouling. During separation, large-molecule solutes and particles (such as colloids and starch) flow tangentially across the membrane surface with the solution, while small-molecule substances and solvents are driven by pressure to pass through the micropores in the dense layer and enter the other side of the membrane. Therefore, ultrafiltration membranes can be used continuously for extended periods while maintaining relatively constant yield and separation efficiency. In ultrafiltration, large-molecule solutes are either adsorbed onto the surface and within the pores of the filter membrane (basic adsorption), retained within the pores, or expelled from the pores (clogging), or mechanically trapped on the surface of the filter membrane. Most of the removed substances are easily removed from the surface.

[0004] The method of determining the integrity of ultrafiltration membrane by detecting the HA value of the permeate requires taking a sample of the permeate from the ultrafiltration membrane for HA detection. The detection procedure is complicated, and the reagents used for HA detection also need to be prepared. The result waiting time is long, about 30 minutes.

[0005] Currently, membrane integrity testing methods rely on conventional testing methods provided by manufacturers: ① The bubble point method is mainly used to identify and isolate damaged membrane fibers. It is based on a capillary model. However, a drawback of the bubble point method is that it produces significant errors at higher pressures and larger membrane areas, as higher pressures alter the original structure of the membrane pores. ② The diffusion airflow method has the disadvantage of requiring offline testing, lacking standardized testing procedures and reliable baseline values, and is not yet a standard method. The diffusion flow in the diffusion airflow test is independent of the membrane pore size, making it suitable for large-area filter membranes, but with larger measurement errors for small-area filter membranes. ③ The pressure decay method is based on the same principle as the bubble point method. It involves introducing gas at a pressure lower than the bubble point pressure into the feed side of the wetted membrane, then sealing it, and observing the pressure decay over a certain period to determine membrane integrity. The pressure decay test has a short response time to membrane module damage and can accurately reflect membrane fiber damage, but it requires offline testing and the bubble point test to determine the specific location of the damage. The above three conventional testing methods can only be used before the ultrafiltration membrane is used. They cannot guarantee that there will be no leakage throughout the entire process of use. The operation is complicated, requires applying pressure to the ultrafiltration membrane, and unstable pressure can easily cause secondary damage to the membrane. The operation has limitations.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide a method for detecting the integrity of an ultrafiltration membrane package, thereby solving the technical problem that existing technologies cannot detect the integrity of an ultrafiltration membrane package during use.

[0008] The second objective of this invention is to provide the application of the above-mentioned detection method in detecting the integrity of ultrafiltration membrane packs during virus production.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for detecting the integrity of an ultrafiltration membrane package, comprising filtering a clarified viral solution through an ultrafiltration membrane package to be tested, wherein if the turbidity value of the permeate is ≥4 NTU, the membrane package is incomplete.

[0010] Furthermore, the method for preparing the clarified virus solution includes taking virus culture medium and culturing it, centrifuging it, and microfiltration it.

[0011] Furthermore, the initial inoculation density of the virus culture is 1.0 × 10⁻⁶. 6 ~1.0×10 7 cells / ml, preferably 8.0 × 10⁻⁶ 6 ~1.0×10 7 cells / ml.

[0012] Furthermore, the virus culture temperature is 32~35℃, preferably 32.5~34℃; Preferably, the virus culture rotation speed is 100-150 rpm, more preferably 100-120 rpm; Preferably, the virus culture time is 48-72 hours, and more preferably 48-60 hours.

[0013] Furthermore, the temperature of the virus culture medium is lowered to 2-8°C before centrifugation.

[0014] Furthermore, the centrifugation speed is 5000~12000 rpm, preferably 6000~8000 rpm; Preferably, the centrifugation time is 3-4 hours, more preferably 3.5 hours.

[0015] Furthermore, the microfiltration process includes hollow fiber column microfiltration.

[0016] Furthermore, the virus includes avian influenza virus.

[0017] Secondly, the present invention provides the application of the above-mentioned detection method in detecting the integrity of ultrafiltration membrane packs during virus production.

[0018] Furthermore, the virus includes avian influenza virus.

[0019] This invention provides a method for detecting the integrity of an ultrafiltration membrane pack. The method determines the presence of incompleteness in the ultrafiltration membrane pack simply by detecting the turbidity of the permeate. The process requires only a turbidity measurement, takes only 3-5 seconds, and does not require stopping production. It can be used for testing single membrane packs, multiple membrane packs, or systems. The method is simple, rapid, and effective. It enables the monitoring of incompleteness in ultrafiltration membranes during use, ensuring effective virus purification and concentration. Furthermore, this method does not affect the physicochemical properties or safety of the viral solution, nor does it affect the structure of the ultrafiltration membrane pack, thus improving detection efficiency. It solves the technical problem in existing technologies where the integrity of ultrafiltration membrane packs cannot be detected during use. Detailed Implementation

[0020] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0021] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, those commonly practiced in the art, or those described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] The present invention provides a method for detecting the integrity of an ultrafiltration membrane package, comprising filtering a clarified viral solution through an ultrafiltration membrane package to be tested; if the turbidity value of the permeate is ≥4 NTU, the membrane package is incomplete.

[0024] The presence of incomplete ultrafiltration membrane packs can be determined by detecting the turbidity of the permeate. This process requires only a turbidity measurement, takes only 3-5 seconds, and does not require stopping production. It can be used for testing single packs, multiple packs, or systems, and the method is simple, rapid, and effective. This allows for the monitoring of incomplete ultrafiltration membranes during use, ensuring effective virus purification and concentration. Furthermore, this detection method does not affect the physicochemical properties or safety of the viral solution, nor does it affect the structure of the ultrafiltration membrane pack, thus improving detection efficiency. It solves the technical problem of existing technologies that cannot perform ultrafiltration membrane pack integrity testing during use.

[0025] In some specific embodiments, the method for preparing the clarified virus solution includes taking a virus culture medium and culturing it, centrifuging it, and microfiltration it.

[0026] To control the turbidity of the clarified virus solution, in some specific embodiments, the initial inoculation density of the virus culture is 1.0 × 10⁻⁶. 6 ~1.0×10 7 cells / ml; in some specific embodiments, the culture temperature of the virus culture medium is 32~35℃; in some specific embodiments, the culture rotation speed of the virus culture medium is 100~150rpm; in some specific embodiments, the culture time of the virus culture medium is 48~72h.

[0027] The initial inoculation density may be, but is not limited to, 1.0 × 10⁻⁶. 6 cells / ml, 2.0×10 6 cells / ml, 3.0×10 6 cells / ml, 4.0×10 6 cells / ml, 5.0×10 6 cells / ml, 6.0×10 6 cells / ml, 7.0×10 6 cells / ml, 8.0×10 6 cells / ml, 9.0×10 6 cells / ml or 1.0×10 7 cells / ml, or 1.0×10 6 ~1.0×10 7 Any value between cells / ml, preferably 8.0 × 10⁻⁶. 6 ~1.0×10 7 cells / ml.

[0028] The culture temperature can be, but is not limited to, 32℃, 32.3℃, 32.5℃, 33℃, 33.3℃, ​​33.5℃ or 35℃, or any value between 32 and 35℃, preferably 32.5 to 34℃.

[0029] The culture rotation speed can be, but is not limited to, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm or 150 rpm, or any value between 100 and 150 rpm, preferably 100 to 120 rpm.

[0030] The culture time can be, but is not limited to, 48h, 50h, 53h, 55h, 58h, 60h, 63h, 65h, 68h or 72h, or any value between 48 and 72h, preferably 48 to 60h.

[0031] In some specific embodiments, the temperature of the virus culture medium is lowered to 2-8°C before centrifugation.

[0032] In some specific embodiments, the centrifugation speed is 5000~12000 rpm; in some specific embodiments, the centrifugation time is 3~4 hours.

[0033] The centrifugation speed can be, but is not limited to, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, 10000 rpm, 11000 rpm or 12000 rpm, or any value between 5000 and 12000 rpm, preferably 6000 to 8000 rpm.

[0034] The centrifugation time can be, but is not limited to, 3h, 3.1h, 3.2h, 3.3h, 3.4h, 3.5h, 3.6h, 3.7h, 3.8h, 3.9h or 4h, or any value between 3 and 4h, preferably 3.5h.

[0035] In some specific embodiments, the microfiltration process includes hollow fiber column microfiltration.

[0036] To further confirm the integrity of the membrane, in some specific implementations, if the turbidity value of the transmissive liquid is <4 NTU, chicken embryo testing is performed.

[0037] In some specific embodiments, the chicken embryo detection includes inoculating SPF chicken embryos with permeate as the test group and uninoculated SPF chicken embryos as the control group, and incubating for 96 hours; if the control group chicken embryo fluid produces HA titer, the chicken embryo detection is repeated; if the control group chicken embryo fluid has no HA titer and the test group chicken embryo fluid has an HA titer ≥ 1:2, the membrane coating is incomplete; if neither the control group chicken embryo fluid nor the test group chicken embryo fluid has HA titer, the test group chicken embryo fluid is inoculated into SPF chicken embryos as the repeat test group, and incubated for 96 hours; if the repeat test group chicken embryo fluid has an HA titer < 1:2, the membrane coating is complete; if the repeat test group chicken embryo fluid has an HA titer ≥ 1:2, the membrane coating is incomplete.

[0038] To ensure the accuracy of the results, in some specific embodiments, the chicken embryo detection further includes recording the number of dead chicken embryos between 24 and 48 hours of incubation or between 24 and 72 hours of incubation. If the number of dead chicken embryos is greater than 50%, the chicken embryo detection is repeated; if the number of dead chicken embryos is ≤50%, incubation continues. In some specific embodiments, the chicken embryo detection also includes excluding chicken embryos that died after 24 hours of incubation, to further improve the accuracy of the results.

[0039] The above-described detection method enables the monitoring of incompleteness during the use of ultrafiltration membranes, ensuring the effectiveness of virus purification and concentration. According to another aspect of the invention, the application of the above-described detection method in detecting the integrity of the ultrafiltration membrane package during virus production is also provided.

[0040] In some specific implementations, the virus includes avian influenza virus.

[0041] The term "avian influenza virus" in this article includes wild-type avian influenza virus and / or recombinant avian influenza virus, specifically including at least one of the H7N9, H5N6, and H5N8 subtypes.

[0042] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0043] The recombinant avian influenza virus strains H7N9 H7-Re4, H5N6 H5-Re13, and H5N8 H5-Re14 all originated from Jilin Guanjie Biotechnology Co., Ltd.

[0044] Example 1 In this embodiment, viral fluid of recombinant avian influenza virus strain H7N9 H7-Re4 was used for ultrafiltration membrane envelope integrity testing: 1. Preparation of Clarified Virus Fluid (1) Recombinant avian influenza virus H7N9 H7-Re4 strain was cultured in a 1000-liter stainless steel reactor using serum-free culture medium (Bean-German, MDCK serum-free medium) at a temperature of 32℃-35℃ and a rotation speed of 110 rpm. Viral HA and cell viability were monitored every 12 hours during the 24-72 hour period. When the virus reached the harvest state, it was cooled to 4℃ and centrifuged. The centrifuged virus solution was concentrated by hollow fiber column microfiltration and then analyzed according to the method of this invention. The initial inoculation density of recombinant avian influenza virus H7N9 H7-Re4 strain was 1.0 × 10⁻⁶. 6 cells / ml, 2.0×10 6 cells / ml, 3.0×10 6 cells / ml, 4.0×10 6 cells / ml, 5.0×10 6 cells / ml, 6.0×10 6 cells / ml, 7.0×10 6 cells / ml, 8.0×10 6 cells / ml, 9.0×10 6 cells / ml and 1.0×10 7The culture speed and temperature have a relatively small impact on the initial turbidity, mainly affecting cell and virus growth. The culture speed is 100-150 rpm, preferably 100 rpm, 110 rpm, or 120 rpm. The culture temperature is controlled at 32℃-35℃, preferably 32.6℃, 33℃, or 34℃. Initial density and culture time directly affect the initial turbidity. The centrifugation speed during pretreatment affects the next processing step. In this embodiment, based on data from large-scale production trials, the culture temperature is fixed at 33℃ and the culture speed is fixed at 110 rpm. The preparation conditions are shown in Table 1. The turbidity values ​​after culture under different conditions are recorded in Table 2, and the HA values ​​after centrifugation at different speeds are recorded in Table 3.

[0045] Table 1

[0046] (2) The culture medium of recombinant avian influenza virus H7N9 H7-Re4 strain was centrifuged. Before centrifugation, the virus solution needed to be cooled to 4°C to protect the virus. The centrifuge speed was 5000rpm-12000rpm. In this example, 6000rpm, 7000rpm and 8000rpm were set respectively, and centrifuged for 3.5h. The turbidity value and corresponding HA value after centrifugation under different speed conditions were recorded, as shown in Tables 2 and 3.

[0047] (3) The recombinant avian influenza virus H7N9 H7-Re4 strain virus solution after centrifugation at different speeds was passed through a hollow fiber column (PALL, 0.65μm, 10.8m). 2 The virus solution was subjected to microfiltration, and the turbidity value of the clarified virus solution after microfiltration was recorded, as shown in Table 2.

[0048] Table 2

[0049] Table 3

[0050] Based on the comprehensive analysis of the data in Tables 1, 2, and 3, as well as the viral titer, the initial culture density was 6.0 × 10⁻⁶. 6 cells / ml - 8.0 × 10 6 During the cell / ml period, the viral HA value was the highest and stable, so the initial density value in this range was taken as the optimal value; the HA value was the highest during 48-60 hours of culture, so this was taken as the optimal culture time; when the centrifugation speed was 8000 rpm, some experimental groups experienced HA loss, with the HA titer decreasing by one, so the centrifugation speed of 6000-7000 rpm was the optimal parameter.

[0051] Based on the above optimal parameters, the preparation process of the clarified virus solution is as follows: First, select an initial culture density of 6.0 × 10⁻⁶. 6 cells / ml up to 8.0 × 10 6 Cells were inoculated and cultured at a density within the range of cells / ml to ensure high viral HA expression and stability. Secondly, the culture time was controlled between 48 and 60 hours to allow the virus to fully proliferate and reach its optimal state. Finally, in the centrifugation stage, a speed of 6000 to 7000 rpm was used to avoid HA loss and ensure the purity and activity of the viral solution. Through these steps, a high-quality, clear viral solution can be prepared.

[0052] 2. Membrane wrapping installation: For damaged membranes (25m...) 2 (The membrane pack is a PALL 300KD, which was found to be damaged by the integrity testing equipment.) Install, clean and sterilize according to the manufacturer's installation standards and operating procedures, start the machine and wait for it to run stably.

[0053] 3. Testing 3.1 The recombinant avian influenza virus strain H7N9 H7-Re4 was passed through a damaged ultrafiltration membrane. The permeate from the rear end of the membrane was collected, and its turbidity was measured. The turbidities were 6.9 NTU, 5.1 NTU, 4.0 NTU, 4.8 NTU, 6.2 NTU, 5.6 NTU, 6.0 NTU, and 5.3 NTU, respectively.

[0054] 3.2 Chicken embryo method for detection: Samples with the minimum turbidity value were tested (since multiple concentration gradients were set in this embodiment, the minimum turbidity value was selected. If the minimum turbidity value passed the chicken embryo test without any problems, the others that were higher than the minimum value were considered qualified. In actual applications, only one concentration setting is required). Ten 10-day-old SPF chicken embryos were taken, with two controls numbered 1# and 2#, and eight embryos were inoculated with membrane permeate solution through the allantoic cavity, 0.1 ml per embryo, numbered 3#, 4#, 5#, 6#, 7#, 8#, 9#, and 10#. After incubation at 33°C for 96 hours, the HA titer of the 10 chicken embryos was tested. The HA titers of the two controls were invalid. The HA titers of the eight chicken embryos inoculated with membrane permeate solution through the allantoic cavity were: 3# (1:4), 4# (1:8), 5# (1:4), 6# (1:4), 7# (1:2), 8# (1:4), 9# (1:8), and 10# (1:4). Based on the results, the integrity of the membrane was deemed unqualified.

[0055] 3.3 Results: The ultrafiltration membrane packing was incomplete, and the turbidity values ​​were all ≥4 NTU. The test results were the same as those of the actual intact membrane packing.

[0056] Example 2 The difference from Example 1 is: 2. Membrane pack installation: First, install the new membrane pack (25m). 2 (The membrane pack is a PALL 300KD, which has been tested and found to be intact.) Install, clean and sterilize the membrane pack according to the manufacturer's installation standards and operating procedures, start the machine and wait for it to run stably.

[0057] 3. Testing 3.1 Recombinant avian influenza virus strain H7N9 H7-Re4 with different initial turbidity values ​​was passed through an ultrafiltration membrane. The permeate from the rear end of the membrane was collected, and its turbidity was measured. The turbidity values ​​were 1.4 NTU, 2.5 NTU, 2.2 NTU, 3.8 NTU, 1.9 NTU, 3.5 NTU, 2.9 NTU, 2.7 NTU, 3.9 NTU, and 3.4 NTU.

[0058] 3.2 Chicken embryo method for detection: For the sample testing the maximum turbidity value (since multiple concentration gradients are set in this example, the maximum turbidity value is selected; if the maximum turbidity value passes the chicken embryo test without problems, the others below the maximum value are considered qualified; in actual applications, only one concentration setting is needed), take 10 10-day-old SPF chicken embryos, 2 as controls, numbered 1# and 2#, and 8 as controls, each inoculated with 0.1 ml of membrane-permeable solution through the allantoic cavity, numbered 3#, 4#, 5#, 6#, and 7#. #, 8, 9, and 10 chicken embryos were incubated at 33℃ for 96 hours in an incubator. The HA titer of the incubator fluid was then tested. Two control embryos had no HA titer. The HA titers of the eight chicken embryos inoculated with the membrane permeate fluid through the allantoic cavity were: 3# (<1:2), 4# (<1:2), 5# (<1:2), 6# (<1:2), 7# (<1:2), 8# (<1:2), 9# (<1:2), and 10# (<1:2). Based on the results, the membrane integrity was deemed acceptable.

[0059] 3.3 Results: The ultrafiltration membrane packing was intact, and the turbidity values ​​were all <4 NTU. The test results were consistent with the actual membrane packing integrity.

[0060] Example 3 Unlike Example 1, the recombinant avian influenza virus strain H5N6 H5-Re13 was selected.

[0061] 1. The optimal parameters in Example 1 were selected for the preparation conditions of the virus solution.

[0062] 2. Membrane installation: First, repair the damaged membrane (25m). 2 (The membrane pack is a PALL 300KD, which was found to be damaged by the integrity testing equipment.) Install, clean and sterilize according to the manufacturer's installation standards and operating procedures, start the machine and wait for it to run stably.

[0063] 3. Testing 3.1 The recombinant avian influenza virus strain H5N6 H5-Re13 was passed through a damaged ultrafiltration membrane. The permeate from the rear end of the membrane was collected, and its turbidity was measured. The turbidity values ​​were 4.9 NTU, 5.6 NTU, 4.4 NTU, 6.9 NTU, 4.7 NTU, 4.1 NTU, 4.0 NTU, 5.3 NTU, 6.1 NTU, and 4.4 NTU.

[0064] 3.2 Chicken embryo method for detection: For the minimum turbidity value test, 10 SPF chicken embryos of 10 days old were used, 2 were controls, numbered 1# and 2#, and 8 were inoculated with membrane permeate solution through the allantoic cavity, 0.1 ml each, numbered 3#, 4#, 5#, 6#, 7#, 8#, 9#, and 10#. After incubation at 33℃ for 96 hours, the HA titer of the 10 chicken embryos was tested. The HA titers of the 2 controls were invalid. The HA titers of the 8 chicken embryos inoculated with membrane permeate solution through the allantoic cavity were: 3# (1:4), 4# (1:2), 5# (1:4), 6# (1:4), 7# (1:2), 8# (1:4), 9# (1:8), and 10# (1:4). Based on the results, the integrity of the membrane was deemed unqualified.

[0065] 3.3 Results: The ultrafiltration membrane packing was incomplete, and the turbidity values ​​were all ≥4 NTU. The test results were the same as those of the actual intact membrane packing.

[0066] Example 4 The difference from Example 3 is: 2. Membrane pack installation: First, install the new membrane pack (25m). 2 (The membrane pack is a PALL 300KD, which has been tested and found to be intact.) Install, clean and sterilize the membrane pack according to the manufacturer's installation standards and operating procedures, start the machine and wait for it to run stably.

[0067] 3. Testing 3.1 Recombinant avian influenza virus strain H5N6 H5-Re13 with different initial turbidity values ​​was passed through an ultrafiltration membrane. The permeate from the rear end of the membrane was collected, and its turbidity was measured. The turbidity values ​​were 2.1 NTU, 3.6 NTU, 2.8 NTU, 3.9 NTU, 2.7 NTU, 1.9 NTU, 3.7 NTU, 2.6 NTU, 2.3 NTU, and 3.4 NTU.

[0068] 3.2 Chicken embryo method for detection: For testing the maximum turbidity value, 10 SPF chicken embryos at 10 days old were used, 2 were controls (numbered 1# and 2#), and 8 were inoculated with membrane permeate solution via the allantoic cavity (0.1 ml per embryo, numbered 3#, 4#, 5#, 6#, 7#, 8#, 9#, and 10#). After incubation at 33℃ for 96 hours, the HA titer of the 10 chicken embryos was tested. The HA titers of the 2 controls were invalid. The HA titers of the 8 chicken embryos inoculated with membrane permeate solution via the allantoic cavity were: 3# (<1:2), 4# (<1:2), 5# (<1:2), 6# (<1:2), 7# (<1:2), 8# (<1:2), 9# (<1:2), and 10# (<1:2). The membrane integrity was determined to be acceptable based on the results.

[0069] 3.3 Results: The ultrafiltration membrane packing was intact, and the turbidity values ​​were all <4 NTU. The test results were consistent with the actual membrane packing integrity.

[0070] Example 5 Unlike Example 1, the recombinant avian influenza virus strain H5N8 H5-Re14 was selected.

[0071] 1. The optimal parameters in Example 1 were selected for the preparation conditions of the virus solution.

[0072] 2. Membrane installation: First, repair the damaged membrane (25m). 2 (The membrane pack is a PALL 300KD, which was found to be damaged by the integrity testing equipment.) Install, clean and sterilize according to the manufacturer's installation standards and operating procedures, start the machine and wait for it to run stably.

[0073] 3. Testing 3.1 The recombinant avian influenza virus strain H5N8 H5-Re14 was passed through a damaged ultrafiltration membrane. The permeate from the rear end of the membrane was collected, and its turbidity was measured. The turbidity values ​​were 6.8 NTU, 6.0 NTU, 4.8 NTU, 5.2 NTU, 4.4 NTU, 4.2 NTU, 6.9 NTU, 5.6 NTU, 4.0 NTU, and 6.3 NTU.

[0074] 3.2 Chicken embryo method for detection: For the minimum turbidity value test, 10 SPF chicken embryos of 10 days old were used, 2 were controls, numbered 1# and 2#, and 8 were inoculated with membrane permeate solution through the allantoic cavity, 0.1 ml each, numbered 3#, 4#, 5#, 6#, 7#, 8#, 9#, and 10#. After incubation at 33℃ for 96 hours, the HA titer of the 10 chicken embryos was tested. The HA titers of the 2 controls were invalid. The HA titers of the 8 chicken embryos inoculated with membrane permeate solution through the allantoic cavity were: 3# (1:8), 4# (1:4), 5# (1:8), 6# (1:8), 7# (1:8), 8# (1:8), 9# (1:8), and 10# (1:4). Based on the results, the integrity of the membrane was deemed unqualified.

[0075] 3.3 Results: The ultrafiltration membrane packing was incomplete, and the turbidity values ​​were all ≥4 NTU. The test results were the same as those of the actual intact membrane packing.

[0076] Example 6 The difference from Example 5 is: 2. Membrane pack installation: First, install the new membrane pack (25m). 2 (The membrane pack is a PALL 300KD, which has been tested and found to be intact.) Install, clean and sterilize the membrane pack according to the manufacturer's installation standards and operating procedures, start the machine and wait for it to run stably.

[0077] 3. Testing 3.1 The recombinant avian influenza virus strain H5N8 H5-Re14 was passed through an ultrafiltration membrane. The permeate from the rear end of the membrane was collected, and its turbidity was measured. The turbidity values ​​were 3.2 NTU, 2.8 NTU, 3.0 NTU, 2.2 NTU, 2.7 NTU, 3.8 NTU, 2.6 NTU, 3.4 NTU, 2.9 NTU, and 3.9 NTU. 3.2 Chicken embryo method: For testing the maximum turbidity value, 10 SPF chicken embryos at 10 days old were used, 2 were controls (numbered 1# and 2#), and 8 were inoculated with membrane permeate solution via the allantoic cavity (0.1 ml per embryo, numbered 3#, 4#, 5#, 6#, 7#, 8#, 9#, and 10#). After incubation at 33℃ for 96 hours, the HA titer of the 10 chicken embryos was tested. The HA titers of the 2 controls were invalid. The HA titers of the 8 chicken embryos inoculated with membrane permeate solution via the allantoic cavity were: 3# (<1:2), 4# (<1:2), 5# (<1:2), 6# (<1:2), 7# (<1:2), 8# (<1:2), 9# (<1:2), and 10# (<1:2). The membrane integrity was determined to be acceptable based on the results.

[0078] 3.3 Results: The ultrafiltration membrane packing was intact, and the turbidity values ​​were all <4 NTU. The test results were consistent with the actual membrane packing integrity.

[0079] Example 7 Unlike Example 1, recombinant avian influenza virus strains H5N6 H5-Re13, H5N8 H5-Re14, and H7N9 H7-Re4 were selected.

[0080] 1. The optimal parameters in Example 1 were selected for the preparation conditions of the virus solution. After culturing, the three strains can be mixed in proportions of 1:1:1, 1:1:2, 1:2:1, and 2:1:1. In this example, a 1:1:1 mixture was used.

[0081] 2. Membrane installation: First, repair the damaged membrane (25m). 2(The membrane pack is a PALL 300KD, which was found to be damaged by the integrity testing equipment.) Install, clean and sterilize according to the manufacturer's installation standards and operating procedures, start the machine and wait for it to run stably.

[0082] 3. Testing 3.1 Three recombinant avian influenza virus strains were passed through a damaged ultrafiltration membrane. The permeate from the rear end of the membrane was collected, and its turbidity was measured. The turbidity values ​​were 6.5 NTU, 5.0 NTU, 4.8 NTU, 4.0 NTU, 5.4 NTU, 4.2 NTU, 4.9 NTU, 5.1 NTU, 5.7 NTU, and 6.2 NTU.

[0083] 3.2 Chicken embryo method for detection: For the minimum turbidity value test, 10 SPF chicken embryos of 10 days old were used, 2 were controls, numbered 1# and 2#, and 8 were inoculated with membrane permeate solution through the allantoic cavity, 0.1 ml each, numbered 3#, 4#, 5#, 6#, 7#, 8#, 9#, and 10#. After incubation at 33℃ for 96 hours, the HA titer of the 10 chicken embryos was tested. The HA titers of the 2 controls were invalid. The HA titers of the 8 chicken embryos inoculated with membrane permeate solution through the allantoic cavity were: 3# (1:8), 4# (1:4), 5# (1:8), 6# (1:8), 7# (1:8), 8# (1:4), 9# (1:8), and 10# (1:4). Based on the results, the integrity of the membrane was deemed unqualified.

[0084] 3.3 Results: The ultrafiltration membrane packing was incomplete, and the turbidity values ​​were all ≥4 NTU. The test results were the same as those of the actual intact membrane packing.

[0085] Example 8 The difference from Example 7 is: 2. Membrane pack installation: First, install the new membrane pack (25m). 2 (The membrane pack is a PALL 300KD, which has been tested and found to be intact.) Install, clean and sterilize the membrane pack according to the manufacturer's installation standards and operating procedures, start the machine and wait for it to run stably.

[0086] 3. Testing 3.1 The three recombinant avian influenza virus strains were passed through an ultrafiltration membrane. The permeate from the rear end of the membrane was collected, and the turbidity of the permeate was measured. The turbidity values ​​were 3.6 NTU, 2.8 NTU, 3.7 NTU, 2.5 NTU, 3.3 NTU, 3.9 NTU, 1.9 NTU, 2.4 NTU, 2.8 NTU, and 3.1 NTU.

[0087] 3.2 Chicken embryo method for detection For testing the maximum turbidity value, 10 SPF chicken embryos at 10 days old were used, 2 were controls (numbered 1# and 2#), and 8 were inoculated with membrane permeate solution via the allantoic cavity (0.1 ml per embryo, numbered 3#, 4#, 5#, 6#, 7#, 8#, 9#, and 10#). After incubation at 33℃ for 96 hours, the HA titer of the 10 chicken embryos was tested. The HA titers of the 2 controls were invalid. The HA titers of the 8 chicken embryos inoculated with membrane permeate solution via the allantoic cavity were: 3# (<1:2), 4# (<1:2), 5# (<1:2), 6# (<1:2), 7# (<1:2), 8# (<1:2), 9# (<1:2), and 10# (<1:2). The membrane integrity was determined to be acceptable based on the results.

[0088] 3.3 Results: The ultrafiltration membrane packing was intact, and the turbidity values ​​were all <4 NTU. The test results were consistent with the actual membrane packing integrity.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the integrity of an ultrafiltration membrane module, characterized by, The method comprises filtering the clarified virus liquid through an ultrafiltration membrane to be detected, and if the turbidity value of the permeate is greater than or equal to 4 NTU, the membrane package is incomplete.

2. The detection method according to claim 1, characterized in that, The method for preparing the clarified virus liquid comprises taking virus culture liquid to be treated by virus culture, centrifugation and microfiltration.

3. The detection method according to claim 2, characterized in that, The initial inoculum density of the virus culture is 1.0 x 10 6 ~1.0 x 10 7 cells / ml, preferably 8.0 x 10 6 ~1.0 x 10 7 cells / ml.

4. The detection method according to claim 3, characterized in that, The culture temperature of the virus culture is 32-35 DEG C, preferably 32.5-34 DEG C. Preferably, the culture rotation speed of the virus culture is 100-150 rpm, preferably 100-120 rpm. Preferably, the culture time of the virus culture is 48-72 h, preferably 48-60 h.

5. The method of claim 2, wherein, The method further comprises reducing the temperature of the virus culture liquid to 2-8 DEG C before the centrifugation.

6. The method of claim 2, wherein, The rotation speed of the centrifugation is 5000-12000 rpm, preferably 6000-8000 rpm. Preferably, the centrifugation time is 3-4 h, preferably 3.5 h.

7. The method of claim 2, wherein The microfiltration treatment comprises hollow fiber column microfiltration.

8. The assay of any one of claims 1 to 7, wherein, The virus comprises avian influenza virus.

9. The use of the detection method according to any one of claims 1-8 in detecting the integrity of an ultrafiltration membrane package in virus production.

10. Use according to claim 9, characterized in that, The virus comprises avian influenza virus.

Images