Multiple stage virus filtration

Abstract

The disclosure herein relates to methods and systems for multiple step virus filtration, where the first virus filter has a greater filtration area than the following virus filter to enable greater virus retention reliability and improved filtration process productivity than single stage filtration.

Description

MULTIPLE STAGE VIRUS FILTRATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 581 ,076, filed September 7, 2023, the entire content of which is incorporated herein by reference.RELEVANT FIELD

[0002] Embodiments described herein relate to methods and systems for multiple stage serial virus filtration. More specifically, some embodiments relate to a method and system for multiple stage virus filtration with at least two virus filter operated in a series, where the virus filter used in a first stage has a greater filtration area than the second virus filter.BACKGROUND

[0003] Biotherapeutic protein manufacturers use virus filtration membranes (also referred to as virus filters) to reduce the risk of product contamination and meet regulatory expectations. Viruses enter manufacturing processes through contaminated raw materials, such as human plasma used to manufacture plasma-derived products or through contaminated cell lines and cell culture media used in the manufacture of cell-derived products. To assure product safety, virus removal steps must be explicitly incorporated in the manufacturing process, along with careful control of the raw materials and testing of the product and product intermediate pools. As part of the overall viral clearance strategy, virus filters provide a size-based removal mechanism complementary to other common physical removal techniques, such as chromatography, or inactivation methods, such as low pH, solvent-detergent, or temperature treatments.

[0004] Currently, virus filters are operated most commonly in a single stage operation resulting in a simple and convenient processing configuration. However, at some ranges of operating conditions, virus retention performance can be diminished using single stage virus filtration. For example, at operation at a low flux less than about 20-100 L / m2h (LMH), the virus log reduction value (LRV) of the filter decreases with decreasing flux, meaning an increasedrisk of virus passage through the filter. In continuous bioprocesses, the virus filter is often operated at low flux to allow the filter to operate for longer periods of time before the filter must be changed out to minimize the risk of virus passage through the filter. For the reasons above, low flux operation is discouraged.

[0005] To avoid virus passage risk associated with low flux operation, the virus filter can be operated at high flux in batch mode instead of a continuous bioprocess, which requires surge tanks to accommodate the continuous operation of the process steps immediately upstream and downstream of the virus filter. However, batch operation of the virus filtration step within an overall continuous process adds cost and reduces the overall advantage of continuous bioprocessing compared to batch bioprocessing.

[0006] Another method for overcoming the increased virus passage at low flux is to add a second stage virus filter in series with a first stage filter. The second stage filter will capture viruses that may have passed through the first stage filter to mitigate the risk of virus passage. Generally, the first and second stage filters are similarly sized and therefore in a 1 :1 ratio although it has been contemplated to use up to a 2:1 ratio. While adding a second stage filter lowers the risk of virus passage, the addition increases the expense by a factor of 1 .5-2 because of the increased number of required virus filters. There is also added flow resistance resulting in either pressure loss across the multiple stages, also referred to in the art as a filtration train, at constant flow operation, or requiring lower flux at constant inlet pressure operation.

[0007] A method and system for multiple stage virus filtration with more than one virus filter without increased risk of virus passage through virus filters that are operated at low flux, or that experience process disruptions, such as flow stoppages due to low pressure, would represent an advance in the art.SUMMARY

[0008] The shortcomings of the prior art are overcome by embodiments described herein.

[0009] Some embodiments include a method of filtration comprising: feeding a stream of liquid contaminated with virus through a first stage filter;and feeding the liquid through a second stage filter after the first stage filter, the ratio of the area of the first stage filter to the area of the second stage filter is about 3:1 or greater.

[0010] In some embodiments, the method further comprises achieving a LRV greater than 4. In some embodiments, the LRV is within a range of 5 to 9. In some embodiments, the method further comprises operating the first stage filter with a flux selected from a range consisting of: about 1 to about 20 LMH. In some embodiments, the ratio is selected from the range selected from about 3:1 to about 50:1. In some embodiments, the method further comprises operating the second stage filter at a flux selected from the range consisting of: about 3 to about 1000 LMH. In some embodiments, the method further comprises operating the second stage filter at a flux 3 to 50 times higher than the operating flux for the first stage filter. In some embodiments, the method further comprises reaching and maintaining a pressure of about 20 psi for the second stage filter. In some embodiments, the area of the first stage filter is selected from the group consisting of 0.22, 0.51 , and 1 .53 m2. In some embodiments, the area of the second stage filter is 0.017 m2or 0.07 m2. In some embodiments, the pore size of the first stage filter and the second stage filter is the same.

[0011] Some embodiments include a system for virus filtration comprising: a filtration train comprising at least two filters, the two filters comprising a first stage filter and a second stage filter, the ratio of the area of the first stage filter to the area of the second stage filter is selected from the range consisting of about 3 to about 50 to an area of a second filter. In some embodiments, the area of the first stage filter is selected from the group consisting of 0.22, 0.51 , and 1 .53 m2. In some embodiments, the area of the second stage filter is 0.017 m2or 0.07 m2.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a graph of the relationship between virus (MVM) LRV and membrane flux for Viresolve® Pro filter membranes. LRV was measured at the point where the membrane was plugged by about 35%.

[0013] FIG. 2A and FIG. 2B is a graph of a transmembrane pressure (psi) in a Viresolve® Pro Modus 1 .3 filter device (effective filtration area of 0.22 m2)labeled as “Stage 1” and Viresolve® Pro Modus 1.1 filter device (effective filtration area of 0.017 m2) labeled as “Stage 2”. FIG. 2A shows the transmembrane pressure separately for Viresolve® Pro Modus 1 .1 filter device and Viresolve® Pro Modus 1.3 filter device versus filtration time. FIG. 2B shows the transmembrane pressure for Viresolve® Pro Modus 1 .1 filter device and Viresolve® Pro Modus 1.3 filter device versus filtration volume per filter area.

[0014] FIGS. 3A and 3B are graphs of the LRV of viruses Phi-X 174 and the Minute virus of mice for some embodiments of a first stage filter (Stage 1 ), which in this case is Viresolve® Pro Modus 1 .3 filter device (effective filtration area of 0.22 m2), and for some embodiments of a filtration train.

[0015] The appended drawings illustrate some embodiments of the disclosure herein and are therefore not to be considered limiting in scope, for the invention may admit to other equally effective embodiments. It is to be understood that elements and features of any embodiment may be found in other embodiments without further recitation and that, where possible, identical reference numerals have been used to indicate comparable elements that are common to the figures.DETAILED DESCRIPTION

[0016] Some embodiments described herein include multiple stages of virus filtration. In some embodiments the system used for the virus filtration comprises two virus filters used in a series. In some embodiments, the filter area of the virus filter in the second stage is substantially lower than the area of the virus filter of the first stage. In some embodiments, the virus filter of the second stage is lower than the area of the virus filter of the first stage by a factor of 3 or more.

[0017] In some embodiments, at a constant flow rate through the filtration train, the flux through the second stage filter compared to the first stage filter will be proportional to the area ratio of the virus filter in the first stage to the virus filter in the second stage. In some embodiments, the virus LRV of the second stage filter is additive to the LRV of the first stage filter, and since the second stage filter operates at higher flux compared to the first stage filter there will be less risk of virus passage. In some embodiments, when operatingthe first stage filter at a low flux in the 1 to 20 LMH range, the second stage filter is operated at a higher flux.

[0018] For example, in some embodiments, the second stage filter is operated a flux about 3-50 times higher than the flux for the first stage filter. In some embodiments, the second stage filter is operated at a flux within the range of 3-50 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux within a range selected from the group consisting of 3-7, 5-9, 7-11 , 9-13, 11-15, 13-17, 15- 19, 17-21 , 19-23, 21-25, 23-27, 25-29, 27-31 , 29-33, 31-35, 33-37, 35-39, 37- 41 , 39-43, 41 -45, 43-47, 45-49, 47-51 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 3 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 4 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 5 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 6 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 7 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 8 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 9 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 10 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 11 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 12 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 13 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 14 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 15 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 16 times higher than the flux of the first stage filter. In some embodiments, the secondstage filter is operated at a flux about 17 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 18 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 19 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 20 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 21 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 22 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 23 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 24 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 25 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 26 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 27 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 28 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 29 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 30 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 31 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 32 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 33 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 34 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 35 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 36 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 37 timeshigher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 38 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 39 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 40 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 41 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 42 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 43 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 44 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 45 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 46 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 47 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 48 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 49 times higher than the flux of the first stage filter. In some embodiments, the second stage filter is operated at a flux about 50 times higher than the flux of the first stage filter.

[0019] In some embodiments, the higher flux in the second stage filter compared to the flux of the first stage filter necessitates a proportionally higher area ratio of the first stage filter to the second stage filter to maintain LRV. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 3:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 4:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 5:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 6:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 7:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 8:1. In someembodiments, the area ratio of the first stage filter to the second stage filter is about 9:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 10:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 11 :1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 12:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 13:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 14:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 15:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 16:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 17:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 18:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 19:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 20:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 21 :1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 22:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 23:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 24:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 25:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 26:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 27:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 28:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 29:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 30:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 31 :1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 32:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 33:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 34:1 . In some embodiments, the area ratio of the first stage filterto the second stage filter is about 35:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 36: 1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 37:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 38:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 39:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 40:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 41 :1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 42:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 43:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 44:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 45:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 46:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 47:1. In some embodiments, the area ratio of the first stage filter to the second stage filter is about 48:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 49:1 . In some embodiments, the area ratio of the first stage filter to the second stage filter is about 50:1.

[0020] In some embodiments, the area ratio of the first stage filter to the second stage filter is within a range selected from the group consisting of: 3:1 to 7:1 , 5:1 to 9:1 , 7:1 to 11 :1 , 9:1 to 13:1 , 11 :1 to 15:1 , 13:1 to 17:1 , 15:1 to 19:1 , 17:1 to 21:1, 19:1 to 23:1 , 21 :1 to 25:1 , 23:1 to 27:1 , 25:1 to 29:1 , 27:1 to 31 :1 , 29:1 to 33:1 , 31 :1 to 35:1 , 33:1 to 37:1 , 35:1 to 39:1 , 37:1 to 41 :1 , 39:1 to 43:1 , 41 :1 to 45:1 , 43:1 to 47:1 , 45:1 to 49:1 , and 47:1 to 51 :1.

[0021] Some embodiments of the method and system described herein have application in multiple commercial products including the Viresolve® Pro devices, also referred to herein as Viresolve® Pro filters, commercially offered by MilliporeSigma.Definitions

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] As used herein, the singular forms "a", "an," and "the" include plural unless the context clearly dictates otherwise.

[0024] As used herein “filtration train” refers to more than one filter used serially in a multiple stage filtration method.

[0025] As used herein, “Viresolve® Pro device” is used to describe the filters including the Modus 1.1 , Modus 1 .2, Modus 1 .3, Magnus 2.1 , and Magnus 2.2 commercially offered by MilliporeSigma.EXAMPLESExample 1. Simulation Results

[0026] Example 1 is a hypothetical example with the following parameters. The continuous flow rate is 10 L / h and the maximum allowable pressure across the filtration train is 20 psi. In this example, commercially available Viresolve Pro® filters is used. Table 1 lists the effective filtration area of commercially available Viresolve® Pro devices.Table 1. Effective Filtration Areas of Viresolve Pro® Devices

[0027] The typical clean water permeability of Viresolve Pro® devices is about 17 LMH / psi.

[0028] In some embodiments, the retention capability of a virus filter can be quantified by its LRV, defined as:

[0029] LRV = - log [C / iitrate

[0030] where Ctutrate is the virus concentration in the filtrate, and Cfeed is the virus concentration in the feed.

[0031] The throughput capacity of Viresolve® Pro filters is highly dependent on the stream characteristics. In this example, a typical throughput value of 1000 L / m2(at which point the permeability has declined by 90% from its unfouled value) is assumed. Since the pore size of the second stage filter is identical to that of the first stage filter, any contaminants that could potentially plug that filter will have been captured by the first stage filter andtherefore it will experience negligible plugging.

[0032] For virus filtration, the desired virus LRV is at least 4 and, preferably, 5 or higher. As shown in Fig. 1 , LRV can be sensitive to flux when the flux is below a certain threshold value. In this example, the relationship between LRV and flux follows the solid line shown in FIG. 1.

[0033] The example further assumes that LRV decreases with membrane plugging, according to:

[0034] LRV = LRV0- log [-1

[0035] Where LRV0is the initial clean membrane LRV, R is the membrane resistance (inverse of permeability), and Ro is the initial membrane resistance when the membrane is clean.

[0036] LRV, throughput per m2of membrane, and changeout time (time at which the filter has reached the end of its useful life and must be changed out, either because it reached the maximum allowable level of plugging, or maximum allowable pressure differential across the filtration train, which was set to 20 psid in this example) were calculated for several scenarios using the prior art and method of this invention.

[0037] The calculation results are summarized in Table 2.Table 2. Calculated performance of a multiple embodiments

[0038] Prior Art 1 employs a single stage filter device that contains sufficient area to reach a changeout time of 153 hours and utilizes the full capacity of the membrane to 1000 L / m2. However, because this filter operates at a flux of only 6.5 LMH, the LRV is only 2.7, which is well below the minimum value of 4.

[0039] Prior Art 2 uses a much smaller area device, which operates at a flux pf 143 LMH resulting in a satisfactory LRV of 6.1. However, the high flux imposes an increasingly large pressure differential across the filter as it plugs, and its useful life is only 3 hours after processing only 377 L / m2.

[0040] In Prior Art 3, two virus filter are operated in series at a 1 :1 area ratio. This results in a satisfactory LRV of 6.3 and a satisfactory changeout time of 153 hours, but the throughput is only 500 L / m2owing to a doubling of the filtration area with the throughput capacity limited by the first stage filter.

[0041] Embodiment 1 uses a first stage filter and a second stage filter used serially wherein the area ratio of the first stage filter to the second stage filter is about 22, with the first stage operating at a flux of 6.5 LMH and the second stage operating at a flux of 143 LMH. The throughput is 953 L / m2at a changeout time of 153 hours, with a high LRV of 9.0. Compared to other evaluation arrangements, Embodiment 1 clearly provides the best combination of high throughput productivity, long changeout time, and high virus retention performance.

[0042] As the LRV for Embodiment 1 is high, there is an opportunity to extend the operation of first stage to beyond 90% plugging, the normal filtration endpoint that had been established in the prior art to avoid excess retention loss.

[0043] Embodiment 2 is similar to Embodiment 1 , except that the maximum allowable plugging of the first stage is set to 95% instead of 90%. The changeout time is increased to 199 hours, along with a higher throughput of 1244 L / m2, with only a slight drop in LRV to 8.7, which is more than the satisfactory level.

[0044] Compared to any of the prior art cases of either single stage operation or dual stage operation at low area ratio, the multiple stage high area ratio method of Embodiment 2 provides higher throughput productivity, longer changeout time, and higher virus retention performance.Example 2. Proof of Concept

[0045] To confirm the validity of the hypothetical example, an experiment was carried out to demonstrate the virus retention advantage and productivity advantage of the filtration train in Embodiment 1 and Embodiment 2. The Cfeed consisted of solution SeraCare® IgG spiked with the viruses Phi-X 174 andMinute virus of mice (MVM) ran at continuous flow rate of 1 .1 L / m2.

[0046] A Modus 1.3 device (effective filtration area of 0.22 m2) was selected as the first stage filter and a Modus 1.1 device (effective filtration area of 0.017 m2) was selected as the second stage filter, operated in series with the first stage filter. This combination resulted in an area ratio of 13, with the first stage filter operating at 5 LMH and the second stage filter operating at 65 LMH. The filtration was conducted continuously for 190 hours, except for some planned depressurization and re-pressurization steps to measure the impact of process disruption on virus passage.

[0047] FIG. 2A and FIG. 2B shows the pressure rise as functions of both filtration time and filtration volume, respectively. Initially, and as expected, the total pressure drop was dominated by the second stage filter which operated at thirteen times the flux of the first stage filter. The pressure drop across the first stage filter increased with throughput (due to membrane plugging) but the second stage filter, which was largely protected from foulants by the first stage filter, experienced only a small increase in pressure drop throughout the run. By the end of the run, determined by reaching the maximum target pressure of 20 psi, the total pressure drop continued to be dominated by the first stage filter. These results demonstrate that adding a second stage filter with much less effective filtration area did not negatively impact total filtration productivity, which is defined as the volume processed per total filtration area.

[0048] As will be shown presently, by enhancing the virus retention performance of the system, adding the second stage filter actually allowed for more extended use of the first stage filter thereby increasing total filter productivity .

[0049] The LRV of Phi-X 174 and MVM as a function of filtration time are shown in FIGS. 3A and 3B. The vertical lines in FIGS. 3A and 3B indicate process disruptions, where the flow to the filters was paused for sixty minutes. Throughout the filtration run, virus was detected in the first stage filtrate with a LRV approaching 4 or lower during parts of the run. However, for both Phi-X 174 and MVM there was no virus detected in the filtrate of the second stage filter at any point in the run. Arrows above symbols indicate no virus was detected in the filtrate, so LRV was at the shown level or higher. Based on the total number of viruses entering the filtration train, and no viruses detected inthe final filtrate pool, a minimum LRV of 6.4 for PhiX-174 and a minimum LRV of 6.7 for MVM was calculated for the series filtration train after a total throughput of 865 L / m2(total volume processed divided by total filtration area). This contrasts with the method with only one stage of filtration, where virus was detected in the filtrate (LRV < 5) as shown in Table 2. The first stage experienced 90% permeability decay, which is typically the end of the useful life of a virus filter in single stage operation, after about 550 L / m2. Therefore, adding a second stage filter increased virus retention and extended the life of the first stage filter resulting in a significant gain in total filter productivity. Since the second stage filter experienced minimum plugging, it could continue to be used after the end of the useful life of the first stage filter in conjunction with a new first stage filter to further increase total filter productivity.EQUIVALENTS

[0050] All ranges for formulations recited herein include ranges therebetween and can be inclusive or exclusive of the endpoints. Optional included ranges are from integer values therebetween (or inclusive of one original endpoint), at the order of magnitude recited or the next smaller order of magnitude. For example, if the lower range value is 0.2, optional included endpoints can be 0.3, 0.4,... 1.1 , 1.2, and the like, as well as 1 , 2, 3 and the like; if the higher range is 8, optional included endpoints can be 7, 6, and the like, as well as 7.9, 7.8, and the like. One-sided boundaries, such as 3 or more, similarly include consistent boundaries (or ranges) starting at integer values at the recited order of magnitude or one lower. For example, 3 or more includes 4, or 3.1 or more.

[0051] Reference throughout this specification to “one embodiment,” “certain embodiments,” “one or more embodiments,” “some embodiments,” or “an embodiment” indicates that a feature, structure, material, or characteristic described is included some embodiments of the disclosure. Therefore, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in one embodiment,” “some embodiments,” or “in an embodiment” throughout this specification are not necessarily referring to the same embodiment.

[0052] Publications of patent applications and patents and other nonpatent references, cited in this specification are herein incorporated by reference in their entirety in the entire portion cited as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in the manner described above for publications and references.

Claims

CLAIMSWhat is claimed is:1 . A method of filtration comprising: feeding a stream of liquid contaminated with virus through a first stage filter; and feeding the liquid through a second stage filter after the first stage filter, a ratio of an area of the first stage filter to an area of the second stage filter is about 3:1 or greater.

2. The method of claim 1 , further comprising achieving a LRV greater than 4.

3. The method of any one of claim 1 and 2, wherein the LRV is within a range of 5 to 9.

4. The method of any one of claims 1-3, further comprising operating the first stage filter with a flux selected from a range consisting of: about 1 to about 20 LMH.

5. The method of any one of claims 1-4, wherein the ratio is selected from a range selected from about 3:1 to about 50:1.

6. The method of any one of claims 1-5, further comprising operating the second stage filter at a flux selected from the range consisting of: about 3 to about 1000 LMH.

7. The method of any one of claims 1-6, further comprising operating the second stage filter at a flux 3 to 50 times higher than the operating flux for the first stage filter.

8. The method of any one of claims 1-7, further comprising reaching and maintaining a pressure of about 20 psi for the second stage filter.

9. The method of any one of claims 1-8, wherein the area of the first stage filter is selected from the group consisting of 0.22, 0.51 , and 1 .53 m2.

10. The method of any one of claims 1 -9, wherein the area of the second stage filter is 0.017 m2or 0.07 m2.11 . The method of any one of claims 1-10, wherein a pore size of the first stage filter and a pore size of the second stage filter are equal.

12. A system for virus filtration comprising: a filtration train comprising at least two filters, the two filters comprising a first stage filter and a second stage filter, a ratio of an area of the first stage filter to an area of the second stage filter is selected from the range consisting of about 3 to about 50 to the area of the second stage filter.

13. The system of claim 12, wherein the area of the first stage filter is selected from the group consisting of 0.22, 0.51 , and 1.53 m2.

14. The system of any one of claims 12 and 13, wherein the area of the second stage filter is 0.017 m2or 0.07 m2.

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