Methods of reducing viral titers in pharmaceuticals

Abstract

Methods and apparatus for optimizing viral removal from solution are disclosed. A method of filtering a process fluid containing a product includes flowing some of the process fluid from a first reservoir to a viral filter; adding a chase fluid to the process fluid in the first reservoir to form a composite fluid; and flowing the composite fluid from the first reservoir to the viral filter to produce an eluate. In some embodiments, the flow through the viral filter is sufficient to avoid significant impairment of viral removal until a preselected event occurs. In some embodiments, the flow of fluid from the first reservoir through the viral filter is maintained without interruption or slowing of the flow for a duration or magnitude sufficient to impair viral removal to a level below the log reduction value of the viral filter.

Description

[0001] This application is a divisional application of the same invention patent application 201780083929.7, filed on December 8, 2017.

[0002] Cross-referencing of related applications

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 432,273, filed December 9, 2016, the entire contents of which are incorporated herein by reference for all purposes. [Field of Invention]

[0004] The present invention relates to methods and apparatus for optimizing the removal of viruses from solutions (e.g., cell culture media). [Background Technology]

[0005] Cell culture products, including recombinant therapeutic proteins, are used in numerous applications, ranging from treating a variety of medical conditions to cancer. These proteins can be synthesized through large-scale cell culture, such as genetically engineered cells containing heterologous nucleic acids encoding the proteins. These protein products must meet stringent regulatory and quality standards before they can be used to treat human patients. Minimizing viral contamination of these products is a critical aspect of product safety and quality. More efficient systems, processes, and methods are needed for the commercial manufacture of recombinant protein therapeutics, meeting Good Manufacturing Practice (GMP) standards at acceptable cost, quality, and in the required quantities. [Invention Overview]

[0006] An important aspect of product production, such as protein products like recombinant protein therapeutics, is the removal of viruses or viral particles after production or fermentation. The methods described herein optimize the inactivation or removal of viruses from product formulations, such as feed from bioreactors.

[0007] While not wishing to be bound by theory, it is believed that interruption of the flow of the product-containing fluid in the virus filter leads to preparation with lower purity, and that maintaining the flow can optimize or reduce the purity of viral contamination.

[0008] According to one aspect of this disclosure, a method for filtering a processing fluid containing products includes: (a) allowing a portion of the processing fluid in a first reservoir to flow out of the first reservoir and through a virus filter to produce an eluent; (b) adding a chasing fluid to the processing fluid in the first reservoir to form a composite fluid; and (c) allowing the composite fluid in the first reservoir to flow out of the first reservoir and through a virus filter to produce an eluent, wherein the fluid flowing through the virus filter is sufficient to avoid significant impairment of virus removal until a preselected event occurs, and wherein the flow of fluid from the first reservoir through the virus filter is maintained without interruption or slowing of the flow for a duration or magnitude sufficient to impair virus removal to a level below the logarithmic reduction value of the virus filter.

[0009] In some embodiments, the product includes an active pharmaceutical ingredient.

[0010] In some implementations, some of the processing fluid in the first reservoir is drained out of the first reservoir and passed through a virus filter to reduce the amount of processing fluid remaining in the first reservoir.

[0011] In some implementations, the addition of chasing fluid is performed before the processing fluid in the first reservoir is emptied.

[0012] In some implementations, when the volume of the processing fluid remaining in the first reservoir is at or within a reference volume, the addition of chasing fluid is performed.

[0013] In some embodiments, the reference volume is equal to or greater than 0.5, 1, 1.5, 2, 3, 5, or 10 times the volume of the component disposed between the first reservoir and the end reservoir.

[0014] In some implementations, the reference volume is equal to or greater than the volume of the component disposed between the first reservoir and the end reservoir.

[0015] In some implementations, the volume of the added chasing fluid is less than or equal to the reference volume of the chasing fluid.

[0016] In some embodiments, the reference volume of the chasing fluid is equal to or greater than 0.5, 1, 1.5, 2, 3, 5, or 10 times the volume of the component disposed between the first reservoir and the final reservoir.

[0017] In some implementations, the chasing fluid reference volume is equal to or greater than the volume of the component disposed between the first reservoir and the final reservoir.

[0018] In some implementations, chasing fluid is added to a first reservoir, while the first reservoir still contains a predetermined amount of processing fluid.

[0019] In some embodiments, the method further includes determining a value as a function of the amount of treatment fluid remaining in the first reservoir.

[0020] In some implementations, the method further includes determining whether the value meets a predetermined reference value.

[0021] In some implementations, the method further includes adding chasing fluid to a first reservoir if the value has a predetermined relationship with a reference value.

[0022] In some implementations, when the chasing fluid is added, the ratio of the chasing fluid volume to the processing fluid volume remaining in the first reservoir is equal to or greater than 1:0.5, 1:1, 1.5:1, 2:1, 3:1, 5:1, or 10:1.

[0023] In some implementations, the flow of fluid from the first reservoir through the virus filter is maintained at a preselected rate.

[0024] In some implementations, a preselected pressure differential is maintained across the virus filter.

[0025] In some implementations, the pressure differential across the virus filter is maintained at or below a preselected maximum value.

[0026] In some implementations, a preselected pressure differential is maintained across the virus filter at a pressure differential of 14 psi, 11 psi, or 13.2 psi.

[0027] In some implementations, the pressure difference across the virus filter is maintained at or above a preselected minimum.

[0028] In some implementations, the pressure difference across the virus filter is sufficient to reduce virus particles by at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times.

[0029] In some implementations, any single stop in the fluid flow from the first reservoir lasts for less than 1, 10, or 60 minutes.

[0030] In some implementations, continuous flow exists during one, two, or all of steps a, b, and c.

[0031] In some implementations, the preselected event includes eliminating the fluid connection between the virus filter and the endpoint reservoir.

[0032] In some implementations, the preselected event includes sending the eluent to subsequent operations.

[0033] In some implementations, the preselected event includes stopping the flow from the virus filter to the accumulated eluent.

[0034] In some implementations, the preselected event includes stopping the flow from the virus filter to the endpoint reservoir.

[0035] In some implementations, the preselected event includes collecting the eluent generated by the fluid flow from the first reservoir.

[0036] In some implementations, the preselected event includes the arrival of the end of the preselected time period.

[0037] In some embodiments, the method further includes providing a system comprising a first reservoir, a virus filter, and an endpoint reservoir, wherein the first reservoir is fluidly connected to the virus filter, and the virus filter is fluidly connected to the endpoint reservoir.

[0038] In some implementations, a virus filter is disposed between the first reservoir and the final reservoir.

[0039] In some implementations, a virus filter is disposed between the first reservoir and the conduit.

[0040] In some implementations, the system includes a pre-filter conduit configured to deliver fluid from a first reservoir to a virus filter.

[0041] In some embodiments, the system includes a conduit configured to deliver fluid to a terminal reservoir or to a terminal.

[0042] In some embodiments, the system includes a first conduit and a second conduit, the first conduit being configured to deliver fluid from a first reservoir to a virus filter, and the second conduit being configured to deliver fluid from the virus filter to a final reservoir.

[0043] In some implementations, the system includes a first valve configured to control fluid flow from a first reservoir to a virus filter.

[0044] In some implementations, the system includes a second valve configured to control fluid flow from the virus filter to the endpoint reservoir.

[0045] In some implementations, the system includes a computer or microprocessor for controlling one or more valves.

[0046] In some implementations, the virus filter is integrated with the wall of the first or second reservoir.

[0047] In some implementations, the flow of fluid from the first reservoir through the virus filter is maintained without any interruption of flow.

[0048] According to another aspect of this disclosure, a method for filtering a processing fluid containing products includes: (a) draining the processing fluid from a first reservoir and passing it through a virus filter to generate an eluent and reduce the amount of processing fluid in the first reservoir; (b) determining a value that is a function of the amount of processing fluid remaining in the first reservoir, and if the value satisfies a predetermined reference value, adding a chasing fluid to the processing fluid in the first reservoir to form a composite fluid, wherein the chasing fluid is added to the first reservoir while the first reservoir still contains a predetermined amount of processing fluid; (c) draining the composite fluid from the first reservoir and passing it through a virus filter to generate an eluent; and (d) stopping the flow from the virus filter, wherein the flow of fluid from the first reservoir through the virus filter is sufficient to avoid significant damage to virus removal until the flow from the virus filter is stopped.

[0049] In one aspect, this disclosure provides a method for filtering a processing fluid containing products. The method includes draining the processing fluid from a first (or delivery) reservoir through a virus filter to produce an eluent. A chasing fluid is added to the processing fluid in the first reservoir to form a composite fluid. The method further includes draining the composite fluid from the first reservoir through the virus filter to produce an eluent. The flow of the fluid through the virus filter in the first reservoir is sufficient to avoid significant impairment of virus removal until a preselected event occurs, such as the collection of accumulated eluent.

[0050] In one embodiment, the product comprises an active pharmaceutical ingredient (API).

[0051] In one embodiment, the processing fluid in the first reservoir is drained from the first reservoir and passed through a filter to reduce the amount of processing fluid remaining in the first reservoir.

[0052] In one embodiment, chasing fluid is added to the processing fluid before the processing fluid in the first reservoir is emptied.

[0053] In one embodiment, chasing fluid is added when the volume of the processing fluid remaining in the first reservoir is at or within a reference volume.

[0054] In one embodiment, the reference volume is equal to or greater than 0.5, 1, 1.5, 2, 3, 5, or 10 times the volume of the components (e.g., conduits and filters) disposed between the first reservoir and the final reservoir.

[0055] In one embodiment, the reference volume is equal to or greater than the volume of the components (e.g., conduits and filters) disposed between the first reservoir and the final reservoir.

[0056] In one implementation, the volume of the added chasing fluid is at or within the reference volume.

[0057] In one embodiment, the reference volume is equal to or greater than 0.5, 1, 1.5, 2, 3, 5, or 10 times the volume of the components (e.g., conduits and filters) disposed between the first reservoir and the final reservoir.

[0058] In one embodiment, the reference volume is equal to or greater than the volume of the components (e.g., conduits and filters) disposed between the first reservoir and the final reservoir.

[0059] In one embodiment, a chasing fluid is added to a first reservoir, which still contains a predetermined amount of processing fluid, for example, at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50% of the original amount of processing fluid.

[0060] In one embodiment, the method includes determining a value as a function of the amount of processing fluid remaining in the first reservoir.

[0061] In one implementation, the method includes determining whether the value meets a predetermined reference value.

[0062] In one implementation, if the value has a predetermined relationship with a reference value (e.g., if it satisfies, exceeds or is less than the reference value), then chasing fluid is added to the first reservoir.

[0063] In one embodiment, when the chasing fluid is added, the ratio of the amount of chasing fluid to the amount of processing fluid remaining in the first reservoir is equal to or greater than 1:0.5, 1:1, 1.5:1, 2:1, 3:1, 5:1 or 10:1.

[0064] In one implementation, the flow of fluid from the first reservoir through the virus filter is maintained without interruption or slowing of the flow for a duration or magnitude sufficient to significantly impair virus removal.

[0065] In one embodiment, the flow has a duration or magnitude sufficient to reduce viral particles by at least 5, 10, 15, 20, 30, 40, 50, 60%, 70%, 80%, 90%, or 100 times.

[0066] In one implementation, the flow of fluid from the first reservoir through the virus filter is maintained at a preselected rate.

[0067] In one implementation, a preselected pressure differential is maintained, such as a preselected pressure differential range across the virus filter.

[0068] In one implementation, the pressure differential across the virus filter is maintained at or below a preselected maximum value.

[0069] In one implementation, a preselected pressure differential is maintained, equal to or not greater than 14 psi, 11 psi, or 13.2 psi.

[0070] In one implementation, the pressure difference across the virus filter is maintained at or above a preselected minimum.

[0071] In one implementation, the pressure difference across the virus filter is sufficient to reduce virus particles by at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times.

[0072] In one implementation, the flow of fluid in the first reservoir is stopped for a total of less than 1 minute, 10 minutes, 60 minutes, 120 minutes, or 180 minutes.

[0073] In one implementation, the duration of any single stop in the flow of fluid into the first reservoir is less than 1, 10, 60, 120, or 180 minutes.

[0074] In one embodiment, continuous flow exists during one, two, or all of the following steps: (a) draining the processing fluid from the first (or sending) reservoir and passing it through a virus filter to produce an eluent; (b) adding a chasing fluid to the processing fluid in the first reservoir to form a composite fluid; and / or (c) draining the composite fluid from the first reservoir and passing it through a virus filter to produce an eluent.

[0075] In one implementation, the preselected event includes eliminating the fluid connection between the virus filter and the second or final reservoir.

[0076] In one implementation, the preselected event includes sending the eluent to the next unit operation.

[0077] In one implementation, the preselected event includes stopping the flow from the virus filter to the accumulated eluent.

[0078] In one implementation, the preselected event includes stopping the flow from the virus filter to a second or final reservoir.

[0079] In one implementation, the preselected event includes collecting the eluent generated by the fluid flow from the first reservoir.

[0080] In one implementation, the preselected event includes the arrival of the end of the preselected time period.

[0081] In one embodiment, the method includes providing a system comprising: a first (or transmitting) reservoir; a virus filter; and (optionally) a second reservoir, such as a receiving container, wherein the first reservoir is fluidly connected to the virus filter, and (optionally) the filter is fluidly connected to the second reservoir.

[0082] In one embodiment, a virus filter is disposed between a first reservoir and a second or endpoint reservoir.

[0083] In one implementation, a virus filter is disposed between a first reservoir and a conduit (e.g., a conduit leading to an endpoint, such as the next unit operation).

[0084] In one embodiment, the system includes a conduit configured to deliver fluid from a first reservoir to a virus filter.

[0085] In one embodiment, the system includes a conduit configured to deliver fluid (e.g., eluent) from a virus filter to a second or receiving reservoir or conduit (e.g., to a conduit at an endpoint, such as the next unit operation).

[0086] In one embodiment, the system includes a conduit configured to deliver fluid from a first reservoir to a virus filter; and a conduit configured to deliver fluid (e.g., eluent) from the virus filter to a second reservoir.

[0087] In one embodiment, the system includes a valve configured to control fluid flow from a first reservoir to a virus filter.

[0088] In one embodiment, the system includes a valve configured to control the flow of fluid (e.g., eluent) from the virus filter to a second reservoir.

[0089] In one implementation, the system includes a computer or microprocessor for controlling the valve.

[0090] In one embodiment, the filter is integrated with the wall of the first or second reservoir.

[0091] In another aspect, this disclosure provides a method for filtering a processing fluid containing products. The method includes draining the processing fluid from a first reservoir through a virus filter to generate eluent and reducing the amount of processing fluid in the first reservoir, determining a value as a function of the amount of processing fluid remaining in the first reservoir, and if the value satisfies a predetermined reference value, adding a chasing fluid to the processing fluid in the first reservoir to form a composite fluid, wherein the chasing fluid is added to the first reservoir while the first reservoir still contains a predetermined amount of processing fluid. The composite fluid in the first reservoir is then drained from the first reservoir through the virus filter to generate eluent. The method further includes stopping the flow from the virus filter into the accumulated eluent, wherein the flow of the first reservoir fluid through the virus filter is sufficient to avoid significant damage to virus removal or removal until the flow from the virus filter into the accumulated eluent is stopped.

[0092] Unless otherwise defined, 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. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, materials, methods, and embodiments are illustrative only and not restrictive. Titles, subheadings, or numbering or letter elements, such as (a), (b), (i), etc., are presented solely for readability. The use of titles, numbering, or letter elements in this document does not require that steps or elements be performed in alphabetical order, or that steps or elements must be discrete from each other. Other features, objects, and advantages of the invention will become apparent from the specification, drawings, and claims. [Brief Description of the Attached Image]

[0093] First, a brief description of the diagram.

[0094] Figure 1 This is a diagram of a system used for virus purification in cell culture medium. [Detailed Description of the Invention]

[0095]

definition

[0096] Unless otherwise defined, 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. While any methods and materials similar or equivalent to those described herein may be used in the practice and / or testing of the invention, preferred materials and methods are described herein. In describing and claiming protection for the invention, the following terms will be used as defined, where definitions are provided. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not restrictive.

[0097] The articles “one” and “a” used here refer to one or more (i.e., at least one) grammatical objects. For example, “cell” can refer to one cell or more cells.

[0098] As used herein, the term "elution buffer" refers to a fluid that has passed through, for example, a virus filter. In embodiments, the amount or concentration of virus in the eluent is less than the amount of virus in the culture medium or composite solution prior to passing through the virus filter.

[0099] The term “first reservoir fluid” as used herein refers to one or more of the following: culture medium, chasing fluid, and complex fluid.

[0100] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds or otherwise. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise a protein or peptide sequence. In one embodiment, a protein may comprise more than one, for example, two, three, four, five, or more polypeptides, each of which is associated with another polypeptide by covalent or non-covalent bonds / interactions. A polypeptide includes any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or otherwise. As used herein, the term refers to a short chain, which is also commonly referred to in the art as peptide, oligopeptide, and oligomer, and relates to longer chains, which are commonly referred to in the art as proteins, of which many exist. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc.

[0101] As used herein, the term "product" refers to a molecule, nucleic acid, polypeptide, or any hybrid thereof produced by cells that have been modified or engineered to produce the product. In one embodiment, the product is either naturally occurring or non-naturally occurring, such as a synthetic product. In one embodiment, a portion of the product is naturally occurring while another portion is non-naturally occurring. In one embodiment, the product is a polypeptide, such as a recombinant polypeptide. In one embodiment, the product is suitable for diagnostic or preclinical use. In another embodiment, the product is suitable for therapeutic use, such as for treating a disease. In one embodiment, the modified or engineered cells contain exogenous nucleic acids that control the expression or encoding of the product. In other embodiments, the modified or engineered cells contain other molecules, such as molecules that are not nucleic acids, that control the expression or construction of the product in the cell.

[0102] As used herein, "recombinant polypeptide" or "recombinant protein" refers to a polypeptide that can be produced by the cells described herein. A recombinant polypeptide is a polypeptide formed by genetic engineering (cells or precursor cells) to contain at least one nucleotide encoding a polypeptide sequence, or at least one nucleotide controlling polypeptide expression. For example, at least one nucleotide is altered, for example, it is introduced into the cell or it is the product of genetic engineering rearrangement. In one embodiment, the sequence of the recombinant polypeptide is identical to that of a naturally occurring isotype of the polypeptide or protein. In one embodiment, the amino acid sequence of the recombinant polypeptide differs from the sequence of a naturally occurring isotype of the polypeptide or protein. In one embodiment, the recombinant polypeptide and the cell originate from the same species. In one embodiment, the recombinant polypeptide is endogenous to the cell; in other words, the cell originates from a first species, and the recombinant polypeptide is natural for the first species. In one embodiment, the amino acid sequence of the recombinant polypeptide is identical or substantially identical to, or differs by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% from polypeptides encoded by an endogenous cellular genome. In one embodiment, the recombinant polypeptide and the cell are from different species; for example, the recombinant polypeptide is a human polypeptide, and the cell is non-human, such as rodent, such as CHO, or insect cells. In one embodiment, the recombinant polypeptide is exogenous to the cell; in other words, the cell is from a first species, and the recombinant polypeptide is from a second species. In one embodiment, the polypeptide is a synthetic polypeptide. In one embodiment, the polypeptide is derived from a non-naturally occurring source. In one embodiment, the recombinant polypeptide is a human polypeptide or protein whose amino acid sequence is not different from the naturally occurring isotype of the human polypeptide or protein. In one embodiment, the recombinant polypeptide differs from the naturally occurring isotype of the human polypeptide or protein by no more than 1, 2, 3, 4, 5, 10, 15, or 20 amino acid residues. In one embodiment, the recombinant polypeptide differs from the naturally occurring isotype of the human polypeptide by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15% of amino acid residues.

[0103] [Optimized system for virus removal]

[0104] According to one aspect of this disclosure, a system for optimizing virus removal includes at least one product tank, at least one chasing fluid reservoir connected to the inlet of the at least one product tank, at least one virus filter connected to the outlet of the at least one product tank, and at least one eluent reservoir connected to the at least one virus filter. In some embodiments, a controller regulates the flow of chasing fluid from the at least one chasing fluid reservoir into the at least one product tank. In some embodiments, the controller regulates a first flow rate of the chasing fluid from the at least one chasing fluid reservoir to match a second flow rate, such as the flow rate of fluid from the at least one product tank or the flow rate through the virus filter downstream of the at least one product tank.

[0105] According to one aspect of this disclosure, a system for filtering a process fluid comprising a product is provided. In some embodiments, the product comprises an active pharmaceutical ingredient. The system can be used in conjunction with any method for filtering process fluids according to this disclosure. Figure 1 An embodiment of a production system, typically denoted by 10, is shown, including a product storage tank (first reservoir) 12 leading to a virus filter 14. Figure 1 In some embodiments, the virus filter 14 leads to the eluent reservoir (second reservoir) 16. In some embodiments, the eluent reservoir 16 is not included. The chasing fluid reservoir leads to the first reservoir 12.

[0106] The first reservoir 12 promotes the proliferation and maintenance of cells or cell lines. For example, in some embodiments, the first reservoir 12 promotes the proliferation and maintenance of the cells or cell lines described herein.

[0107] The first reservoir 12 may include or be connected to a bioreactor and / or culture module. The bioreactor and / or culture module may be rotated or agitated throughout the device by controlled actuators. Rotation of the first reservoir 12 about an axis may enable the beneficial use of gravity to achieve specific biological treatment sequences, such as sedimentation-based cell inoculation and fluid exchange within the bioreactor.

[0108] In one embodiment, the bioreactor includes a bioreactor shell having one or more inlet ports and one or more outlet ports for media flow, and at least one chamber defined within the bioreactor shell for receiving cells and promoting cell culture and protein production. The chamber may be selected from a cell culture / proliferation chamber and / or a protein production chamber. Furthermore, the chamber houses one or more substrates and / or scaffolds. In another embodiment of the invention, the bioreactor includes two chambers operatively connected within the bioreactor. Alternatively, the two chambers may operate independently or operatively. In a further aspect, the chambers and / or the bioreactor may be operatively connected to provide fluid, cell, and / or tissue exchange between the chambers and / or the bioreactor. The scaffold used in the invention is selected from porous scaffolds, porous scaffolds with gradient pores, porous mesh scaffolds, fiber scaffolds, membrane-enclosed scaffolds, and combinations thereof. Funnels or similar channels may be provided between the chambers within the bioreactor. Additionally, one or more filters may be provided at any location within the bioreactor.

[0109] The cell culture apparatus (or culture module) described herein in various embodiments is controlled by one or more microprocessors, which can be pre-programmed so that a user can select a specific type of environment (or environmental sequence) within the bioreactor, such as cell proliferation, cell maintenance, protein production, or protein secretion. This eliminates operator intervention and reduces the possibility of unintentional contamination.

[0110] Associated with the first reservoir 12 is a sensor assembly 20 configured to monitor the amount of fluid in the first reservoir 12. In some embodiments, the sensor assembly 20 may also sense the rate of change of the amount of fluid in the first reservoir 12. In some embodiments, the sensor assembly 20 is embedded in or fixed to the wall of the first reservoir 12. In some embodiments, the sensor assembly 20 may include a mechanical sensor, an electrical sensor, an optical sensor, and / or another sensor.

[0111] The product storage tank (first reservoir) 12 is fluidly connected to the virus filter 14. Figure 1 In this configuration, a virus filter 14 is disposed between a first reservoir 12 and a second reservoir 16, so that fluid flows from the first reservoir 12 to the virus filter 14 and then to the second reservoir 16. Specifically, a pre-filter conduit (first conduit) 22 is configured to deliver fluid from the first reservoir 12 to the virus filter 14. In some embodiments, the pre-filter conduit 22 is a conduit. The pre-filter conduit 22 may include standard conduits used for biological treatment operations. In some embodiments, the pre-filter conduit 22 has an outer diameter of 2 inches. In some embodiments, the pre-filter conduit 22 is a sanitary conduit. In some embodiments, the pre-filter conduit 22 is a disposable tube.

[0112] To control the flow from the first reservoir 12 to the virus filter 14, a first valve 24 is disposed between the first reservoir 12 and the virus filter 14 along a pre-filter conduit 22. The first valve 24 fluidly connects the first reservoir 12 and the virus filter 14. The first valve 24 can be controlled by a processor 26 to regulate the fluid flow from the first reservoir 12 to the virus filter 14. The first valve 24 can be opened to allow fluid to flow out. In some embodiments, the first valve 24 can be adjusted between an open position and a closed position to throttle the fluid flow from the first reservoir 12 to the virus filter 14.

[0113] although Figure 1 The virus filter 14 is shown to be separate from the first reservoir 12 and the second reservoir 16, but in some embodiments, the virus filter 14 is integrated with the wall of the first reservoir 12 or the second reservoir 16.

[0114] A virus filter 14 is disposed between a pre-filter conduit 22 and a post-filter conduit 28, which connects the virus filter 14 to a second reservoir 16 and is configured to deliver fluid (e.g., eluent) from the virus filter 14 to the second reservoir 16. In some embodiments, the post-filter conduit 28 is a conduit. The post-filter conduit 28 may include standard tubing for biological processing operations. In some embodiments, the post-filter conduit 28 has an outer diameter of 2 inches. In some embodiments, the post-filter conduit 28 is a sanitary conduit. The virus filter 14 is in fluid connection to the eluent reservoir (second reservoir) 16 via the post-filter conduit 28.

[0115] Virus filter 14 has a certain virus removal capacity, such as a logarithmic reduction value. If the processed fluid contains more material to be removed than its effective capacity, multiple virus filters can be used in parallel.

[0116] A second valve 30 is disposed along the post-filter conduit 28 and configured to regulate (stop or allow) fluid flow between the virus filter 14 and the eluent reservoir 16. The second valve 30 can be controlled by the processor 26 to regulate the flow of fluid (e.g., eluent) from the virus filter 14 to the second reservoir 16. The second valve 30 can be opened to allow fluid to flow from the virus filter 14 to the second reservoir 16. In some embodiments, the second valve can regulate the fluid shown in FIG. 30 between an open position and a closed position to throttle the fluid flow from the virus filter 14 to the second reservoir 16. In some embodiments, the second reservoir 16 is referred to as a terminal reservoir.

[0117] When system 10 is used to perform subsequent biological treatment operations that do not require the second reservoir 16, the post-filter conduit 28 may be connected to an endpoint or an endpoint reservoir other than the second reservoir 16. In various embodiments, the post-filter conduit 28 is configured to deliver fluid (e.g., eluent) to the second reservoir 16, another endpoint, or another conduit to the endpoint.

[0118] A chasing fluid reservoir 18 is connected to a first reservoir 12 via a chasing fluid conduit 32, allowing chasing fluid to flow from the chasing fluid reservoir 18 into the first reservoir 12. A third valve 34 is disposed along the chasing fluid conduit 32 between the chasing fluid reservoir 18 and the first reservoir 12. The third valve 34 can be controlled by a processor 26 to regulate the flow of chasing fluid from the chasing fluid reservoir 18 to the first reservoir 12. The third valve 34 can be opened to allow fluid to flow from the chasing fluid reservoir 18 to the first reservoir 12. In some embodiments, the third valve 34 can be adjusted to throttle the fluid flow from the chasing fluid reservoir 18 to the first reservoir 12.

[0119] Processor 26 can independently or simultaneously control the first valve 24, the second valve 30, and the third valve 34. Processor 26 is configured to control the fluid flow through system 10. As shown, processor 26 is connected to the first valve 24 via a first lead 36, to the second valve 30 via a second lead 38, and to the third valve 34 via a third lead 40. The first lead 36, the second lead 38, and the third lead 40 each allow signal communication, such as via a conductor, optical fiber, or wireless transmission.

[0120] In some implementations, the first valve 24, the second valve 30, and the third valve 34 can be manually operated to override the processor 26.

[0121] In some implementations, processor 26 is a microprocessor. In some implementations, processor 26 is integrated into a general-purpose computer.

[0122] System 10 can be configured for a variety of specialized applications, such as, but not limited to, one or more of the following: aseptic reception / storage of cells; automated mixing and delivery of reagents for protein expression, production, modification (e.g., post-translational modification) and / or secretion; automated monitoring of protein expression, production, modification and / or secretion; cell sorting and selection, including secure waste collection; cell washing and cell collection; cell seeding on or within a proliferation matrix or scaffold; automated mixing and delivery of proliferation reagents; cell proliferation to expand cell populations; automated monitoring of cell status, including detection of confluence or growth phase; controlled release of cells from a proliferation matrix or scaffold; repeating proliferation steps on selected surface areas to increase cell numbers; cell seeding on or within a culture scaffold or matrix; automated monitoring of cell / tissue culture conditions; automated monitoring of protein expression or secretion; mechanical and / or biochemical stimulation to promote proliferation; protein purification and / or protein recovery; and storage and transport of cells and / or protein products.

[0123] According to one aspect of this disclosure, a method for filtering a process fluid containing a product is provided. In some embodiments, the product contains an active pharmaceutical ingredient. The method can be performed by, for example... Figure 1 The system execution of system 10. Some implementations of this method include providing a system, such as Figure 1 The system.

[0124] refer to Figure 1 The system, the method comprising a first step of allowing some of the processing fluid in the product storage tank (first reservoir) 12 to flow out of the first reservoir 12 and through a virus filter 14 to produce eluent. Upon completion of fermentation in the first reservoir 12, the processor 26 opens a first valve 24 and a second valve 30, allowing cell culture medium to flow from the first reservoir 12 through the virus filter 14 to produce eluent. The eluent flows from the virus filter 14 into an eluent reservoir 16.

[0125] The method includes a second step: adding a chasing fluid from the chasing fluid reservoir 18 to the processed fluid in the first reservoir 12 to form a composite fluid in the first reservoir 12. In some embodiments, the amount of fluid in the first reservoir 12 is sensed by the sensor assembly 20, and when a predetermined amount of fluid is reached in the first reservoir 12, the processor 26 opens the third valve 34, allowing the chasing fluid to flow from the chasing fluid reservoir 18 into the first reservoir 12 to form the composite fluid in the first reservoir 12.

[0126] When a signal from sensor assembly 20 reaches a preselected volume of composite fluid in first reservoir 12, processor 26 closes third valve 34, causing chasing fluid to no longer be added from chasing fluid reservoir 18 to first reservoir 12.

[0127] In some implementations, the second step of adding the chasing fluid is performed before emptying the first reservoir 12 of the treatment fluid. It is undesirable that air in the virus filter 14 could stop flowing through the system when air enters the virus filter 14. By maintaining a sufficient amount of fluid in the first reservoir 12 before adding the chasing fluid, the liquid level in the first reservoir will not be at a low level that would allow air to enter the virus filter 14.

[0128] The fluid volume in the first reservoir 12 can be selected precisely before the addition of the chasing fluid, such that air does not enter the virus filter 14. In some embodiments, the fluid volume in the first reservoir 12 prior to the addition step is related to a component downstream of the first reservoir 12, such as the combined volume of the virus filter 14, the pre-filter conduit 22, and the post-filter conduit 28. In some embodiments, the fluid volume in the first reservoir 12 precisely before the addition of the chasing fluid is related to a component downstream of the first reservoir 14, such as the combined volume of the virus filter 14 and the pre-filter conduit 22.

[0129] In some embodiments, some of the processing fluid in the first reservoir 12 is drained out of the first reservoir 12 and passed through the virus filter 14 to reduce the amount of processing fluid remaining in the first reservoir 12.

[0130] In some embodiments, when the volume of the processing fluid remaining in the first reservoir 12 is at or within a reference volume range, a second step of adding chasing fluid to the first reservoir 12 is performed. In some embodiments, the reference volume is equal to or greater than 0.5, 1, 1.5, 2, 3, 5, or 10 times the volume of the components disposed between the first reservoir 12 and the endpoint reservoir. For example, in some embodiments, the reference volume is equal to or greater than 0.5, 1, 1.5, 2, 3, 5, or 10 times the combined volume of the virus filter 14, the pre-filter conduit 22, and the column. In some embodiments, the reference volume is equal to or greater than the volume of the components disposed between the first reservoir 12 and the endpoint reservoir. For example, in some embodiments, the reference volume is equal to or greater than the combined volume of the virus filter 14, the pre-filter conduit 22, and the post-filter conduit 28.

[0131] In some embodiments, the volume of the chasing fluid added to the first reservoir 12 in the second step is equal to, less than, or equal to the chasing fluid reference volume. In some embodiments, the chasing fluid reference volume is equal to or greater than 0.5, 1, 1.5, 2, 3, 5, or 10 times the volume of the component disposed between the first reservoir 12 and the terminal reservoir. For example, in some embodiments, the chasing fluid reference volume is equal to or greater than 0.5, 1, 1.5, 2, 3, 5, or 10 times the combined volume of the virus filter 14, the pre-filter conduit 22, and the post-filter conduit 28.

[0132] In some embodiments, the chasing fluid reference volume is equal to or greater than the volume of the component disposed between the first reservoir and the terminal reservoir. For example, in some embodiments, the chasing fluid reference volume is equal to or greater than the combined volume of the virus filter 14, the pre-filter conduit 22, and the post-filter conduit 28.

[0133] In some embodiments, a chasing fluid is added to a first reservoir 12, while the first reservoir 12 still contains a predetermined amount of processing fluid. For example, in some embodiments, a chasing fluid is added to the first reservoir 12, while the first reservoir 12 still contains at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50% of the original amount of processing fluid.

[0134] In some embodiments, the method includes operating processor 26 to determine a value as a function of the amount of process fluid remaining in the first reservoir 12. In some embodiments, the method includes operating processor 26 to determine whether the value satisfies the predetermined reference value. In some embodiments, if the value has a predetermined relationship with the reference value, chasing fluid is added to the first reservoir 12. In some embodiments, the predetermined relationship is that the value satisfies, exceeds, or is less than the reference value.

[0135] In some embodiments, when the chasing fluid is added, the ratio of the amount of chasing fluid to the amount of processing fluid remaining in the first reservoir 12 is equal to or greater than 1:0.5, 1:1, 1.5:1, 2:1, 3:1, 5:1 or 10:1.

[0136] The method includes a third step of allowing the composite fluid in the first reservoir 12 to flow out of the first reservoir 12 and through the virus filter 14 to produce an eluent.

[0137] In some embodiments, during one, two, or all of the first, second, and third steps of the above method, there is a continuous flow from the first reservoir 12 to the virus filter 14.

[0138] In some embodiments, the flow of the first reservoir fluid through the virus filter 14 is maintained without interruption or slowing of the flow for a duration or magnitude sufficient to cause significant impairment to virus removal. In some embodiments, the flow has a duration or magnitude sufficient to cause a reduction in viral particles of at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times. In some embodiments, the flow of the first reservoir fluid through the virus filter 14 is maintained at a preselected rate.

[0139] In some embodiments, the chasing fluid is added from the chasing fluid reservoir 18 to the first reservoir 12 without interrupting the flow of material from the first reservoir 12 through the virus filter 14 to the eluent reservoir 16 or another endpoint. Higher purity formulations can be obtained by avoiding interruption of the flow of product-containing fluid from the first reservoir 12 through the virus filter 14. The purity or reduction of viral contamination in the eluent can be optimized by maintaining the flow from the first reservoir 12 to the virus filter 14 and from the virus filter 14 to the eluent reservoir 16. In some cases, if the flow from the first reservoir 12 to the eluent reservoir 16 stops, it may be necessary to reprocess a new batch of processing fluid.

[0140] In some embodiments, the flow of fluid from the first reservoir through the virus filter is maintained without interruption or slowing of the flow sufficient to substantially impair the duration or magnitude of virus removal. In some embodiments, the flow of fluid from the first reservoir through the virus filter is maintained without cessation of flow.

[0141] In some implementations, the flow of fluid from the first reservoir through the virus filter is maintained for a duration or magnitude that is not interrupted or slowed enough to impair virus removal to a level below the logarithmic reduction value of the virus filter. Typical virus filters have associated logarithmic reduction values ​​reported or listed by the manufacturer. These values ​​vary depending on the virus filter used and represent the ability of each virus filter to remove virus content from the fluid as fluid and viral contents pass through it. If air enters the virus filter, the virus filtration process will prematurely stop. If the virus filtration process stops prematurely, the results of the virus filtration step are invalid.

[0142] In some embodiments, the method allows for continuous flow of fluid from the first reservoir 12 to the virus filter 14. In some embodiments, the total cessation of fluid flow in the first reservoir is less than 1 minute, 10 minutes, or 60 minutes. In some embodiments, any single cessation of fluid flow in the first reservoir lasts less than 1, 10, or 60 minutes. The method of this disclosure increases the yield of product from the first reservoir.

[0143] The flow of fluid through the first reservoir of the virus filter is sufficient to avoid significant damage to the removal of the virus from the first reservoir 12 until a preselection event occurs. In some embodiments, the preselection event includes severing the fluid connection between the virus filter and the second or endpoint reservoir. For example, the preselection event may include closing a second valve. In some embodiments, the preselection event includes sending the eluent to a subsequent operation, such as the next unit operation. In some embodiments, the next unit operation includes liquid chromatography or ultrafiltration. In some embodiments, the preselection event includes stopping the flow from the virus filter to the accumulated eluent. In some embodiments, the preselection event includes stopping the flow from the virus filter to the second or endpoint reservoir. In some embodiments, the preselection event includes collecting the eluent generated by the fluid flow from the first reservoir. In some embodiments, the preselection event is a collection of accumulated eluent. In some embodiments, the preselection event includes reaching the end of a preselection time period.

[0144] In some embodiments, a preselected pressure difference is maintained, such as a preselected pressure difference range across virus filter 14. In some embodiments, the pressure difference across virus filter 14 is maintained at or below a preselected maximum value. In some embodiments, the maintained preselected pressure difference across virus filter 14 is equal to or no greater than 14 psi, 11 psi, or 13.2 psi. In some embodiments, the pressure difference across virus filter 14 is maintained at or above a preselected minimum value. In some embodiments, the pressure difference across virus filter 14 is sufficient to reduce viral particles by at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times. In some embodiments, the pressure difference across virus filter 14 can be measured as the difference between a first pressure sensed by a first pressure sensor at the inlet of virus filter 14 and a second pressure sensed by a second pressure sensor at the outlet of virus filter 14.

[0145] In some embodiments, the flow rate of the transviral filter is in the range of 1 to 10 L / min while maintaining the preselected pressure differential range of the transviral filter 14. In some embodiments, the flow rate of the transviral filter is 0.1, 0.2, 0.5, 1, 2, 4, 8, 10, 12 or 20 L / min while maintaining the preselected pressure differential range of the transviral filter 14.

[0146] Example Method

[0147] In one exemplary method, upon reaching the end of fermentation, processor 26 opens first valve 24 and second valve 30, allowing cell culture medium to flow from first reservoir 12 through virus filter 14 and into eluent reservoir 16. Sensor assembly 20 senses the fluid volume in first reservoir 12. In some embodiments, first reservoir 12 has a volume of 5,000 L. When a predetermined amount of fluid, such as 200 kg, is reached in first reservoir 12, processor 26 opens third valve 34, allowing chasing fluid to flow from chasing fluid reservoir 18 into first reservoir 12 to form a complex in first reservoir 12. When a preselected volume of complex fluid is reached in first reservoir 12, processor 26 closes third valve 34, as indicated by sensor assembly 20. In some embodiments, the preselected volume of complex fluid in first reservoir is 100 L, 150 L, 200 L, 250 L, or 300 L. After further emptying of the first reservoir 12, and upon reaching a preselected amount of composite fluid in the first reservoir 12, the sensor assembly 20 signals the processor 26 to close the second valve 30. The processor 26 is configured to maintain a minimum flow rate from the first reservoir 12 through the virus filter 14 from the opening of the first valve 24 to drain the first reservoir 12 until eluent collection is complete. The processor 26 may be configured such that once the flow through the virus filter 14 reaches a minimum rate, e.g., stops, eluent is not allowed to flow into the eluent reservoir 16.

[0148] According to one aspect of this disclosure, a method for filtering a processing fluid comprising products includes a first step of draining the processing fluid from a first reservoir through a virus filter to produce eluent and reducing a predetermined amount of processing fluid in the first reservoir. The method includes a second step of determining a value that is a function of the amount of processing fluid remaining in the first reservoir, and if the value satisfies a predetermined reference value, adding a chasing fluid to the processing fluid in the first reservoir to form a composite fluid. In some embodiments, a chasing fluid is added to the first reservoir while the first reservoir still contains a predetermined amount of processing fluid. The method includes a third step of draining the composite fluid from the first reservoir through a virus filter to produce eluent. The method includes a fourth step of stopping the flow from the virus filter into the accumulated eluent. In some embodiments, the flow of fluid from the first reservoir through the virus filter is sufficient to avoid significant impairment of virus removal or fluid removal from the first reservoir until the flow from the virus filter into the accumulated eluent stops.

[0149] [Virus Filter]

[0150] Various filtration methods can be used to remove viral particles from cultures or other fluid-containing products for medical and veterinary applications. Size exclusion filtration offers many advantages. Typically, it does not alter the structure or functional properties of the peptide product and provides non-specific virus removal.

[0151] Hollow fiber membrane filters are particularly well-suited for removing viral particles. A hollow fiber membrane filter comprises bundles of parallel hollow fibers, roughly forming a straw. The walls of each hollow fiber have a three-dimensional network structure of pores interconnected by fine capillaries. While not wishing to be bound by theory, it is believed that through size exclusion mechanisms, the membrane allows proteins to easily pass through the hollow fiber walls while retaining viruses. During the filtration of the protein solution, viruses are removed and proteins permeate through the membrane. This method can provide high protein recovery rates without adsorption or denaturation.

[0152] An exemplary hollow fiber filter includes a hydrophilically modified polyvinylidene fluoride (PVDF) hollow fiber membrane. Filter elements can be disposed in inert cartridges configured at each end for insertion into a production line. An exemplary virus filter includes Planova. TM 15N, 20N, 35N and 75N filters, Planova TM BioEX filters and filters that are essentially similar to these.

[0153] In one embodiment, the method described herein provides a product solution having a virus reduction of at least 5, 10, 15, 20, 30, 40, 50, or 100 times (after passing through a filter).

[0154] In some embodiments, the methods described herein provide -12 to 18 log to endogenous retroviruses. 10 The logarithmic reduction in clearance provides approximately 6 log for foreign viruses. 10 The removal.

[0155] Other filters that can be used in the methods described herein include, for example, tangential flow filters.

[0156] Reactor

[0157] The optimized virus removal methods disclosed herein can be used with a bioreactor, or more generally with any feed source. Typically, the bioreactor is not immediately upstream of the filter, but rather the filter's feed originates from a downstream container, such as a product tank. The apparatus, facilities, and methods may include any suitable reactor, including but not limited to stirred tanks, airlift reactors, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or jet bed bioreactors. The reactor may be directly connected to the virus filter or may have other elements, such as other containers positioned between the reactor and the virus filter, for example, a vessel. As used herein, "reactor" may include a fermenter or fermentation unit, or any other reaction vessel or suitable reservoir; the term "reactor" is used interchangeably with "fermenter."

[0158] The apparatus, facilities, and methods described herein are suitable for culturing any desired cell lines, including prokaryotic and / or eukaryotic cell lines. Furthermore, in embodiments, the apparatus, facilities, and methods are suitable for culturing suspension cells or adherent cells, and are suitable for configuring manufacturing operations for producing pharmaceutical and biopharmaceutical products, such as peptide products, nucleic acid products (for example, DNA or RNA), or cells and / or viruses, such as cells and / or viruses for cell and / or viral therapies.

[0159] In embodiments, cells express or produce products, such as recombinant therapeutic or diagnostic products. Examples of cell-produced products, as described in more detail below, include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimics (peptide molecules that bind specifically to antigens but are not structurally independent of antibodies, such as DARPins, affibodies, adnectin, or IgNARs), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapeutics (e.g., anticancer oncolytics), viruses, viral vectors for gene therapy, and viral immunotherapy), cell therapies (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), vaccines, or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA) or DNA (e.g., plasmid DNA), antibiotics, or amino acids. In embodiments, the apparatus, facilities, and methods can be used to produce biosimilars.

[0160] As described above, in the embodiments, the apparatus, facilities, and methods allow the production of eukaryotic cells, such as mammalian cells or lower eukaryotic cells, such as yeast cells or filamentous fungal cells, or prokaryotic cells, such as Gram-positive or Gram-negative cells, and / or products of eukaryotic or prokaryotic cells, such as proteins, peptides, antibiotics, amino acids, nucleic acids (such as DNA or RNA), synthesized by eukaryotic cells in a large-scale manner. Unless otherwise stated herein, the apparatus, facilities, and methods may include any desired volume or production capacity, including but not limited to laboratory-scale, pilot-scale, and full-scale production capacity.

[0161] Furthermore, unless otherwise stated herein, the apparatus, facilities, and methods described may include any suitable reactor, including but not limited to stirred tanks, airlift reactors, fiber reactors, microfiber reactors, hollow fiber reactors, ceramic matrix reactors, fluidized bed reactors, fixed bed reactors, and / or jet bed reactors. As used herein, “reactor” may include a fermenter or fermentation unit, or any other reaction vessel, and the term “reactor” may be used interchangeably with “fermentation tank.” For example, in some aspects, a bioreactor unit may perform one or more of the following: feeding of nutrients and / or carbon sources, injection of suitable gases (e.g., oxygen), inlet and outlet flow of fermentation or cell culture media, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. Exemplary reactor units, such as fermentation units, may contain multiple reactors within a unit. For example, the unit may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors in each unit, and / or the equipment may be contained within multiple units having single or multiple reactors within a facility. In various embodiments, the bioreactor may be suitable for batch, semi-feed batch, fed batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the bioreactor may have a volume of from about 100 mL to about 50,000 L. Non-limiting examples include 100mL, 250mL, 500mL, 750mL, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 15L, 20L, 25L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​200L, 250L, 300L, 350L, 400L, 450L, 500L, 550L, 6 Volumes of 00L, 650L, 700L, 750L, 800L, 850L, 900L, 950L, 1000L, 1500L, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 6000L, 7000L, 8000L, 9000L, 10,000L, 15,000L, 20,000L, and / or 50,000L. Additionally, suitable reactors can be reusable, disposable, single-use, or non-disposable, and can be formed from any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics, and / or glass.

[0162] In implementations and unless otherwise stated herein, the apparatus, facilities, and methods described herein may also include any suitable unit operations and / or devices not otherwise mentioned, such as operations and / or devices for separating, purifying, and isolating such products. Any suitable facility and environment may be used, such as conventional pole-mounted facilities, modular, mobile, and temporary facilities, or any other suitable structure, facility, and / or layout. For example, in some implementations, a modular cleanroom may be used. Additionally, unless otherwise stated, the apparatus, systems, and methods described herein may be housed and / or performed in a single location or facility, or optionally in one or more locations and / or facilities.

[0163] By way of non-limiting example and not limitation, U.S. Publications 2013 / 0280797; 2012 / 0077429; 2009 / 0305626; and U.S. Patent Nos. 8,298,054; 7,629,167 and 5,656,491 describe exemplary facilities, devices and / or systems that may be suitable.

[0164] In this embodiment, the cells are eukaryotic cells, such as mammalian cells. Mammalian cells can be, for example, human, rodent, or bovine cell lines or cell strains. Examples of such cells, cell lines, or cell strains are, for example, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (juvenile hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, such as COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EB1, EB2, EB3, oncolytic or hybridoma cell lines. Preferably, the mammalian cells are CHO cell lines. In one embodiment, the cells are CHO cells. In one embodiment, the cells are CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS, CHO GS knockout cells, CHOFUT8 GS knockout cells, CHOZN, or CHO-derived cells. CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1SV GS knockout cells. CHO FUT8 knockout cells are, for example, Potelligent® CHOK1 SV (Lonza Biologies, Inc.). Eukaryotic cells may also be avian cells, cell lines, or cell strains, such as EBx® cells, EB14, EB24, EB26, EB66, or EBv13.

[0165] In one implementation, the eukaryotic cell is a stem cell. Stem cells can be, for example, pluripotent stem cells, including embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs).

[0166] In one embodiment, the cell is a differentiated form of any cell described herein. In one embodiment, the cell is a cell derived from any primary cell in culture.

[0167] In this implementation, the cells are hepatocytes, such as human hepatocytes, animal hepatocytes, or non-parenchymal cells. For example, the cells could be transferable metabolically qualified human hepatocytes, transferable induced-qualified human hepatocytes, or transferable QualystTransporter Certified cells. TM Human hepatocytes, suspension-qualified human hepatocytes (including hepatocytes from 10 and 20 donor pools), human hepatic Kupffer cells, human hepatic stellate cells, canine hepatocytes (including single and pooled Beagle hepatocytes), mouse hepatocytes (including CD-I and C57BI / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including Cynomolgus or Rhesus monkeyhepatocytes), cat hepatocytes (including domestic short-haired cell hepatocytes), and rabbit hepatocytes (including New Zealand white hepatocytes). Sample hepatocytes are commercially available from Triangle Research Labs, LLC, 6 Davis Drive Research Triangle Park, North Carolina, USA 27709.

[0168] In one embodiment, the eukaryotic cell is a lower eukaryotic cell, such as a yeast cell, for example, a Pichia pastoris genus (…). Peach (e.g., Pichia pastoris) Shepherd's pie ), Pichia pastoris ( Peach methanol ), Pichia kluflii ( Pichia kluyveri ), and Angus Pichia pastoris ( Peach narrow )), Komagataella Genus (e.g.) Shepherd's Komagataella , Komagataella pseudo-shepherd or Komagataella phaffii ), saccharomyces ( Saccharomyces (e.g., beer sugar yeast) Saccharomyces cerevisiae ), Klefleurone sugar yeast ( Saccharomyces kluyveri), grape juice yeast ( Saccharomyces vitis Kluyveromyces ( )), Kluyveromyces ( Kluyveromyces (e.g., Kluyveromycin) Kluyveromyces lactis ), Max Kluwer yeast ( Kluyveromyces marxianus ), Candida genus ( White (e.g., Candida utilis) Useful Candida ), Candida cocoa ( Candida cacaoi ), Candida boydinis ( Candida boidinii )), Geotrichum ( Geotrichum (e.g., fermented terrestris) Geotrichum fermenting ), Hansenula polymorpha ( Hansenula polymorpha ), Yarrowia lipolytica ( Yarrow lipolytic ), or millet wine schizophytes ( Schizosaccharomyces pombe Pichia pastoris is preferred. Shepherd's pie Pichia pastoris ( ). Shepherd's pie Examples of strains are X33, GS115, KM71, KM71H; and CBS7435.

[0169] In one implementation, the eukaryotic cell is a fungal cell, such as Aspergillus ( ). Aspergillus (such as Aspergillus niger ( A. niger Aspergillus fumigatus ( A. fumigatus Aspergillus oryzae ( A. oryzae Aspergillus oryzae ( ), A. nidulans ), genus Apocynum ( Acremonium (such as thermophilic apothecia) A. thermophilum ), Mucor genus ( Chaetonium (such as thermophilic chamomile) C. thermophilum ), genus Aureospora ( Chrysosporium (such as thermophilic aureospores) C. thermophilic )), Cordyceps (such as) C. military ), Corynascus , Ctenomyces Fusarium ( Fusarium (such as Fusarium oxysporum) F. oxysporum ), genus *Pathobacter* ( Glomerulus (such as Gramineae) G. grass-like plant ), Sarcoptes genus ( Hypocritical (such as Rhodophyta rubrum) H. jecorina ), genus *Oryza sativa* ( Magnaporth (such as prickly spores ( M. oryzae ), genus *Hypericum* ( Mycelium (such as thermophilic filamentous fungus) M. thermophile ), genus *Cryptotympany* ( Nectarine (such as Red Globe Fungus (Red Globe Fungus) N. haematococci ), genus *Neurospora* ( Neurospora (such as *Neurospora rubra*) N. crassa Penicillium ()), Penicillium genus ( Penicillium ), Sporothrix ( Sporotrichum (such as thermophilic spores) S. thermophile ), genus Rhizopus ( Thielavia (such as terrestrial grass rhizomyces ( T. terrestris ), heterophyllophoric combination of herbaceous rhizomycin ( T. heterothallica Trichoderma ( ) , Trichoderma genus ( Trichoderma (such as Trichoderma reesei) T. reesei ), or Verticillium ( Verticillium wilt (such as Verticillium dahliae) V. dahlias )).

[0170] In one implementation, the eukaryotic cell is an insect cell (e.g., Sf9, Mimic). TM SF9, SF21, HighFive TM (BT1-TN-5B 1-4) or BT1-Ea88 cells), algal cells (e.g., genus...) Amphora , Bacillariophyceae , Dunaliella , Chlorella , Chlamydomonas , Cyanophyta (Cyanobacteria), *Micrococcus* genus ( Nannochloropsis ), Spirulina or Ochromonas ), or plant cells (e.g., cells from monocotyledonous plants (such as corn, rice, wheat, or foxtail grass), or cells from dicotyledonous plants (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, sphagnum moss) Physcomitrella patens ) or Arabidopsis genus ( Arabidopsis )).

[0171] In one implementation, the cell is a bacterium or other prokaryotic cell.

[0172] In this embodiment, the prokaryotic cells are Gram-positive cells, such as Bacillus spp. ( Bacillus Streptomyces ( Streptomyces Streptococcus ( ) Streptococcus Staphylococcus spp. Staphylococcus ) or Lactobacillus spp. Lactobacillus Bacillus species that can be used ( ). Bacillus Yes, for example, Bacillus subtilis ( B. subtle ), Bacillus amyloliquefaciens ( B. amyloliquefaciens ), Bacillus licheniformis ( B. licheniformis), Bacillus natto ( B. natto ) or Bacillus megaterium ( B. megatherium In this embodiment, the cells are Bacillus subtilis (…). B. subtilis ), such as Bacillus subtilis ( B. subtilis )3NA and Bacillus subtilis ( B. subtle 168. Bacillus ( ) Bacillus (It can be obtained, for example, from Bacillus Genetic Stock Center, Biological Sciences 556, 484 West 12th Avenue, Columbus OH 43210-1214.)

[0173] In one implementation, the prokaryotic cell is a Gram-negative cell, such as Salmonella spp. Salmonella ssp. ) species or Escherichia coli ( Escherichia coli Examples include TGI, TG2, W3110, DH1, DHB4, DH5a, HMS174, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue, and Origami, as well as those derived from Escherichia coli. E. coli B-strains, such as BL-21 or BL21(DE3), are all commercially available.

[0174] Suitable host cells are commercially available, for example from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0175] In some embodiments, the cultured cells are used to produce proteins, such as antibodies, including monoclonal antibodies and / or recombinant proteins, for therapeutic purposes. In others, the cultured cells produce peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites. For example, in some embodiments, molecules with molecular weights ranging from about 4,000 Daltons to greater than about 140,000 Daltons can be prepared. In others, these molecules may have a range of complexities and may include post-translational modifications, including glycosylation.

[0176] In the implementation, the protein is, for example, BOTOX, Myobloc, Neurobloc, Dysport (or other botulinum neurotoxin serotypes), aglucosidase α, daptomycin, YH-16, human chorionic gonadotropin α, filgrastim, cetrorex, interleukin-2, adelin, teceleulin, dnisolone interleukin-toxin conjugate, interferon α-n3 (injection), interferon α-nl, DL-8234, interferon, Suntory (γ-1a), interferon γ, thymosin α1, tasonemine, DigiFab, Viper. aTAb, EchiTAb, CroFab, Nesiritide, Abatacept, Alfasate, Rebif, Etotamine Alpha, Teriparatide (Osteoporosis), Calcitonin Injectable (Bone Disease), Calcitonin (Nasal, Osteoporosis), Etanercept, Polyglutaraldehyde Hemoglobin 250 (Bovine), Triclopramide Alpha, Collagenase, Capecitide, Recombinant Human Epidermal Growth Factor (Topical Gel, Wound Healing), DWP401, Dabepoetin Alpha, Epoetin ω, Epoetin β, Epoetin Alpha, Diasiludin, Lepirudine, Bivalirudin, Nonacog Alpha, Mononine, Eptacog Alpha (Activated), Recombinant Factor VIII+VWF, Recombinate, Recombinant Factor VIII, Factor VIII (Recombinant), Alphnmate, Octocog α, Factor VIII, Palivmin, Indikinase, Tenexerase, Alteplase, Pamiplase, Reteplase, Nateplase, Monteteplase, Follicle-stimulating factor α, rFSH, hpFSH, Micafungin, Pefexus, Levofloxacin, Natosine, Semorelin, Glucagon, Ezenatide, Pramyltide, Iniglucerase, Thionase, Leucotropin, Molgramostirn, Triptorelin Acetate, Histamine Relin (subcutaneous implant, Hydron), Lorerelin, Histamine Relin, Nafarelin, Leuprorelin Extended-Release Depot (ATRIGEL), Leuprorelin Implant (DUROS), Goserelin, Eutropin, KP-102 Program, Growth Hormone, Mecaprelin (Growth Disorder), Enlfavirtide, Org-33408, Insulin Glargine, Insulin Glucosides, Insulin (Inhalation), Insulin Lispro, Insulin Detemir, Insulin (Oral, RapidMist), Mecaprelin Linfiber, Analepidocanine, Simo-Interleukin, 99 mTc-apcitide Injection, Myelopid, Betaseron, Grammer Acetate, Gepon, Saxaglastine, Olepidocanine, Human Leukocyte-Derived Alpha Interferon, Bilive, Insulin (Recombinant), Recombinant Human Insulin, Insulin Aspart, Mecasenin, Roferon-A, Interferon-α2, Alfaferone, Interferon Alfacon-1Interferon Alpha, Avonex' Recombinant Human Luteinizing Hormone, Levofloxacin Alpha, Triflunomide, Ziconopeptide, Tatirelin, Deborahamine Alpha, Atosiban, Becaprolactam, Etibatide, Zemaira, CTC-111, Shanvac-B, HPV Vaccine (Quadrivalent), Octreotide, Lanreotide, Anselstatin, Agassizine Beta, Agassizine Alpha, Laronibase, Prezatide Copper Acetate (Topical Gel), Raburicase, Ranibizumab, Actimmune, PEG-Intron, Tricomin, Recombinant House Dust mite allergy desensitization injection, recombinant human parathyroid hormone (PTH) 1-84 (sc, osteoporosis), epoetin δ, transgenic antithrombin III, allicin, Vitrase, recombinant insulin, interferon-α (oral tablets), GEM-21S, valproate, iduxitase, omatracheline, recombinant serum albumin, serzumab, glucarpidase, recombinant human CI esterase inhibitor (angioedema), lanteplase, recombinant human growth hormone, enfuviride (needle-free injection, Biojector) 2000), VGV-1, Interferon (α), Lucinatan, Atetidil (inhalation, lung disease), Itiband, Icaratide, Omega-7, Aurograb, Perzeganam acetate, ADI-PEG-20, LDI-200, Degarelix, Cintredelinbesudotox, Favld, MDX-1379, ISATX-247, Liraglutide, Teriparatide (osteoporosis), Tifacogin, AA4500, T4N5 liposome lotion, Caputoxumab, DWP413, ART-123, Chrysalin, Desmoplase, Amideplase, Corifollitropin Alpha, TH-9507, Tiduglutide, Diamyd, DWP-412, Growth Hormone (Continuous Release Injection), Recombinant G-CSF, Insulin (Inhalation, AIR), Insulin (Inhalation, Techno Ball), Insulin (Inhalation, AERx), RGN-303, DiaPep277, Interferon β (Hepatitis C Virus Infection (HCV)), Interferon α-n3 (Oral), Beracip, Transdermal Insulin Patch, AMG-531, MBP-8298, Xerecept, Opebacan, A IDSVAX, GV-1001, LymphoScan, ranpirnase, Lipoxysan, Rusuptide, MP52 (β-tricalcium phosphate carrier, bone regeneration), melanoma vaccine, sipuleucel-T, CTP-37, Insegia, Vitespen, human thrombin (for cryotherapy and surgical bleeding), thrombin, TransMID, alfimeprase, Puricase, terlipressin (for intravenous administration and hepatorenal syndrome), EUR-1008MRecombinant FGF-I (injection, vascular disease), BDM-E, rotigaptide, ETC-216, P11, MBI-594AN, nifedipine (inhalation, cystic fibrosis), SCV-07, OPI-45, endostatin, angiostatin, ABT-510, Bowman Birk inhibitor concentrate, XMP-629, 99 mTc-Hynic-annexin V, kahalalide F, CTCE-9908, Teverek (extended release), ozarelix, rornidepsin, BAY-504798, interleukin-4, PRX-321, Pepscan, iboctadekin, rh lactoferrin, TRU-015, IL-21, ATN-161, silengitide, Albuferon, Biphasix, IRX-2, ω-interferon, PCK-3145, CAP-232, pasireotide, huN901-DMI, ovarian cancer immunotherapy vaccine, SB-249553, Oncovax-CL, OncoVax-P, BLP-25, CerVax-16, multi-epitope peptide melanoma vaccine (MART-1, gp100, tyrosinase) nemifitide, rAAT (inhalation), rAAT (dermatology), CGRP (inhalation, asthma), pegsunercept, thymosin β4, pril-tide, GTP-200, ramoranine, GRASPA, OBI-1, AC-100, salmon calcitonin (oral, eligen), calcitonin (oral, osteoporosis), esarilin, caprelin, Cardeva, velafermin, 131I-TM-601, KK-220, T-10, uralipide, delestatin, hematide, Chrysalin (topical), rNAPc2, recombinant factor VIII (PEGylated liposomes), bFGF, PEGylated recombinant staphylococcal kinase variant, V-10153, SonoLysis Prolyse, NeuroVax, CZEN-002, Islet Cell Regeneration Therapy, rGLP-1, BIM-51077, LY-548806, Exenatide (Controlled Release, Medisorb), AVE-0010, GA-GCB, avorelin, ACM-9604, linaclotid eacetate, CETi-1, Hemospan, VAL (Injectable), Rapid-Acting Insulin (Injectable, Viadel), Intranasal Insulin, Insulin (Inhalation), Insulin (Oral, Eligen), Recombinant Methionine Leptin, Pitrakinra (Subcutaneous Injection, Eczema), Pitrakinra (Inhaled Dry Powder, Asthma), Multikine, RG-1068MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10 (autoimmune diseases / inflammation), talactoferrin (topical), rEV-131 (ophthalmology), rEV-131 (respiratory diseases), oral recombinant human insulin (diabetes), RPI-78M, Oprein interleukin (oral), CYT-99007 CTLA4-Ig, DTY-001, valategrast, interferon α-n3 (topical), IRX-3, RDP-58, Tauferon, bile salt-stimulated lipase, Meripase, alaline phosphatase, EP-2104R, Melanotan-II, bremelanotide, ATL-104, recombinant human microcellulose, AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, dynorphin A, SI-6603, LAB GHRH, AER-002, BGC-728, Malaria Vaccine (Vitro, PeviPRO), ALTU-135, Parvovirus B19 Vaccine, Influenza Vaccine (Recombinant Neuraminidase), Malaria / HBV Vaccine, Anthrax Vaccine, Vacc-5q, Vacc-4x, HIV Vaccine (Oral), HPV Vaccine, Tat Toxoid, YSPSL, CHS-13340, PTH (l-34) Liposome Cream (Novasome), Ostab Olin-C, PTH analogue (topical, psoriasis), MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85A vaccine (tuberculosis), FARA04, BA-210, recombinant plague FIV vaccine, AG-702, OxSODrol, rBetV1, Der-pl / Der-p2 / Der-p7 allergen-targeted vaccine (house dust mite allergy), PR1 peptide antigen (leukemia), mutant ras vaccine, HPV-16 E7 lipopeptide vaccine, labyrinthine toxin vaccine (adenocarcinoma), CML vaccine, WT1-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, icrocaptide, telbermin (dermatology, diabetic foot ulcer), rupine, reticulose, rGRF, HA, α-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensin-converting therapeutic vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral, tuberculosis), DRF-7295, ABT-828, ErbB2 specific immunotoxin (anti-cancer), DT3SSIL-3, TST-10088, PRO-1762, CombotoxCholecystokinin-β / gastrin receptor-binding peptide, 111In-hEGF, AE-37, trasnizumab-DM1, antagonist G, IL-12 (recombinant), PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647 (topical), L-19-based radioimmunotherapy agent (cancer), Re-188-P-2045, AMG-386, D C / 1540 / KLH vaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1 vaccine (peptide), NA17.A2 peptide, melanoma vaccine (pulse antigen therapy), prostate cancer vaccine, CBP-501, recombinant human lactoferrin (dry eye), FX-06, AP-214, WAP-8294A (injectable), ACP-HIP, SUN-11031, peptide YY [3-36] (Obesity, Intranasal), FGLL, atacicept, BR3-Fc, BN-003, BA-058, Human Parathyroid Hormone 1-34 (Nose, Osteoporosis), F-18-CCR1, AT-1100 (Celiac Disease / Diabetes), JPD-003, PTH (7-34) Liposome Cream (Novasome), Nejumycin (Ophthalmology, Dry Eye), CAB-2, CTCE-0214, Glycosylated PEGylated Erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, Factor XIII, Aminodoxine, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, Teverek (Immediate Release), EP-51216, hGH (Controlled Release, Biosphere), O GP-1, Sifuviride, TV4710, ALG-889, Org-41259, rhCCIO, F-991, Thymopentin (lung disease), r(m)CRP, liver-selective insulin, Suyalin, L19-IL-2 fusion protein, elafin, NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist (thrombocytopenia), AL-108, AL-208, nerve growth factor antagonist (pain), SLV-317, CGX-1007, INNO-105, oral teriparatide (eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion vaccine (Therapore), EP-1043, pneumococcal pediatric vaccine, malaria vaccine, Neisseria meningitidis (… Neisseria meningitidisGroup B vaccines, neonatal group B streptococcal vaccine, anthrax vaccine, HCV vaccine (gpE1 + gpE2 + MF-59), treatment for otitis media, HCV vaccine (core antigen + ISCOMATRIX), hPTH (1-34) (transdermal, ViaDerm), 768974, SYN-101, PGN-0052, aviscumnine, BIM-23190, tuberculosis vaccine, multi-epitope tyrosinase peptide, cancer vaccine, enkastim, APC-8024, GI-5005, ACC-001, TTS-CD3, vascular-targeted TNF (solid tumors), desmopressin (oral controlled release), onercept and TP-9201.

[0177] In some embodiments, the polypeptide is adalimumab (HUMIRA) or infliximab (REMICADE). TM rituximab TM / MAB THERA TM Enbrel TM ), bevacizumab (AVASTIN) TM trastuzumab (HERCEPTIN) TM ), pegrilgrastim (NEULASTA TM ) or any other suitable polypeptide, including biosimilars and biomimetic drugs.

[0178] Other suitable polypeptides are those listed below and in Table 1 of US2016 / 0097074:

[0179] Table 1

[0180]

[0181]

[0182]

[0183] In the embodiments, the polypeptide is a hormone, coagulation / coagulation factor, cytokine / growth factor, antibody molecule, fusion protein, protein vaccine or peptide, as shown in Table 2.

[0184] Table 2: Exemplary Products

[0185]

[0186]

[0187] In this implementation, the protein is a multispecific protein, such as the bispecific antibodies shown in Table 3.

[0188] Table 3: Bispecific Forms

[0189]

[0190]

[0191]

[0192] Each patent, patent application, and publication disclosure cited herein is incorporated herein by reference in its entirety. While the invention has been disclosed with reference to specific aspects, it will be apparent to those skilled in the art that other aspects and variations of the invention can be devised without departing from the true spirit and scope thereof. The appended claims are intended to be construed as encompassing all such aspects and equivalent variations.

[0193] [Some embodiments of the present invention]

[0194] 1. A method for filtering a process fluid containing products, the method comprising:

[0195] (a) Allow some of the processing fluid in the first reservoir to flow out of the first reservoir and through the virus filter to produce an eluent;

[0196] (b) Adding a chasing fluid to the processing fluid in the first reservoir to form a composite fluid; and

[0197] (c) Allow the composite fluid in the first reservoir to flow out of the first reservoir and through a virus filter to produce an eluent.

[0198] The fluid flowing through the virus filter is sufficient to avoid significant damage to virus removal until a preselected event occurs, and

[0199] The flow of fluid from the first reservoir through the virus filter is maintained without interruption or slowing of the flow for a duration or magnitude sufficient to impair virus removal to a level below the logarithmic reduction value of the virus filter.

[0200] 2. The method of embodiment 1, wherein the product comprises an active pharmaceutical ingredient.

[0201] 3. The method of any one of embodiments 1 to 2, wherein some of the processing fluid in the first reservoir is discharged from the first reservoir and the amount of processing fluid remaining in the first reservoir is reduced by passing through the virus filter.

[0202] 4. The method of any one of embodiments 1 to 3, wherein the addition of the chasing fluid is performed before the processing fluid of the first reservoir is drained.

[0203] 5. The method of any one of embodiments 1 to 4, wherein the addition of the chasing fluid is performed when the volume of the processing fluid remaining in the first reservoir is within or at a reference volume range.

[0204] 6. The method of embodiment 5, wherein the reference volume is equal to or greater than 0.5, 1, 1.5, 2, 3, 5 or 10 times the volume of the component disposed between the first reservoir and the end reservoir.

[0205] 7. The method of embodiment 5, wherein the reference volume is equal to or greater than the volume of the component disposed between the first reservoir and the end reservoir.

[0206] 8. The method of any one of embodiments 1 to 7, wherein the volume of the added chasing fluid is less than or equal to the reference volume of the chasing fluid.

[0207] 9. The method of embodiment 8, wherein the reference volume of the chasing fluid is equal to or greater than 0.5, 1, 1.5, 2, 3, 5 or 10 times the volume of the component disposed between the first reservoir and the endpoint reservoir.

[0208] 10. The method of embodiment 8, wherein the chasing fluid reference volume is equal to or greater than the volume of the component disposed between the first reservoir and the endpoint reservoir.

[0209] 11. The method of any one of embodiments 1 to 10, wherein the chasing fluid is added to the first reservoir, and the first reservoir still contains a predetermined amount of processing fluid.

[0210] 12. The method of any one of embodiments 1 to 11 further includes determining a value as a function of the amount of processing fluid remaining in the first reservoir.

[0211] 13. The method of embodiment 12 further includes determining whether the value satisfies a predetermined reference value.

[0212] 14. The method of embodiment 12 further includes: adding chasing fluid to the first reservoir if the value has a predetermined relationship with the reference value.

[0213] 15. The method of any one of embodiments 1 to 14, wherein when the chasing fluid is added, the ratio of the amount of chasing fluid to the amount of processing fluid remaining in the first reservoir is equal to or greater than 1:0.5, 1:1, 1.5:1, 2:1, 3:1, 5:1 or 10:1.

[0214] 16. The method of any one of embodiments 1 to 15, wherein the flow of fluid from the first reservoir through the virus filter is maintained at a preselected rate.

[0215] 17. The method of any one of embodiments 1 to 16, wherein a preselected pressure difference across the virus filter is maintained.

[0216] 18. The method of any one of embodiments 1 to 17, wherein the pressure difference across the virus filter is maintained at or below a preselected maximum value.

[0217] 19. The method of any one of embodiments 1 to 18, wherein a preselected pressure difference is maintained at or above 14 psi, 11 psi or 13.2 psi across the virus filter.

[0218] 20. The method of any one of embodiments 1 to 19, wherein the pressure difference across the virus filter is maintained at or above a preselected minimum value.

[0219] 21. The method of any one of embodiments 1 to 20, wherein the pressure difference across the virus filter is sufficient to reduce virus particles by at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times.

[0220] 22. The method of any one of embodiments 1 to 21, wherein the duration of any single stop in the fluid flow from the first reservoir is less than 1, 10, or 60 minutes.

[0221] 23. The method of any one of embodiments 1 to 22, wherein continuous flow exists during one, two, or all of steps a, b, and c.

[0222] 24. The method of any one of embodiments 1 to 23, wherein the preselected event includes eliminating the fluid connection between the virus filter and the endpoint reservoir.

[0223] 25. The method of any one of embodiments 1 to 23, wherein the preselected event includes sending the eluent to a subsequent operation.

[0224] 26. The method of any one of embodiments 1 to 23, wherein the preselected event includes stopping the flow from the virus filter to the accumulated eluent.

[0225] 27. The method of any one of embodiments 1 to 23, wherein the preselected event includes stopping the flow from the virus filter to the endpoint reservoir.

[0226] 28. The method of any one of embodiments 1 to 23, wherein the preselected event includes collecting eluent generated from the fluid flow from the first reservoir.

[0227] 29. The method of any one of embodiments 1 to 23, wherein the preselected event includes the arrival of the end of the preselected time period.

[0228] 30. The method of any one of embodiments 1 to 29 further includes providing a system, the system comprising:

[0229] First storage container,

[0230] Virus filters, and

[0231] End-point storage,

[0232] The first reservoir is fluidly connected to the virus filter, and the virus filter is fluidly connected to the endpoint reservoir.

[0233] 31. The method of embodiment 30, wherein the virus filter is disposed between the first reservoir and the endpoint reservoir.

[0234] 32. The method of any one of embodiments 30 to 31, wherein the virus filter is disposed between the first reservoir and the conduit.

[0235] 33. The method of any one of embodiments 30 to 32, wherein the system includes a pre-filter conduit configured to deliver fluid from the first reservoir to the virus filter.

[0236] 34. The method of any one of embodiments 30 to 33, wherein the system includes a conduit configured to deliver fluid to the endpoint reservoir or a conduit to the endpoint.

[0237] 35. The method of any one of embodiments 30 to 32, wherein the system comprises:

[0238] A first conduit configured to deliver fluid from the first reservoir to the virus filter; and

[0239] A second conduit is configured to deliver fluid from the virus filter to the terminal reservoir.

[0240] 36. The method of any one of embodiments 30 to 35, wherein the system includes a first valve configured to control fluid flow from the first reservoir to the virus filter.

[0241] 37. The method of any one of embodiments 30 to 36, wherein the system includes a second valve configured to control fluid flow from the virus filter to the endpoint reservoir.

[0242] 38. The method of any one of embodiments 30 to 37, wherein the system includes a computer or microprocessor for controlling one or more valves.

[0243] 39. The method of any one of embodiments 30 to 38, wherein the virus filter is integral with the wall of the first or second reservoir.

[0244] 40. The method of embodiment 1, wherein the flow of fluid from the first reservoir through the virus filter is maintained without the flow stopping.

[0245] 41. A method for filtering a process fluid containing products, comprising:

[0246] (a) Allow the processing fluid in the first reservoir to flow out of the first reservoir and through the virus filter to generate eluent and reduce the amount of processing fluid in the first reservoir;

[0247] (b) Determine a value that is a function of the amount of processing fluid remaining in the first reservoir, and if the value satisfies a predetermined reference value, add chasing fluid to the processing fluid in the first reservoir to form a composite fluid, wherein chasing fluid is added to the first reservoir while the first reservoir still contains a predetermined amount of processing fluid;

[0248] (c) Allow the composite fluid in the first reservoir to flow out of the first reservoir and through a virus filter to produce an eluent; and

[0249] (d) Stop the flow from the virus filter.

[0250] The flow of fluid from the first reservoir through the virus filter is sufficient to avoid significant damage to virus removal until the flow from the virus filter stops.

Claims

1. A method for reducing the viral titer of a processing fluid containing products, the method comprising: (a) Allowing some of the processing fluid in the first reservoir to drain out of the first reservoir to reduce the amount of processing fluid remaining in the first reservoir and passing through a size-restricted virus filter to produce a first eluent; (b) Add chasing fluid to the processing fluid in the first reservoir to form a composite fluid. The chasing fluid is added to the first reservoir, which still contains a predetermined amount of processing fluid. When the chasing fluid is added, the ratio of the amount of chasing fluid to the amount of processing fluid remaining in the first reservoir is equal to or greater than 1:1; and (c) Allow the composite fluid in the first reservoir to flow out of the first reservoir and pass through a size-restricted virus filter to produce a second eluent. The composite fluid does not include the first or second eluent. The process fluid and / or composite fluid are maintained flowing through the size-block virus filter to prevent air from entering the size-block virus filter until a preselected event occurs. Wherein, without interruption or slowing of the flow for a duration or magnitude sufficient to impair virus removal to a level below the logarithmic reduction value of the size-restricted virus filter, the flow of fluid from the first reservoir through the size-restricted virus filter is maintained, and The flow of fluid from the first reservoir through the size-blocking virus filter is maintained by one or more valves set between the first reservoir and the size-blocking virus filter, which are manually or automatically controlled by the processor.

2. The method of claim 1, wherein the product comprises an active pharmaceutical ingredient.

3. The method of any one of claims 1 to 2, wherein the addition of the chasing fluid is performed before the processing fluid in the first reservoir is drained.

4. The method according to any one of claims 1 to 3, wherein, When the volume of the processing fluid remaining in the first reservoir is at or within the reference volume range, the chasing fluid is added.

5. The method of claim 4, wherein the reference volume is equal to or greater than 0.5, 1, 1.5, 2, 3, 5, or 10 times the volume of the component disposed between the first reservoir and the end reservoir.

6. The method of claim 4, wherein the reference volume is equal to or greater than the volume of the component disposed between the first reservoir and the end reservoir.

7. The method of any one of claims 1 to 6, wherein the volume of the added chasing fluid is less than or equal to the reference volume of the chasing fluid.

8. The method of claim 7, wherein the chasing fluid reference volume is equal to or greater than 0.5, 1, 1.5, 2, 3, 5, or 10 times the volume of the component disposed between the first reservoir and the endpoint reservoir.

9. The method of claim 7, wherein the chasing fluid reference volume is equal to or greater than the volume of the component disposed between the first reservoir and the endpoint reservoir.

10. The method of any one of claims 1 to 9, further comprising determining a value as a function of the amount of processing fluid remaining in the first reservoir.

11. The method of claim 10, further comprising determining whether the value satisfies a predetermined reference value.

12. The method of claim 10, further comprising: If the value has a predetermined relationship with the reference value, then chasing fluid is added to the first reservoir.

13. The method of any one of claims 1 to 12, wherein when the chasing fluid is added, the ratio of the amount of chasing fluid to the amount of processing fluid remaining in the first reservoir is equal to or greater than 1.5:1, 2:1, 3:1, 5:1 or 10:

1.

14. The method of any one of claims 1 to 13, wherein the flow of fluid from the first reservoir through the size-blocking virus filter is maintained at a preselected rate.

15. The method of any one of claims 1 to 14, wherein a preselected pressure differential is maintained across the size exclusion virus filter.

16. The method of any one of claims 1 to 15, wherein the pressure differential across the size-blocking virus filter is maintained at or below a preselected maximum value.

17. The method of any one of claims 1 to 16, wherein a preselected pressure differential is maintained equal to or not greater than the pressure differential of a cross-size exclusion virus filter of 14 psi, 11 psi, or 13.2 psi.

18. The method of any one of claims 1 to 17, wherein the pressure differential across the size-blocking virus filter is maintained at or above a preselected minimum.

19. The method of any one of claims 1 to 18, wherein the pressure differential across the size-blocking virus filter is sufficient to reduce virus particles to at least 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90 or 1 / 100.

20. The method of any one of claims 1 to 19, wherein any single stop in the flow of fluid from the first reservoir lasts for less than 1, 10, or 60 minutes.

21. The method of any one of claims 1 to 20, wherein continuous flow exists during one, two, or all of steps a, b, and c.

22. The method of any one of claims 1 to 21, wherein the preselected event comprises eliminating the fluid connection between the size-blocking virus filter and the endpoint reservoir.

23. The method of any one of claims 1 to 21, wherein the preselected event includes sending the first or second eluent to a subsequent operation.

24. The method of any one of claims 1 to 21, wherein the preselected event includes stopping the flow from the size exclusion virus filter to the accumulated first or second eluent.

25. The method of any one of claims 1 to 21, wherein the preselected event includes stopping the flow from the size-blocking virus filter to the endpoint reservoir.

26. The method of any one of claims 1 to 21, wherein the preselected event includes collecting a first or second eluent generated from a fluid flow from the first reservoir.

27. The method of any one of claims 1 to 21, wherein the preselected event includes the arrival of the end of the preselected time period.

28. The method of any one of claims 1 to 27, further comprising providing a system, the system comprising: First storage container, Size-controlled virus filter, and End-point storage, The first reservoir is fluidly connected to the size exclusion virus filter, and the size exclusion virus filter is fluidly connected to the endpoint reservoir.

29. The method of claim 28, wherein the size-blocking virus filter is disposed between the first reservoir and the endpoint reservoir.

30. The method of any one of claims 28 to 29, wherein the size-blocking virus filter is disposed between the first reservoir and the conduit.

31. The method of any one of claims 28 to 30, wherein the system includes a pre-filter conduit configured to deliver fluid from the first reservoir to the size-blocking virus filter.

32. The method of any one of claims 28 to 31, wherein the system comprises a conduit configured to deliver fluid to the endpoint reservoir or a conduit to the endpoint.

33. The method of any one of claims 28 to 32, wherein the system comprises: A first conduit is configured to deliver fluid from the first reservoir to the size-restricted virus filter; as well as A second conduit is configured to deliver fluid from the size-blocking virus filter to the terminal reservoir.

34. The method of any one of claims 28 to 33, wherein the system further comprises a second valve configured to control fluid flow from the size-blocking virus filter to the endpoint reservoir.

35. The method of any one of claims 28 to 34, wherein the system comprises a computer or microprocessor for controlling one or more valves.

36. The method of any one of claims 28 to 35, wherein the size-blocking virus filter is integral with the wall of the first or second reservoir.

37. The method of claim 1, wherein the flow of fluid from the first reservoir through the size-blocking virus filter is maintained without the flow stopping.

38. A method for reducing the viral titer in a processing fluid containing products, comprising: (a) Allow the processing fluid in the first reservoir to flow out of the first reservoir and through a size-restricted virus filter to produce a first eluent and reduce the amount of processing fluid in the first reservoir; (b) Determine a value as a function of the amount of processed fluid remaining in the first reservoir, and if this value satisfies a predetermined reference value, add chasing fluid to the processed fluid in the first reservoir to form a composite fluid. The chasing fluid is added to the first reservoir, which still contains a predetermined amount of processing fluid, and When the chasing fluid is added, the ratio of the amount of chasing fluid to the amount of processing fluid remaining in the first reservoir is equal to or greater than 1:

1. (c) Allow the composite fluid in the first reservoir to drain from the first reservoir and pass through a size-restricted virus filter to produce a second eluent; and (d) Stop the flow from the size-blocking virus filter. The composite fluid does not include the first or the second eluent, and The flow of fluid from the first reservoir through the size-blocking virus filter is maintained such that air does not enter the size-blocking virus filter until the flow from the size-blocking virus filter is stopped. The flow of fluid from the first reservoir through the size-blocking virus filter is maintained by one or more valves set between the first reservoir and the size-blocking virus filter, which are manually or automatically controlled by the processor.

Images