Verification device and verification method for verifying filterability of virus removal filter

Abstract

The invention relates to a verification device and a verification method for verifying filterability of a virus removal filter. The verification device comprises a sample introduction part, a pre-filter, a mixing part, a to-be-verified virus removal filter and a collection part which are sequentially connected in series, the verification device further comprises a virus adding part and a sampling part, the virus adding part is communicated with an inlet of the mixing part and used for adding viruses into the mixing part, and the sampling part is communicated with an outlet of the pre-filter. The sampling part is communicated with the outlet of the mixing part and is used for sampling the mixed liquid flowing out of the mixing part; the sampling component is further provided with a pressure balancing component used for balancing the pressure difference between the mixing component and the sampling component when sampling is started. According to the verification device and the verification method, the problem of re-aggregation in the verification process can be avoided, so that more real and credible filterable data support is provided for application of the virus removal filter.

Description

Technical Field

[0001] The present invention relates to the technical field of virus filtration, and particularly to a verification device and a verification method for verifying the filterability of a virus removal filter. Background Art

[0002] All kinds of biological agents have developed vigorously because they can prevent, treat, and diagnose various infectious diseases, immune diseases, and other diseases that are difficult to prevent and control by conventional methods. Biological agents are generally processed from microorganisms (such as bacteria, rickettsiae, viruses, etc.), their metabolite effective antigen components, animal toxins, human or animal blood or tissues, etc. In addition to general production requirements, the production of vaccines, broad-spectrum biological agents, etc. also has a series of characteristics. For example, they generally require the cultivation of microorganisms, viruses, living cells, etc., and then subsequent processing of the obtained biological substances. At the same time, there are also operations such as purification, cleaning, inactivation, extraction, freezing, freeze-drying, etc.

[0003] During the production process of biological agents, it is difficult to avoid introducing various viruses, which poses a challenge to the virus safety of biological agents. Considering the important impact of viruses on the safety of biological agents, relevant documents such as the Chinese Pharmacopoeia 2020 Edition and ICH Q5A "Viral Safety Evaluation of Biologics" have clearly defined the virus safety requirements for biological agents. When applying for drug approval, the content of the virus safety assessment test report of biological agents will also directly affect the review results. In fact, at present, for various biological and pharmaceutical enterprises, the production of biological agents almost inevitably includes virus removal and / or virus inactivation processes to ensure the virus safety of the prepared biological agents.

[0004] Therefore, filtration using a virus removal filter is an essential process. Due to the advantages of high separation efficiency, low energy consumption, no need for external chemical reagents, the ability to separate systems that cannot be separated by conventional methods (such as azeotropic systems), not easily causing denaturation of active substances, and being able to be directly scaled up for large-scale production, membrane separation technology is applied by various biological and pharmaceutical enterprises to the virus removal process in the production process of biological agents. The virus removal filter removes viruses in the fluid flowing through the filter membrane through the particle size exclusion mechanism. In order to understand the virus removal ability of the virus removal membrane purification process, it is necessary to first add viruses to verify the filterability of the virus removal filter and calculate the virus removal rate to ensure that the virus removal results can be applied to biological therapy products.

[0005] Traditional virus removal verification methods include: mixing the feed liquid with the virus liquid, then performing batch pre-filtration treatment, and then verifying through the virus removal filter. However, the mixed liquid after batch pre-filtration is extremely prone to re-aggregation problems in a short time, resulting in the flux and load of virus filtration verification deviating from the true data. Summary of the Invention

[0006] Based on this, it is necessary to provide a verification device and a verification method for verifying the filterability of a virus removal filter for the above problems. The verification device and the verification method can avoid the problem of re-aggregation during the verification process, thereby providing more authentic and reliable filterability data support for the application of the virus removal filter.

[0007] The present invention discloses a verification device for verifying the filterability of a virus removal filter, including a sample injection component, a pre-filter, a mixing component, a virus removal filter to be verified, and a collection component connected in series in sequence. The verification device further includes a virus addition component and a sampling component. Among them, the virus addition component is communicated with the inlet of the mixing component for adding virus into the mixing component, and the sampling component is communicated with the outlet of the mixing component for sampling the mixed liquid flowing out of the mixing component; the sampling component is further provided with a pressure balancing component for balancing the pressure difference between the mixing component and the sampling component at the start of sampling.

[0008] In one embodiment, the sampling component includes a first container communicated with the mixing component through a first conduit. The first container is communicated with the first conduit through a first three-way valve, and the side port of the first three-way valve is connected with the pressure balancing component.

[0009] In one embodiment, the virus addition component and the sampling component are driven by a bidirectional pump.

[0010] In one embodiment, the pre-filter satisfies at least one of the following conditions:

[0011] (1) The filter material of the pre-filter includes a depth filtration medium or a filter membrane;

[0012] (2) The pore size of the filter material of the pre-filter is 0.2 μm - 0.45 μm.

[0013] In one embodiment, the mixing component is selected from a static mixer or a spiral pipeline mixer.

[0014] In the verification device for verifying the filterability of a virus removal filter provided by the present invention, first, the sample injection component, the pre-filter, the mixing component, the virus removal filter to be verified, and the collection component are connected in series in sequence. At the same time, the virus addition component is communicated with the inlet of the mixing component, capable of mixing the virus with the pre-filtered liquid in real time and directly filtering it through the virus removal filter to be verified, thus eliminating the step of batch pre-filtering after the liquid and the virus are mixed, avoiding the occurrence of re-aggregation problems, and further making the data such as the flux or loading capacity of the virus removal filter obtained by verification close to the real data. Secondly, due to the pressure difference between the mixing component and the sampling component, at the initial stage of sampling, the mixed liquid downstream of the mixing component will be ejected into the sampling component, resulting in a smaller filtered volume in the collection component. This will not only make the calculated flux of the virus removal filter smaller, affecting the authenticity of the flux decay curve, but also reduce the virus concentration in the collection component, resulting in a smaller calculated loading capacity of the virus removal filter. To avoid the situation of liquid ejection, the sampling component is provided with a pressure balancing component, which can balance the pressure difference between the mixing component and the sampling component at the start of sampling, avoiding the situation of liquid ejection caused by the pressure difference, thereby improving the authenticity of the filterability report. Thus, it provides more real and reliable filterability data support for the application of the virus removal filter.

[0015] The present invention also discloses a verification method for verifying the filterability of a virus removal filter, including the following steps:

[0016] S10, Provide the verification device as described above, and perform pre-treatment on the sample injection component, the pre-filter, the mixing component, the virus removal filter, and the collection component;

[0017] S20, Use the pressure balancing component to balance the pressure difference between the sampling component and the mixing component;

[0018] S30, Add liquid to the sample injection component for sample injection, record the filtration time, the filtration volume, and the real-time flow rate of the liquid;

[0019] S40, Operate the virus addition component, add the virus to the mixing component and adjust the virus addition speed according to the real-time flow rate of the liquid, and at the same time operate the sampling component to sample the mixed liquid downstream of the mixing component;

[0020] S50, Draw the flux decay curve of the virus removal filter according to the filtration time and the filtration volume, and output a filterability report.

[0021] In an embodiment, step S20 includes the following steps:

[0022] Use the pressure balancing component to make the pressure of the sampling component equal to the injection pressure minus the partial pressure of the pre-filter.

[0023] In one embodiment, step S20 includes the following steps:

[0024] First, adjust the first three-way valve to connect the first container with the pressure balancing component, and through the pressure balancing component, make the pressure in the first container equal to the injection pressure minus the partial pressure of the pre-filter. Then, adjust the first three-way valve to connect the first conduit with the first container.

[0025] In one embodiment, the partial pressure of the pre-filter in step S20 is calculated according to the injection pressure and the regression equation of the injection pressure and the partial pressure of the pre-filter, wherein the regression equation is obtained by testing the partial pressure of the pre-filter under different injection pressures and performing linear fitting.

[0026] In one embodiment, the partial pressure of the pre-filter in step S20 is measured by setting a pressure gauge between the pre-filter and the mixing component.

[0027] In one embodiment, in step S30, the real-time flow rate of the feed liquid is calculated according to the filtration time and the filtration volume. The real-time flow rate of the feed liquid = △filtration volume / t, where △filtration volume is equal to the filtration volume minus the filtration volume t seconds ago, and t is less than or equal to 30 min.

[0028] In one embodiment, the addition rate of the virus is equal to the real-time flow rate of the feed liquid multiplied by the theoretical spiking percentage of the virus, or the addition rate of the virus = the real-time flow rate of the feed liquid × (total planned virus addition amount / total planned feed liquid addition amount).

[0029] In one embodiment, the theoretical spiking percentage of the virus is less than or equal to 10%, and the value of (total planned virus addition amount / total planned feed liquid addition amount) is less than or equal to 10%.

[0030] In one embodiment, step S50 includes the following steps:

[0031] After the filtration is completed, calculate the flux of the virus removal filter according to the filtration time and the filtration volume. Take the filtration time as the horizontal axis and the flux of the virus removal filter as the vertical axis to plot the attenuation curve of the virus removal filter, and output a filterability report, where the flux of the virus removal filter = the filtration volume / (the filtration time × the membrane area of the virus removal filter).

[0032] In one embodiment, step S10 includes the following steps:

[0033] S101, connect the injection component, the pre-filter, the mixing component, the virus removal filter, and the collection component in series;

[0034] S102, perform exhaust treatment, wetting treatment, and balancing treatment on the sample introduction component, the pre-filter, the mixing component, the virus removal filter, and the collection component in sequence;

[0035] S103, connect the virus addition component to the inlet of the mixing component, and connect the sampling component to the outlet of the mixing component.

[0036] The verification method for verifying the filterability of the virus removal filter provided by the present invention is carried out by using the verification device provided by the present invention. The virus addition component can continuously add virus into the mixing component, and after being mixed with the pre-filtered liquid in real time, it is directly filtered through the virus removal filter to be verified, eliminating the step of batch pre-filtration after the liquid is mixed with the virus, avoiding the occurrence of re-aggregation problems, so that the data such as the flux or load of the virus removal filter obtained by verification is close to the real data; at the same time, before operating the sampling component, first use the pressure balancing component to balance the pressure difference between the sampling component and the mixing component, avoiding the situation of liquid jet caused by the pressure difference between the mixing component and the sampling component at the beginning of sampling, further improving the authenticity of the filterability report. Thus, it provides more real and reliable filterability data support for the application of the virus removal filter.

[0037] In addition, for the verification method for verifying the filterability of the virus removal filter provided by the present invention, adding virus at the inlet of the mixing component and sampling the effluent downstream of the mixing component can better represent the tandem production process of the pre-filter and the virus removal filter in the actual production process, better ensure the virus safety of biological products, improve the declaration success rate of biological products, and accelerate the listing process of biological products. Description of the Drawings

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0039] Figure 1 It is a schematic structural diagram of a verification device for verifying the filterability of a virus removal filter according to an embodiment provided by the present invention;

[0040] Figure 2 It is a schematic structural diagram of a verification device for verifying the filterability of a virus removal filter according to another embodiment provided by the present invention;

[0041] Figure 3 It is a regression curve graph of the injection pressure and the partial pressure of the pre-filter in Example 1;

[0042] Figure 4 It is a comparison graph of the flux decay curves output by Example 1 and Comparative Example 1;

[0043] Figure 5 It is a comparison graph of the total amount of virus at the collection end of Example 1 and Comparative Example 2.

[0044] In the figure, 10 is a sample injection component; 20 is a pre-filter; 30 is a mixing component; 40 is a virus removal filter; 50 is a collection component; 60 is a virus addition component; 601 is a second container; 602 is a second conduit; 70 is a sampling component; 701 is a first container; 702 is a first conduit; 703 is a first three-way valve; 704 is a pressure balance component; 80 is a two-way pump; 90 is a second three-way valve; 100 is a third three-way valve; 110 is a two-way valve. Detailed implementation manners

[0045] For the convenience of understanding the present invention, the present invention will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments or examples and are not intended to limit the present invention. The optional range of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include any two related listed items, any more related listed items, or the combination of all related listed items.

[0047] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to an integer, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0048] The present invention provides a verification device for verifying the filterability of a virus removal filter, such as Figure 1As shown in the figure, the verification device includes a sample injection component 10, a pre-filter 20, a mixing component 30, a virus removal filter 40 to be verified, and a collection component 50 connected in series in sequence. The verification device also includes a virus addition component 60 and a sampling component 70. Among them, the virus addition component 60 is communicated with the inlet of the mixing component 30 for adding virus into the mixing component 30, and the sampling component 70 is communicated with the outlet of the mixing component 30 to sample the mixed liquid flowing out of the mixing component 30. The sampling component 70 is also provided with a pressure balancing component 704 for balancing the pressure difference between the mixing component 30 and the sampling component 70 at the start of sampling.

[0049] The verification device for verifying the filterability of the virus removal filter 40 provided by the present invention can provide more authentic and reliable filterability data support for the application of the virus removal filter 40.

[0050] First of all, the sample injection component 10, the pre-filter 20, the mixing component 30, the virus removal filter 40 to be verified, and the collection component 50 are connected in series in sequence. At the same time, the virus addition component 60 is communicated with the inlet of the mixing component 30, which can mix the virus with the pre-filtered liquid in real time and directly filter it through the virus removal filter 40 to be verified, thus eliminating the step of batch pre-filtration after the liquid and the virus are mixed, avoiding the occurrence of re-aggregation problems, and further making the data such as the flux or loading capacity of the virus removal filter 40 obtained by verification close to the real data.

[0051] The sample injection component 10 is used to store the liquid to be sampled. In one embodiment, the sample injection component 10 is selected from a sleeve.

[0052] In one embodiment, the sample injection component 10 and the pre-filter 20 are communicated through a two-way valve 110.

[0053] The pre-filter 20 is used to remove the precipitates other than viruses in the liquid. In order to better remove the insoluble impurities in the liquid and avoid the insoluble impurities in the liquid from clogging the virus removal filter 40 and affecting the authenticity of the filterability report, in one embodiment, the filter material of the pre-filter 20 includes a deep filtration medium or a filter membrane. Preferably, the pore size of the filter material of the pre-filter 20 is 0.2μm - 0.45μm, including but not limited to 0.2μm, 0.25μm, 0.3μm, 0.35μm, 0.4μm or 0.45μm.

[0054] In one embodiment, the pre-filter 20 and the mixing component 30 are communicated through a second three-way valve 90, and the side port of the second three-way valve 90 is communicated with the virus addition component 60.

[0055] The mixing component 30 is used to mix the liquid material with the virus. In order to mix the liquid material with the added virus more fully and quickly and better avoid the occurrence of re-aggregation problems, in one embodiment, the mixing component 30 is selected from a static mixer or a spiral pipe mixer.

[0056] In one embodiment, the mixing component 30 and the virus removal filter 40 are connected through the third three-way valve 100, and the side port of the third three-way valve 100 is connected to the sampling component 70.

[0057] The collection component 50 is used to receive the liquid flowing out of the virus removal filter 40. In one embodiment, the collection component 50 is selected from a beaker.

[0058] In one embodiment, the virus removal filter 40 and the collection component 50 are connected through the two-way valve 110.

[0059] Since the luer connector has the advantages of simple structure, high reliability, flexible use, and being able to withstand the one-way or two-way flow of high-pressure liquid, preferably, the two-way valve 110 is selected from a two-way luer connector, and the second three-way valve 90 and the third three-way valve 100 are both selected from three-way luer connectors.

[0060] In one embodiment, the virus adding component 60 includes a second container 601 connected to the mixing component 30 through a second conduit 602. It can be understood that at this time, the second conduit 602 is connected to the side port of the second three-way valve 90.

[0061] In one embodiment, the sampling component 70 includes a first container 701 connected to the mixing component 30 through a first conduit 702. It can be understood that at this time, the first conduit 702 is connected to the side port of the third three-way valve 100.

[0062] Secondly, due to the pressure difference between the mixing component 30 and the sampling component 70, at the initial stage of sampling, the mixed liquid downstream of the mixing component 30 will be ejected into the sampling component 70, resulting in a smaller filtration volume in the collection component 50. This will not only make the calculated flux of the virus removal filter 40 smaller, affecting the authenticity of the flux decay curve, but also reduce the virus concentration in the collection component 50, resulting in a smaller calculated loading capacity of the virus removal filter 40. In order to avoid the situation of liquid ejection, the sampling component 70 is provided with a pressure balancing component 704. The pressure balancing component 704 can balance the pressure difference between the mixing component 30 and the sampling component 70 at the start of sampling, avoiding the situation of liquid ejection caused by the pressure difference, thereby improving the authenticity of the filterability report. Thus, it provides more authentic and reliable filterability data support for the application of the virus removal filter 40.

[0063] In one embodiment, the first container 701 is communicated with the first conduit 702 through a first three-way valve 703. A pressure balancing component 704 is connected to the side port of the first three-way valve 703. According to different injection pressures, the pressure balancing component 704 can fill different pressures into the first container 701, better avoiding the situation of liquid spraying caused by pressure difference. Since the luer connector has the advantages of simple structure, high reliability, flexible use, and being able to withstand the unidirectional or bidirectional flow of high-pressure liquid, therefore, preferably, both the first three-way valve 703 and the third three-way valve 100 are selected from three-way luer connectors.

[0064] The bi-directional pump 80 can accurately control the addition ratio of the virus. During the verification process, it can adjust the virus addition speed according to the attenuation of the feed liquid flow rate. Therefore, preferably, the virus addition component 60 and the sampling component 70 are driven by the bi-directional pump 80. The virus addition component 60 is driven by the advancing end of the bi-directional pump 80, and the sampling component 70 is driven by the collecting end of the bi-directional pump 80. Further preferably, as Figure 2 shown, the virus addition component 60 and the sampling component 70 are driven by the same bi-directional pump 80. It can be understood that at this time, when the first container 701 and the second container 601 have the same specifications, the first virus addition speed is the same as the sampling speed, that is, the addition amount added to the mixing component 30 is the same as the sampling amount from the downstream mixed liquid in terms of mass. Thus, it can better ensure that there is no additional partial pressure caused by different addition amounts and sampling amounts, so that the partial pressure of the virus removal filter 40 remains stable, and better improve the authenticity of the filterability report.

[0065] The present invention also discloses a verification method for verifying the filterability of the virus removal filter 40, including the following steps:

[0066] S10, provide the verification device as described above, and perform pretreatment on the sample injection component 10, pre-filter 20, mixing component 30, virus removal filter 40, and collection component 50;

[0067] S20, use the pressure balancing component 704 to balance the pressure difference between the sampling component 70 and the mixing component 30;

[0068] S30, add feed liquid to the sample injection component 10 for sample injection, and record the filtration time, filtration volume, and the real-time flow rate of the feed liquid;

[0069] S40, operate the virus addition component 60, add the virus to the mixing component 30 and adjust the virus addition speed according to the real-time flow rate of the feed liquid, and at the same time operate the sampling component 70 to sample the downstream mixed liquid of the mixing component 30;

[0070] S50, draw the flux attenuation curve of the virus removal filter 40 according to the filtration time and filtration volume, and output the filterability report.

[0071] In the verification method for verifying the filterability of the virus removal filter 40 provided by the present invention, each step cooperates with each other to greatly improve the authenticity of the filterability report. Specifically:

[0072] Step S10 provides the verification device as described above. When sampling the verification device provided in step S10, the virus addition component 60 can continuously add the virus into the mixing component 30, and directly filter it through the virus removal filter 40 to be verified after being mixed with the pre-filtered liquid in real time. The step of batch pre-filtering after the liquid and the virus are mixed is eliminated, avoiding the occurrence of re-aggregation problems, so that the data such as the flux or loading capacity of the virus removal filter 40 obtained by verification is close to the real data;

[0073] Step S20 uses the pressure balancing component 704 to balance the pressure difference between the sampling component 70 and the mixing component 30, avoiding the situation of liquid jet caused by the pressure difference between the mixing component 30 and the sampling component 70 at the beginning of sampling, and further improving the authenticity of the filterability report;

[0074] Step S30 obtains the filtration time, filtration volume, and the real-time flow rate of the liquid, and thus determines the virus addition speed according to the real-time rate and the virus spiking strategy;

[0075] Step S40 enables the virus to be evenly mixed with the liquid according to the planned addition amount;

[0076] Step S50 outputs a flux decay curve according to the filtration time and filtration volume, and outputs a filterability report, so as to intuitively understand the filterability of the virus removal filter 40.

[0077] Therefore, steps S10 - S50 provide more authentic and reliable filterability data support for the application of the virus removal filter 40.

[0078] Step S10 provides the verification device as described above and pre-treats the sample injection component 10, pre-filter 20, mixing component 30, virus removal filter 40, and collection component 50, so as to stabilize the partial pressure of the virus removal filter 40. In one embodiment, step S10 includes the following steps:

[0079] S101, connect the sample injection component 10, pre-filter 20, mixing component 30, virus removal filter 40, and collection component 50 in series;

[0080] S102, perform exhaust treatment, wetting treatment, and balancing treatment on the sample injection component 10, pre-filter 20, mixing component 30, virus removal filter 40, and collection component 50 in sequence;

[0081] S103. Connect the virus to the inlet of component 60 and the mixing component 30, and connect the sampling component 70 to the outlet of the mixing component 30.

[0082] Step S101 connects the sample injection component 10, the pre-filter 20, the mixing component 30, the virus removal filter 40, and the collection component 50 in series, which can simplify the experimental operation and shorten the experimental time.

[0083] In one embodiment, step S101 includes the following steps: connect the sample injection component 10 and the pre-filter 20 using a two-way valve 110, connect the pre-filter 20 and the mixing component 30 using a second three-way valve 90, connect the mixing component 30 and the virus removal filter 40 using a third three-way valve 100, and connect the virus removal filter 40 and the collection component 50 using a two-way valve 110.

[0084] Preferably, step S101 is carried out in a biosafety cabinet, thus providing a cleaner environment, which not only avoids introducing germs into the verification device, but also effectively protects the experimental personnel and the environment from potential hazards.

[0085] In step S102, it is preferable to use water for exhaust treatment and wetting treatment. The balance treatment can avoid the aggregation of the liquid material during the sample injection process, resulting in the blockage of the virus removal filter 40 and affecting the authenticity of the virus filtration verification. It is preferable to use the buffer solution of the liquid material for the balance treatment.

[0086] In one embodiment, step S103 includes the following steps: connect the side port of the second three-way valve 90 to the virus addition component 60, and connect the side port of the third three-way valve 100 to the sampling component 70.

[0087] When the virus addition component 60 and the sampling component 70 are driven by a two-way pump 80, and the virus addition component 60 includes a second container 601 connected to the mixing component 30 through a second conduit 602, the step of connecting the side port of the second three-way valve 90 to the virus addition component 60 includes: placing the second container 601 containing the virus at the advancing end of the two-way pump 80, and connecting the second conduit 602 to the side port of the second three-way valve 90; in order to avoid the residual air in the virus addition component 60 from entering the mixing component 30 and affecting the authenticity of the virus filtration verification, preferably, before the step of connecting the second conduit 602 to the side port of the second three-way valve 90, first evacuate the air in the second conduit 602, and then connect the second conduit 602 to the side port of the second three-way valve 90.

[0088] In step S20, there is no limitation on the step of using the pressure balancing component 704 to balance the pressure difference between the sampling component 70 and the mixing component 30, as long as the pressures between the sampling component 70 and the mixing component 30 are equal; in one embodiment, step S20 includes the following steps: using the pressure balancing component 704 to make the pressure of the sampling component 70 equal to the injection pressure minus the partial pressure of the pre-filter 20. The pressure charged into the first container 701 by the pressure balancing component 704 can be adjusted according to different injection pressures, so as to better avoid the situation of liquid injection caused by the pressure difference under different injection pressures.

[0089] In one embodiment, the partial pressure of the pre-filter 20 is calculated according to the injection pressure and the regression equation, where the regression equation is obtained by testing the partial pressure of the pre-filter 20 under different injection pressures. This method is more applicable to the scenario where the same pre-filter 20 is used repeatedly. After obtaining the regression equation of this pre-filter 20, if the injection pressure changes, the partial pressure of the pre-filter 20 can be calculated according to the injection pressure without the need to additionally install a pressure gauge.

[0090] In one embodiment, the partial pressure of the pre-filter 20 is measured by setting a pressure gauge between the pre-filter 20 and the mixing component 30, so as to obtain the partial pressure of the pre-filter 20 more quickly. This method is more applicable to the case where the pre-filter 20 is used only once.

[0091] When the sampling component 70 includes a first container 701 connected to the mixing component 30 through a first conduit 702, and the first container 701 and the first conduit 702 are connected through a first three-way valve 703, and a pressure balancing component 704 is connected to the side port of the first three-way valve 703, step S30 includes the following steps: first adjust the first three-way valve 703 to connect the first container 701 and the pressure balancing component 704, and make the pressure in the first container 701 equal to the injection pressure minus the partial pressure of the pre-filter 20 through the pressure balancing component 704. Then adjust the first three-way valve 703 to connect the first conduit 702 and the first container 701. According to different injection pressures, the pressure balancing component 704 can charge different pressures into the first container 701, better avoiding the situation of liquid injection caused by the pressure difference.

[0092] Step S30 does not limit the measurement of the real-time flow rate of the liquid material, which can be obtained by testing with a flowmeter or calculated from the recorded filtration volume and filtration time.

[0093] When the real-time flow rate of the feed liquid is calculated based on the recorded filtration volume and filtration time, the real-time flow rate of the feed liquid = △filtration volume / t, where △filtration volume is equal to the filtration volume minus the filtration volume t seconds ago, and t is less than or equal to 30 min; it should be noted that during the injection process, t can be changed. At the initial stage of injection, since the real-time flow rate of the feed liquid changes rapidly, the smaller t is, the better, preferably less than or equal to 5 min, including but not limited to 0.25 min, 0.5 min, 0.75 min, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min or 5 min. However, as the injection progresses, the change in the real-time flow rate of the feed liquid is smaller. Therefore, t can be appropriately increased, preferably 5 min - 30 min, including but not limited to 5 min, 10 min, 15 min, 20 min, 25 min or 30 min.

[0094] In step S40, in order to enable the virus to be uniformly mixed with the feed liquid according to the planned addition amount, it is necessary to adjust the virus addition speed according to the real-time rate of the feed liquid.

[0095] The virus addition speed depends on the virus spiking strategy. The virus spiking strategy mainly has two modes: virus percentage spiking and total virus amount spiking. The choice of the virus spiking strategy depends on the virus removal filter 40 and its application in actual production.

[0096] When the virus spiking strategy is virus percentage spiking, the virus retention capacity of the virus removal filter 40 can be calculated by comparing the virus percentage difference before and after filtration. The calculation is simple and convenient. The virus addition speed is equal to the real-time flow rate of the feed liquid multiplied by the theoretical virus spiking percentage, and the theoretical virus spiking percentage is preferably less than or equal to 10%.

[0097] When the virus spiking strategy is total virus amount spiking, since the total virus amount is less than the loading capacity of the virus removal filter 40, it can well avoid the occurrence of side leakage of the virus removal filter 40. At this time, the virus addition speed = the real-time flow rate of the feed liquid × (total planned virus addition amount / total planned feed liquid addition amount), and (total planned virus addition amount / total planned feed liquid addition amount) is preferably less than or equal to 10%.

[0098] When the virus addition component 60 and the sampling component 70 are driven by the bidirectional pump 80, the bidirectional pump 80 can accurately control the virus addition ratio. When the virus addition component 60 and the sampling component 70 are driven by the same bidirectional pump 80, and the specifications of the second container 601 in the virus addition component 60 and the first container 701 in the sampling component 70 are the same, the virus addition speed is equal to the sampling speed, that is, the addition amount added to the mixing component 30 is the same as the sampling amount from the downstream mixed liquid in terms of mass. Thus, it can better ensure that there is no additional partial pressure caused by different addition amounts and sampling amounts, so that the partial pressure of the virus removal filter 40 remains stable, and the authenticity of the filterability report can be better improved.

[0099] In order to more intuitively understand the filterability of the virus removal filter 40, in one embodiment, step S50 includes the following steps: after the filtration is completed, calculate the flux of the virus removal filter 40 according to the filtration time and the filtration volume. Taking the filtration time as the horizontal axis and the flux of the virus removal filter 40 as the vertical axis, plot the attenuation curve of the virus removal filter 40, and output the filterability report, where the flux of the virus removal filter 40 = filtration volume / (filtration time × membrane area of the virus removal filter 40).

[0100] In addition, the verification method for verifying the filterability of the virus removal filter 40 provided by the present invention adds virus at the inlet of the mixing component 30 and samples the effluent downstream of the mixing component 30, which can better represent the series production process of the pre-filter 20 and the virus removal filter 40 in the actual production process, better ensure the virus safety of biological products, improve the application success rate of biological products, and accelerate the listing process of biological products.

[0101] Hereinafter, the verification device and verification method for verifying the filterability of the virus removal filter 40 will be further described through the following specific embodiments.

[0102] Embodiment 1

[0103] S101. Use a sleeve as the sampling component 10, a two-way Luer connector as the two-way valve 110, a Viruclear pre-filter as the pre-filter 20, a second three-way Luer connector as the second three-way valve 90, a static mixer as the mixing component 30, a third three-way Luer connector as the third three-way valve 100, and a Viruclear PlusX virus removal filter as the virus removal filter 40. In a biosafety cabinet, connect the sleeve, the two-way Luer connector, the Viruclear pre-filter, the second three-way Luer connector, the static mixer, the third three-way Luer connector, the Viruclear PlusX virus removal filter 40 and the collection component 50 in series in sequence.

[0104] S102. Add 200 ml of ultrapure water into the sleeve, evacuate the air in the whole pipeline, perform wetting treatment at an injection pressure of 2.07 Bar. After sufficient wetting, replace the water with the buffer solution of the protein feed liquid, and fully equilibrate the Viruclear prefilter and the Viruclear PlusX virus filter. After equilibration, the flow rate of the protein feed liquid is 1.1 mL / min.

[0105] S103. Use the first screw syringe and the second screw syringe of the same specification as the first container 701 and the second container 601 respectively, use the first three-way Luer connector as the first three-way valve 703, and use the two-way micro-injection pump as the two-way pump 80. Use the second screw syringe to aspirate 7 mL of X-MuLV virus dilution solution, place it at the advancing end of the two-way micro-injection pump 80, place another first screw syringe of the same specification at the collecting end of the two-way micro-injection pump. Install an interface at the injection port of the second screw syringe and connect it to the side port of the second three-way Luer connector in front of the static mixer. After evacuating the gas in the second catheter 602, connect it to the side port of the second three-way Luer connector after the static mixer. Install a first three-way Luer connector at the injection port of the first screw syringe, connect it to the side port of the third three-way Luer connector after the static mixer through the first catheter 702, and a pressure balance component 704 is connected to the side port of the first three-way Luer connector. Turn on the power of the two-way micro-injection pump. Set the inner diameters of both the first screw syringe and the second screw syringe in the two-way micro-injection pump to be 14.45 mm. In Example 1, the virus spiking strategy adopts virus percentage spiking. Therefore, the virus addition speed = the real-time flow rate of the protein feed liquid × the theoretical spiking percentage of the virus. According to the theoretical spiking percentage of 2.5% and the initial flow rate of the protein feed liquid of 1.1 mL / min, the initial virus addition speed is 27.5 μL / min.

[0106] S20. As Figure 3 shown is the regression curve graph of the injection pressure and the partial pressure of the Viruclear prefilter. The partial pressure of the Viruclear prefilter = 0.9275 × injection pressure + 0.02. The injection pressure is 2.07 Bar. It can be seen from this that the partial pressure of the Viruclear prefilter is 1.94 Bar; adjust the first three-way Luer connector to connect the first screw syringe with the pressure balance component 704, fill 0.1301 Bar of helium gas into the first screw syringe through the pressure balance component 704, and then adjust the first three-way Luer connector to connect the first catheter 702 with the first screw syringe.

[0107] S30. Add 75 m of protein feed liquid into the sleeve, adjust the pressure at the upper end of the sleeve to 2.07 Bar, and open the Viruclear The two-way Luer connector 110 before the pre-filter adjusts the first three-way Luer connector 90 and the second three-way Luer connector 100 to the three-way state for sample injection, and the filtration time, the filtration volume, and the flow rate of the protein feed liquid are recorded in real time.

[0108] S40. Operate the two-way micro-injection pump 80, adopt the virus percentage spike mode, add the virus dilution to the static mixer, and adjust the virus addition speed according to the real-time flow rate of the protein feed liquid. The theoretical spike percentage is 2.5%. Therefore, when the real-time flow rate of the protein feed liquid decreases by 0.04 mL / min, the flow rate of the two-way micro-injection pump decreases by 1 μL / min. At the same time, the sampling component 70 samples the mixed liquid downstream of the static mixer.

[0109] S50. After the filtration is completed, according to the recorded filtration time and the real-time filtration volume, with the filtration time as the horizontal axis and the flux of the Viruclear PlusX virus removal filter as the vertical axis, draw the attenuation curve of the Viruclear PlusX virus removal filter, and output the filterability report. Among them, Viruclear PlusX virus removal filter = filtration volume / (filtration time × membrane area of the Viruclear PlusX virus removal filter).

[0110] Example 2

[0111] Example 2 is carried out with reference to Example 1. The difference is that the virus spike strategy is the total virus spike. Therefore, in step S40, the virus addition speed = real-time flow rate of the feed liquid × (total planned virus addition amount / total planned feed liquid addition amount), and the value of (total planned virus addition amount / total planned feed liquid addition amount) is 10%.

[0112] Comparative Example 1

[0113] Refer to the conditions of Example 1 to fully wet and balance the Viruclear pre-filter at 2.07 Bar, and then pre-filter the protein feed liquid at 0.52 Bar; fully wet and balance the Viruclear PlusX virus removal filter at 2.07 Bar, and carry out virus removal filtration on the protein feed liquid that has been added with 2.5% X-MuLV virus and has been pre-filtered at 2.07 Bar, and record the filtration time and the real-time filtration volume. After the filtration is completed, draw the flow rate attenuation curve and output the filterability report.

[0114] The comparison chart of the flux attenuation curves output by Example 1 and Comparative Example 1 is as Figure 4As shown, it can be seen from the figure that the initial flux of Example 1 is 285.60 LMH, the final flux is 140.16 LMH, and the attenuation is 51.0%; the initial flux of Comparative Example 1 is 266.40 LMH, the final flux is 5.28 LM, and the attenuation is 98.0%. It can be seen that the protein feed liquid after batch pre-filtration is very likely to have the problem of re-aggregation in a short time, resulting in a relatively fast reduction in the flux of virus filtration verification and deviation from the true data. The verification device and verification method provided by the present invention can avoid the problem of re-aggregation during the verification process, thereby providing more authentic and reliable filterability data support for the application of the virus removal filter 40.

[0115] Comparative Example 2

[0116] Comparative Example 2 was carried out with reference to Example 1, except that step S20 was not performed.

[0117] Example 1 and Comparative Example 2 were repeated twice, and the virus titers in the collection component 50 of Example 1 and Comparative Example 2 were measured respectively. The test results are as Figure 5 shown. It can be seen from Figure 5 that if the pressure difference between the sampling component 70 and the mixing component 30 is not balanced before operating the sampling component 70, due to the pressure difference between the mixing component 30 and the sampling component 70, a part of the mixed liquid downstream of the mixing component 30 will be sprayed into the sampling component 70, resulting in a decrease in the virus concentration in the collection component 50.

[0118] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0119] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A verification device for verifying the filterability of a virus removal filter, characterized in that: The invention comprises a sampling component, a pre-filter, a mixing component, a virus removal filter to be verified and a collecting component which are connected in series in sequence. The verification device also comprises a virus adding component and a sampling component, wherein the virus adding component is connected to the inlet of the mixing component for adding the virus into the mixing component, and the sampling component is connected to the outlet of the mixing component for sampling the mixed liquid flowing out of the mixing component; the sampling component is also provided with a pressure balancing component for balancing the pressure difference between the mixing component and the sampling component when sampling starts.

2. The verification device according to claim 1, characterized in that: The sampling component comprises a first container connected to the mixing component through a first conduit, the first container and the first conduit are connected through a first three-way valve, and a side port of the first three-way valve is connected to the pressure balancing component.

3. The verification device according to claim 1, characterized in that: The virus adding component and the sampling component are driven by a bidirectional pump.

4. The verification device according to any one of claims 1 to 3, characterized in that: The pre-filter satisfies at least one of the following conditions: (1) The filter material of the pre-filter includes a deep filter medium or a filter membrane; (2) The pore size of the filter material of the pre-filter is 0.2 μm-0.45 μm.

5. The verification device according to any one of claims 1 to 3, characterized in that: The mixing element is selected from a static mixer or a spiral pipeline mixer.

6. A method for verifying the filterability of a virus removal filter, characterized in that: The following steps are involved: S10, providing a verification device as described in any one of claims 1 to 5, and pre-treating a sampling component, a pre-filter, a mixing component, a virus removal filter, and a collection component; S20, balancing the pressure difference between the sampling component and the mixing component by using a pressure balancing component; S30, adding feed liquid into the sampling component for sampling, and recording the filtration time and filtration volume as well as the real-time flow rate of the feed liquid; S40, operating the virus adding component to add the virus into the mixing component and adjusting the virus adding speed according to the real-time flow rate of the feed liquid, and operating the sampling component to sample the mixed liquid downstream of the mixing component; S50, plotting a flux attenuation curve of the virus removal filter according to the filtration time and the filtration volume, and outputting a filterability report.

7. The verification method according to claim 6, characterized in that: Step S20 includes the following steps: The pressure of the sampling component is made equal to the injection pressure minus the partial pressure of the pre-filter by using the pressure balancing component.

8. The verification method according to claim 7, characterized in that: Step S20 includes the following steps: First, adjust the first three-way valve to connect the first container with the pressure balancing component, and make the pressure in the first container equal to the injection pressure minus the partial pressure of the pre-filter through the pressure balancing component, and then adjust the first three-way valve to connect the first conduit with the first container.

9. The verification method according to claim 7 or 8, characterized in that: In step S20, the partial pressure of the pre-filter is calculated according to the injection pressure and the regression equation of the injection pressure and the partial pressure of the pre-filter, wherein the regression equation is obtained by testing the partial pressure of the pre-filter under different injection pressures and performing linear fitting.

10. The verification method according to claim 7 or 8, characterized in that: The partial pressure of the pre-filter in step S20 is measured by installing a pressure gauge between the pre-filter and the mixing element.

11. The verification method according to any one of claims 6 to 8, characterized in that: In step S30, the real-time flow rate of the feed liquid is calculated according to the filtration time and the filtration volume, and the real-time flow rate of the feed liquid = △filtration volume / t, wherein △filtration volume is equal to the filtration volume minus the filtration volume t seconds ago, and t is less than or equal to 30 minutes.

12. The verification method according to any one of claims 6 to 8, characterized in that: In step S40, the virus addition rate is equal to the real-time flow rate of the feed liquid multiplied by the theoretical addition percentage of the virus, or the virus addition rate = the real-time flow rate of the feed liquid × (total planned virus addition amount / total planned feed liquid addition amount).

13. The verification method according to claim 12, characterized in that: The theoretical virus addition percentage is less than or equal to 10%, and the value of (total planned virus addition amount / total planned liquid addition amount) is less than or equal to 10%.

14. The verification method according to any one of claims 6 to 8, characterized in that: Step S50 includes the following steps: After the filtration is completed, the flux of the virus removal filter is calculated according to the filtration time and the filtration volume, and the attenuation curve of the virus removal filter is plotted with the filtration time as the horizontal axis and the flux of the virus removal filter as the vertical axis, and a filterable report is output, wherein the flux of the virus removal filter = the filtration volume / (the filtration time × the membrane area of ​​the virus removal filter).

15. The verification method according to any one of claims 6 to 8, characterized in that: Step S10 includes the following steps: S101, connecting a sample injection component, a pre-filter, a mixing component, a virus removal filter, and a collection component in series; S102, performing exhaust treatment, wetting treatment and balancing treatment on the sample injection component, the pre-filter, the mixing component, the virus removal filter and the collection component in sequence; S103, connecting the virus adding component with the inlet of the mixing component, and connecting the sampling component with the outlet of the mixing component.

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