Virus removal verification and evaluation method suitable for continuous nanofiltration process and application of virus removal verification and evaluation method

Abstract

The invention provides a virus removal verification evaluation method suitable for a continuous nanofiltration process and application thereof, the method comprises the following steps: a virus sample enters a pressure detector and an online mixing pool through a pipeline I, and the pipeline I is provided with a pump B; an intermediate sample enters the pressure detector and the online mixing pool through a pipeline II, and a pump A and a prefilter are arranged on the pipeline II; the virus sample and the intermediate sample are mixed in the on-line mixing pool through a pipeline; after mixing, enabling the mixture to sequentially flow through a conductance cell and an ultraviolet (UV) detection cell, and enabling a detected sample to enter a virus removal filter through a column position valve to be filtered; collecting a filtered sample, recording the filtering pressure, the flow velocity and the filtering volume of the sample by utilizing a protein purification instrument, detecting the virus quantity in the collected sample in an off-line manner to obtain a logarithmic decline value of the virus after nanofiltration, and judging the virus removal effect of the continuous nanofiltration process according to the parameter results.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a virus clearance verification and evaluation method suitable for a continuous nanofiltration process and an application thereof. Background Art

[0002] With the implementation of continuous perfusion cell culture, biopharmaceutical production has gradually evolved from traditional batch processing to continuous flow production. Compared to batch production, continuous flow production involves a continuous input of raw materials and a continuous output of product. This model not only improves production efficiency, reduces production time and costs, but also enhances product quality consistency.

[0003] Virus removal filtration, also known as nanofiltration, is a key downstream step in biopharmaceutical production. It intercepts viral particles through the use of specialized nanofiltration membranes, making it an important step in ensuring the safety of biopharmaceuticals. The traditional method for evaluating the removal effect of virus removal filtration is to use an air source and a sleeve to perform pre-filtration and virus removal filtration operations separately. By adding viruses to the pre-filtered sample, the virus removal ability of the nanofiltration membrane is evaluated separately. This evaluation method can eliminate the interference factors that may interfere with the removal of viruses by the pre-filtration membrane, but the disadvantage is that as the filtration time increases, the sample spiked with viruses increases the risk of aggregate formation. The formation of aggregates may cause the nanofiltration flow rate to decay too quickly, making it impossible to achieve the actual expected process load. At the same time, the aggregates of viruses and samples are more easily retained by the nanofiltration membrane, thereby overestimating the removal effect of the process. Therefore, the limitations of traditional evaluation methods are not applicable to continuous flow modes with long-term filtration. For continuous nanofiltration processes, more suitable evaluation methods for virus clearance verification need to be developed.

[0004] Currently, research on the viral clearance efficacy of continuous flow production models focuses primarily on continuous chromatography and continuous inactivation steps, with relatively little research on continuous nanofiltration. Therefore, providing a method for validating and evaluating viral clearance in continuous nanofiltration processes is of significant application value. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention aims to provide a method and application for the validation and evaluation of virus clearance in continuous nanofiltration processes. By modifying the protein purification system and changing the virus spike method used in traditional evaluation methods, the present invention establishes a scaled-down model for online continuous spiked virus removal filtration. This allows for real-time online mixing of viruses and pre-filtration intermediates. This specific mixing method can mitigate complications such as protein aggregation and decreased viral titer caused by prolonged filtration. The present invention is expected to provide a new solution for the validation and evaluation of virus clearance in continuous nanofiltration processes.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a virus clearance verification and evaluation method suitable for a continuous nanofiltration process, the method comprising: a virus sample enters a pressure detector and an online mixing pool through a first pipeline, wherein a B pump is provided on the first pipeline; an intermediate product sample enters a pressure detector and an online mixing pool through a second pipeline, wherein a A pump and a pre-filter are provided on the second pipeline; the virus sample and the intermediate product sample are mixed in the online mixing pool through the pipeline; after mixing, the samples flow through a conductivity cell and an ultraviolet (UV) detection cell in sequence, and the samples after detection enter a virus removal filter through a column valve for filtration; the filtered samples are collected, the filtration pressure, flow rate and filtration volume of the samples are recorded using a protein purification instrument, the amount of virus in the collected samples is detected offline, and a virus logarithmic reduction value (log 10 The virus removal effect of the continuous nanofiltration process was judged based on the above parameter results.

[0008] The present invention first changes the virus spiking procedure in the traditional evaluation method. With the help of the binary pump channel and mixing tank of the protein purification system, an online spiking method is used to mix the virus and the pre-filtered intermediate in real time online. The constant pressure control mode of the protein purification system is utilized, and a virus removal filter is connected at the column valve to complete the nanofiltration operation.

[0009] The design ideas of the present invention include:

[0010] 1. Modify the pipeline of pump A of the system, connect a 0.22μm syringe filter (optional) and a pre-filter. The sample passes through the liquid inlet of pump A and then flows through the pre-filter to produce the intermediate product.

[0011] 2. Select the pipeline of system B pump as the virus inlet.

[0012] 3. Set the gradient ratio of the A / B pump so that the intermediate and virus pass through the online mixing pool at a fixed gradient ratio and a certain flow rate to achieve the purpose of real-time online addition.

[0013] 4. Streamline the redundant branches of the protein purifier so that the online mixing pool is directly connected to the conductivity cell and the UV detection cell, and the column valve is connected after the UV cell to retain the function of real-time monitoring of conductivity and UV values.

[0014] 5. Select the column valve as the receiving position of the virus removal filter, and use the column switching function to realize the conversion of the nanofiltration membrane offline (Off-line) and online (Inline) state.

[0015] 6. Use the protein purifier's own pressure control mode to replace the traditional gas pressure control mode.

[0016] 7. The attenuation trend of nanofiltration can be intuitively presented through the graph and operation log of the protein purifier.

[0017] Preferably, the B pump and the A pump are used to control the mixing ratio of the virus sample and the intermediate sample.

[0018] Preferably, the ratio of the B pump to the A pump is set to 2.0-10.0% by the B pump and the rest by the A pump.

[0019] In the present invention, 2% and 10% are used as upper and lower limits, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, etc. The B pump ratio can be set to any value in the interval, and the rest are A pump ratios.

[0020] In the present invention, the ratio of pump B (virus) is optimized to 2.0-10.0%. A lower ratio requires higher precision and flow rate for pump B, which can increase injection volume errors. A higher ratio increases the impact of the virus solution on the sample system, potentially deviating from the nanofiltration process in terms of physical and chemical properties and concentration. After optimization, a ratio of 2.0-10.0% is more suitable. This optimized ratio can reduce sample dilution by the virus solution, preventing its impact on the sample buffer system (such as significant fluctuations in pH and conductivity) and significantly reducing sample concentration, which could deviate from the nanofiltration process.

[0021] Preferably, the pre-filter provided after the A pump is used to remove impurities or contaminants, such as protein aggregates.

[0022] Preferably, the pressure detector is used to set a constant nanofiltration pressure to control the nanofiltration flow rate.

[0023] Preferably, the pressure range is set to be less than or equal to the upper limit of the allowable working pressure of the virus removal filter.

[0024] In the present invention, the pressure value is set according to the upper limit of the allowable pressure when the virus removal filter is in the working state.

[0025] Preferably, the conductivity cell is used to monitor whether the buffer in the membrane rinsing step and the top wash step pipeline is full of the system; and to monitor whether the sample and virus in the protein purification system are mixed and stable.

[0026] Preferably, the ultraviolet (UV) detection cell is used to monitor whether the sample and virus in the protein purification system are mixed and stable.

[0027] Preferably, a 0.22 μm or 0.45 μm syringe filter is provided after the UV detection cell, and the syringe filter is used to remove particulate impurities, such as aggregates.

[0028] In the present invention, a 0.22 μm syringe filter or a 0.45 μm syringe filter is generally selected. A finer pore size may cause clogging of the membrane pores, and a higher pore size may not achieve the desired filtering effect.

[0029] Preferably, the column position valve serves as the receiving position of the virus removal filter, and utilizes the column position switching function to realize the conversion of the nanofiltration membrane between the closed and open states.

[0030] Preferably, the nanofiltration operation steps of the virus removal filter include: the sample flowing out of the online mixing pool enters the conductivity cell and the ultraviolet UV detection cell in sequence, operates under constant pressure control mode, and after the readings of the ultraviolet UV detection cell and the conductivity cell are stable, bypass sampling is performed to take a spiked sample (Spiked control) and a control sample (Hold control) respectively; using the column position switching function to switch to the nanofiltration membrane inline state for nanofiltration, and filter and collect samples.

[0031] Preferably, after the nanofiltration is completed, the method further comprises removing the 0.22 μm syringe filter or pre-filter at the front end of the pre-filter, setting the A pump ratio to 100%, and connecting the A pump inlet to the buffer solution to start top washing.

[0032] Preferably, the step of determining virus clearance verification of the continuous nanofiltration process comprises:

[0033] (1) Determine the real-time attenuation trend of the nanofiltration process based on the pressure curve, flow rate curve, and volume curve presented by the protein purification system;

[0034] (2) Offline detection of the amount of virus in the collected samples (log 10 ), and then the virus log reduction value (LRV) value before and after nanofiltration was obtained to judge the virus removal effect of the continuous nanofiltration process.

[0035] In the present invention, the virus removal effect of the nanofiltration process depends on the process conditions, such as the selection of membrane packages, process loading capacity, buffer system, etc. In the virus removal validation evaluation, an LRV ≥ 4 is generally considered to be a good effect.

[0036] In one embodiment of the present invention, the protein purification system is based on the present invention to achieve virus removal filtration operation.

[0037] The protein purification system includes: power pumps (pump A and pump B), a pressure detector, a mixing cell, a conductivity cell, a UV detection cell, and a column valve. The protein purification system is modified as follows:

[0038] 1. Modify the system A pump pipeline, use an adapter to connect the syringe filter (microfiltration, optional) and pre-filter at the pump outlet, connect the sample bottle to the pump inlet, and use the power of the pump to complete the pre-filtration operation to generate a pre-filtered intermediate product.

[0039] 2. Remove the redundant pipelines behind the pump and connect the mixing cell, conductivity cell, UV detection cell and column valve directly in series to reduce the dead volume of the system.

[0040] 3. Use pipelines and adapters to install the virus removal filter at the column valve.

[0041] The structure after transformation is as follows Figure 1 and Figure 2 As shown, Figure 1 It is a schematic diagram of the structure in the closed state; Figure 2 This is a schematic diagram of the structure in the open state.

[0042] Figure 1 Displays the offline status of the virus removal filter. In the current state, the bypass path realizes the rinsing of the pre-filtration membrane and the nanofiltration membrane, the stable mixing of the sample and the virus, and the sampling of the spiked sample and the control sample; Figure 2 Displays the online status of the virus removal filter, during which nanofiltration and topwash operations are performed. The entire design and construction process was completed by Suzhou WuXi Detection's Virus Removal Research Laboratory.

[0043] In a second aspect, the present invention provides the application of the virus clearance verification and evaluation method suitable for continuous nanofiltration process described in the first aspect in the continuous flow production of biological products.

[0044] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) Compared with traditional evaluation methods, the online spiking mode of this method can reduce complications such as protein aggregation and virus titer decrease caused by long-term filtration, avoid the rapid attenuation of virus removal filtration flow rate after spiking, and improve filtration efficiency and filtration capacity.

[0047] (2) The filtration flux is no longer limited by the volume of the liquid storage tank used in traditional methods, and virus removal filtration experiments with larger spiked sample volumes can be challenged.

[0048] (3) This method is easy to operate and eliminates the use of gas source and sleeve in traditional methods, avoiding repeated disassembly and reducing cross contamination of samples and viruses.

[0049] (4) This method uses the electronic recording function of the protein purification system to intuitively present the experimental process, achieve result traceability, and meet the audit tracking requirements of industry regulations, which is impossible to achieve with traditional evaluation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a schematic diagram of the structure in the closed state.

[0051] Figure 2 It is a schematic diagram of the structure in the open state.

[0052] Figure 3 It is a structural diagram of the traditional evaluation experiment.

[0053] Figure 4 This is a comparison of the nanofiltration attenuation trends between Comparative Example 1 and Example 1.

[0054] Figure 5 This is a comparison of the nanofiltration attenuation trends between Example 2 and Example 2. DETAILED DESCRIPTION

[0055] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0056] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0057] Comparative Example 1

[0058] Validation and evaluation of traditional methods for the clearance of xenotropic murine leukemia virus (X-MuLV)

[0059] 1. Experimental Materials

[0060] The viruses in this comparative example were sourced from Suzhou WuXi Testing & Inspection Co., Ltd.; the pre-filter and virus removal filter were conventional products commercially available through formal channels.

[0061] 2. Experimental process

[0062] Traditional evaluation experiment:

[0063] Figure 3 This is a schematic diagram of the traditional evaluation experiment. Figure 3 As shown, prepare the gas source, pressure regulator and sleeve, assemble the pre-filter device, and rinse the pre-filter membrane with water and buffer at a constant pressure of 20.0 psi; remove the sleeve, add 190.65 mL of microfiltration sample (0.2 μm membrane filtration) into the sleeve, pre-filter at a pressure of 7.0 psi, and collect 190.59 mL of pre-filtered sample; remove the sleeve, assemble the virus removal filter device, and rinse the nanofiltration membrane with water and buffer at a constant pressure of 30.0 psi; add X-MuLV virus solution (virus amount 7.2 log10 ), mix well and take spiked samples and control samples; pour the experimental sample after adding virus into the sleeve, perform nanofiltration at a constant pressure of 30.0 psi, and collect the sample volume by filtration. The filtration time is 69 min; remove the sleeve, add buffer solution thereto, and perform top wash at a constant pressure of 30.0 psi, and the top wash collection volume is 10.74 mL.

[0064] 3. Experimental results

[0065] This comparative example compares the nanofiltration attenuation trend, the viral load of spiked samples and control samples, and the nanofiltration LRV results. The experimental process of the traditional evaluation method is shown in Table 1.

[0066] Table 1

[0067] Time (min) Volume (mL) <![CDATA[Filter loading (L / m 2 )]]> Flow rate (mL / min) Relative flux (%) 2 5.83 17.15 2.92 100.00 5 13.89 40.85 2.69 92.17 10 26.56 78.12 2.53 86.93 15 38.77 114.03 2.44 83.77 20 50.59 148.79 2.36 81.10 30 73.31 215.62 2.27 77.94 45 105.91 311.50 2.17 74.56 60 136.99 402.91 2.07 71.08 69 155.05 456.03 2.01 68.84

[0068] Example 1

[0069] Validation and evaluation of the method of the present invention for clearing X-MuLV virus

[0070] 1. Experimental Materials

[0071] The virus in this example was sourced from Suzhou WuXi Testing Co., Ltd.; the pre-filter and virus removal filter were conventional products commercially available through formal channels.

[0072] 2. Experimental process

[0073] First, according to the design ideas of the present invention, the protein purification system was modified; the constant pressure control mode was set throughout the process (column front pressure 0.21MPa); the pre-filtration membrane was rinsed with water, the pre-filtration membrane and nanofiltration membrane were rinsed with water, and the pre-filtration membrane and nanofiltration membrane were rinsed with buffer; the inlet of pump A was connected to the sample, and the inlet of pump B was connected to the virus dilution solution (virus amount 7.3log 10 ), set the ratio of pump A to 90.0% and pump B to 10.0%; Figure 1 When the UV reading is stable, perform bypass sampling and take spiked samples and control samples respectively; Figure 2 Switch to the nanofiltration membrane online state for nanofiltration, and collect the sample volume of 161.34 mL, with a filtration time of 78.1 min; remove the 0.22 μm syringe filter and pre-filter, set the A pump ratio to 100%, connect the A pump inlet to the buffer solution to start top wash, and the top wash collection volume is 11.04 mL.

[0074] 3. Experimental results

[0075] In this example, the attenuation trend of nanofiltration, the viral log of spiked samples and control samples were analyzed. 10The results were compared with the nanofiltration LRV results in three aspects, wherein the experimental process of the method of the present invention is shown in Table 2.

[0076] Table 2

[0077] Time (min) Volume (mL) <![CDATA[Filter loading (L / m 2 )]]> Flow rate (mL / min) Relative flux (%) 2 5.25 15.44 2.63 100.00 5 12.82 37.70 2.52 96.13 10 24.76 72.82 2.39 90.97 15 36.24 106.57 2.30 87.47 20 47.37 139.33 2.23 84.80 30 69.09 203.20 2.17 82.74 45 99.61 292.96 2.03 77.51 60 128.21 377.09 1.91 72.63 69 144.96 426.34 1.86 70.90 78.1 161.34 474.53 1.80 68.57

[0078] It can be seen from the experimental results of Comparative Example 1 and Example 1 that when performing virus removal verification and evaluation on a conventional virus removal filtration process, the method of the present invention can achieve the same evaluation effect as the traditional method.

[0079] Figure 4 This is a comparison chart of nanofiltration attenuation trends. Figure 4 It can be seen that for conventional virus removal filtration processes, the nanofiltration processes of the method of the present invention and the traditional method have similar attenuation trends.

[0080] Table 3 shows the virus load and nanofiltration LRV results.

[0081] Table 3

[0082]

[0083] As can be seen from Table 3, for conventional virus removal filtration processes, there are no significant differences in the virus load and nanofiltration LRV results of spiked samples and control samples between the method of the present invention and the traditional method.

[0084] In summary, the experimental results show that compared with the traditional evaluation method, the overall attenuation trend of the present invention is relatively close, and there is no significant difference in the spiking effect and virus removal effect, which indicates that the present method can achieve the same evaluation effect as the traditional method.

[0085] Comparative Example 2

[0086] Evaluation of the Validation of Traditional Methods for the Clearance of Murine Parvovirus (MVM)

[0087] 1. Experimental Materials

[0088] The viruses in this comparative example were sourced from Suzhou WuXi Testing & Inspection Co., Ltd.; the pre-filter and virus removal filter were conventional products commercially available through formal channels.

[0089] 2. Experimental process

[0090] Traditional evaluation experiments: Figure 3As shown, prepare the gas source and sleeve, assemble the pre-filtration device, and rinse the pre-filtration membrane with water and buffer at a constant pressure of 29.0 psi; remove the sleeve, add 252.44 mL of microfiltration sample (0.2 μm membrane filtration), pre-filter at 5.0 psi, and collect 244.60 mL of pre-filtered sample; remove the sleeve, assemble the virus removal filter device, and rinse the nanofiltration membrane with water and buffer at a constant pressure of 29.0 psi; add MVM virus solution (virus amount 7.2 log 10 ), mix well and take spiked samples and control samples; pour the experimental sample after adding virus into the sleeve, perform nanofiltration at a constant pressure of 29.0 psi, and collect the sample with a volume of 230.30 mL. The filtration time is 115.6 min; remove the sleeve, add buffer solution, and perform top wash at a constant pressure of 29.0 psi. The top wash collection volume is 10.47 mL.

[0091] 3. Experimental results

[0092] This example compares the nanofiltration attenuation trend, the viral load of spiked samples and control samples, and the nanofiltration LRV results. The experimental process of the traditional evaluation method is shown in Table 4.

[0093] Table 4

[0094] Time (min) Volume (mL) <![CDATA[Filter loading (L / m 2 )]]> Flow rate (mL / min) Relative flux (%) 1.0 3.18 9.35 3.18 100.00 5.0 12.94 38.06 2.44 76.73 10.0 25.06 73.71 2.42 76.23 15.0 36.75 108.09 2.34 73.52 20.0 48.07 141.38 2.26 71.19 30.0 69.95 205.74 2.19 68.81 50.0 111.33 327.44 2.07 65.06 70.0 150.02 441.24 1.93 60.83 90.0 186.49 548.50 1.82 57.34 115.6 230.30 677.35 1.71 53.82

[0095] Example 2

[0096] Evaluation of the Clearance Verification of MVM by the Method of the Invention

[0097] 1. Experimental Materials

[0098] The viruses in this comparative example were sourced from Suzhou WuXi Testing & Inspection Co., Ltd.; the pre-filter and virus removal filter were conventional products commercially available through formal channels.

[0099] 2. Experimental process

[0100] The experiment of the present invention: First, according to the design idea of ​​the present invention, the protein purification system was modified; the constant pressure control mode was set throughout the process (column front pressure 0.20 MPa); the pre-filtration membrane was rinsed with water, the pre-filtration membrane and nanofiltration membrane were rinsed with water, and the pre-filtration membrane and nanofiltration membrane were rinsed with buffer; the inlet of pump A was connected to the sample, and the inlet of pump B was connected to the virus dilution solution (virus amount 7.8log 10 ), set the ratio of pump A to 97.0% and pump B to 3.0%; Figure 1 Run at constant pressure under the state, and after the UV and conductivity readings are stable, perform bypass sampling and take spiked samples and control samples respectively; Figure 2Switch to the nanofiltration membrane online state for nanofiltration, and collect the sample volume by filtration. The filtration time is 116.3 min. Remove the 0.22 μm syringe filter and pre-filter, set the A pump ratio to 100%, and perform buffer top wash. The top wash collection volume is 11.07 mL.

[0101] 3. Experimental results

[0102] This example compares the nanofiltration attenuation trend, the viral load of spiked samples and control samples, and the nanofiltration LRV results. The experimental process of the present invention is shown in Table 5.

[0103] Table 5

[0104]

[0105]

[0106] Comparative Example 2 and Example 2 verify that the present method has a greater performance advantage than the traditional method when the filtration time is longer and the filtration volume is larger.

[0107] Figure 5 For the comparison of nanofiltration attenuation trend, Figure 5 It can be seen that when the filtration time is longer and the filtration volume is larger, the nanofiltration attenuation trend of this method is slower than that of the traditional method.

[0108] Table 6 shows the virus load and nanofiltration LRV results.

[0109] Table 6

[0110]

[0111] As shown in Table 6, when the filtration time and filtration volume are longer, there is no significant difference in the virus amount and nanofiltration LRV results of the spiked samples and the control samples between the method of the present invention and the traditional method.

[0112] The experimental results show that compared with the traditional method, the nanofiltration attenuation trend of this method is slower, and there is no significant difference in the spike effect and virus removal effect. Figure 5 From the nanofiltration attenuation trend, it can be seen that compared with the evaluation results of Comparative Example 2, the results of Example 2 show that under longer filtration time and more filtration volume, the present method has more performance advantages than the traditional method.

[0113] In summary, the present invention is authentic and reliable. Compared with the limitations of traditional methods that are prone to protein aggregation and virus inactivation during long-term filtration, the present invention adopts an online spiking mode to alleviate this situation. Therefore, it is more suitable for continuous nanofiltration processes with continuous filtration. It can also be used to verify and evaluate virus clearance for unstable samples or nanofiltration processes that attenuate too quickly using traditional spiking methods.

[0114] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A virus clearance validation and evaluation method suitable for continuous nanofiltration process, characterized in that: The method includes: a virus sample enters a pressure detector and an online mixing pool through a first pipeline, wherein a pump B is provided on the first pipeline; an intermediate product sample enters a pressure detector and an online mixing pool through a second pipeline, wherein a pump A and a pre-filter are provided on the second pipeline; the virus sample and the intermediate product sample are mixed in the online mixing pool through the pipeline; after mixing, the samples flow through a conductivity cell and an ultraviolet (UV) detection cell in sequence, and the samples after detection enter a virus removal filter through a column valve for filtration; the filtered samples are collected, the filtration pressure, flow rate and filtration volume of the samples are recorded using a protein purifier, the virus amount in the collected samples is detected offline, and a virus logarithmic reduction value after nanofiltration is obtained, and the virus removal effect of the continuous nanofiltration process is judged based on the above parameter results.

2. The virus clearance verification and evaluation method suitable for continuous nanofiltration process according to claim 1, characterized in that: The B pump and the A pump are used to control the mixing ratio of the virus sample and the intermediate sample; Preferably, the ratio of the B pump to the A pump is set to 2.0-10.0% by the B pump, and the rest by the A pump; Preferably, the pre-filter provided after the A pump is used to remove impurities or pollutants.

3. The virus clearance verification and evaluation method suitable for continuous nanofiltration process according to claim 1 or 2, characterized in that: The pressure detector is used to set a constant nanofiltration pressure to control the nanofiltration flow rate; Preferably, the pressure range is set to be less than or equal to the upper limit of the allowable working pressure of the virus removal filter.

4. The virus clearance validation and evaluation method applicable to a continuous nanofiltration process according to any one of claims 1 to 3, characterized in that: The conductivity cell is used to monitor whether the buffer solution in the membrane rinsing step and the top wash step pipeline is full of the system; and to monitor whether the sample and virus in the protein purification system are mixed and stable.

5. The virus clearance validation and evaluation method applicable to a continuous nanofiltration process according to any one of claims 1 to 4, characterized in that: The ultraviolet UV detection pool is used to monitor whether the sample and virus in the protein purification system are mixed and stable; Preferably, a 0.22 μm or 0.45 μm syringe filter is provided after the ultraviolet detection cell, and the syringe filter is used to remove particulate impurities.

6. The virus clearance validation and evaluation method applicable to a continuous nanofiltration process according to any one of claims 1 to 5, characterized in that: The column position valve serves as the receiving position of the virus removal filter and utilizes the column position switching function to realize the conversion between the closed and open states of the nanofiltration membrane.

7. The virus clearance validation and evaluation method for continuous nanofiltration process according to any one of claims 1 to 6, characterized in that: The nanofiltration operation steps of the virus removal filter include: the sample flowing out of the online mixing pool enters the conductivity cell and the ultraviolet UV detection cell in sequence, operates under a constant pressure control mode, and after the readings of the ultraviolet UV detection cell and the conductivity cell are stable, bypass sampling is performed to take a spiked sample and a control sample respectively; the column position switching function is used to switch to the nanofiltration membrane online state for nanofiltration, and the sample is filtered and collected.

8. The virus clearance validation and evaluation method applicable to a continuous nanofiltration process according to any one of claims 1 to 7, characterized in that: After the nanofiltration is completed, the 0.22 μm syringe filter or pre-filter at the front end of the pre-filter is removed, the ratio of pump A is set to 100%, and the inlet of pump A is connected to the buffer solution to start top washing.

9. The virus clearance validation and evaluation method applicable to a continuous nanofiltration process according to any one of claims 1 to 8, characterized in that: The step of determining virus clearance verification of the continuous nanofiltration process includes: (1) Determine the real-time attenuation trend of the nanofiltration process based on the pressure curve, flow rate curve, and volume curve presented by the protein purification system; (2) Offline detection of the amount of virus in the collected samples was performed to obtain the logarithmic reduction value of the virus after nanofiltration, thereby determining the virus removal effect of the continuous nanofiltration process.

10. Use of the virus clearance validation and evaluation method suitable for a continuous nanofiltration process according to any one of claims 1 to 9 in the continuous flow production of biological products.

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