METHOD OF PROCESSING A MAMMALIAN CELL CULTURE

专利摘要:
methods and systems for processing a cell culture. The present invention relates to methods of processing a cell culture and to open loop filtration systems. the filtration systems comprise a tangential flow (tff) filtration unit that has an independent inlet and outlet ports connected to the cell culture bioreactor and a pump adapted to reverse the flow of fluid through the filtration unit.
公开号:BR112016005545B1
申请号:R112016005545-4
申请日:2014-09-16
公开日:2021-06-01
发明作者:Hang Zhou；Benjamin Wright；Marcella Yu；Jin Yin；Konstantin Konstantinov
申请人:Genzyme Corporation；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED PATENT APPLICATIONS
[0001] The present patent application claims priority to U.S. Provisional Patent Application no. 611878,502, filed on September 16, 2013, the full content of which is incorporated by reference in this document. TECHNICAL FIELD
[0002] The present invention relates to methods for processing a cell culture and biotechnology, and more specifically to methods for continuously processing a cell culture in a perfusion bioreactor. BACKGROUND
[0003] Mammalian cells are often used to produce therapeutic proteins. In some processing methods, mammalian cells are cultured in a perfusion bioreactor, a volume of the cell culture containing the recombinant protein is removed from the bioreactor, and fresh culture medium is added to replace the volume. In such perfusion culture methods, the removed cell culture is often filtered to retain the mammalian cells in the bioreactor for additional recombinant protein production, whereas the culture medium (sometimes referred to as "spent medium") which contains a recombinant protein is recovered.
[0004] Conventional methods and devices for filtering cell culture from a perfusion bioreactor have several disadvantages. For example, the closed system alternating tangential flow filtration (TAF) results in cell cultures that spend a long period of time outside the controlled growth conditions in the bioreactor (long external residence time), and traditional unidirectional filtration in the bioreactor. Tangential flow (TFF), with no reverse flow, causes the filter to become dirty. Thus, conventional perfusion bioreactor methods often involve culturing cells by spending a long period of time outside of controlled growth conditions in the bioreactor, leading to decreases in viable cell density, percentage of viability, and specific productivity of culture and volumetric. In addition, the preceding methods often result in an incomplete discharge of the system filters which results in filter fouling. SUMMARY
[0005] Applicants have found that an open loop filtration system that provides reversible tangential fluid flow across a surface of a crossflow filter, as opposed to conventional unidirectional open loop or bidirectional closed loop filtration systems, provides a higher viable cell density, a higher percentage of viable cell, a higher specific and/or volumetric productivity, a higher specific consumption of glucose, and less dirt on the filter.
[0006] The open circuit filtration systems provided in this document provide ideal conditions for the production and yield of recombinant proteins, such as one or more of the reduced external volume of the cell culture (outside the reservoir), the fraction of increased exchange (eg within the first conduit, TFF unit and second conduit), decreased cell culture external residence time (outside the reservoir), decreased shear stress during cell culture filtration, higher cell viability in cell culture, high viable cell density in cell culture, and/or less filter dirt (due to better discharge of the filter(s)), for example, in comparison to other unidirectional open-loop filtration systems (eg, unidirectional TFF systems) or bidirectional closed-loop filtration systems (closed-loop ATF™ systems). Therefore, in the present document, open circuit filtration systems are provided which include a reservoir (e.g. a bioreactor), a tangential flow filtration unit (TFF) having first and second inlets, a first conduit in fluid communication between the reservoir and the second inlet of the TFF unit, and a second conduit in fluid communication between the reservoir and the second inlet of the TFF unit, and at least one pump disposed within the system such that the drive of at least one pump propels fluid reversibly through the system from the reservoir, through the first conduit, the TFF unit, the second conduit, and back to the reservoir. Methods for processing a cell culture are also provided which include (a) the provision of an open loop filtration system (e.g. any of the open loop filtration systems described herein), (b) o thrusting the cell culture from the reservoir through the TFF unit in a first flow direction for a first period of hour, (c) reversing the first flow direction and thrusting the cell culture through the TFF unit in a second flow direction for a second period of time, (d) reversing the second flow direction and pushing the crop through the TFF unit in the first flow direction for a third period of time, (e) repeating the steps ( c) - (d) at least twice, and (f) collecting the filtered material.
[0007] Methods for processing a cell culture are provided herein. These methods include the steps of: (a) providing an open circuit filtration system that includes a reservoir comprising a cell culture, a tangential flow (TFF) filtration unit having first and second inlets, a first conduit in fluid communication between the reservoir and the first inlet of the TFF unit, and a second conduit in fluid communication between the reservoir and the second inlet of the TFF unit, and at least one pump disposed within the system to propel fluid through the system. , wherein the system is configured such that fluid can be reversibly propelled through the system to or from the reservoir and through the first and second conduits and the TFF unit through at least one pump, and the material filtered. can be collected from the TFF unit; (b) pushing the cell culture from the reservoir through the TFF unit in a first flow direction for a first hour period, (c) reversing the first flow direction and pushing the cell culture through the TFF unit in a second flow direction for a second period of time; (d) reversing the second flow direction and pushing the crop through the TFF unit in the first flow direction for a third period of time; (e) repeating steps (c) - (d) at least twice; and (f) collecting the filtered material. In some examples, the reservoir is a bioreactor or a refrigerated containment tank. In some examples, one or both of the first and second conduits include biocompatible tubing. The TFF unit can include a single cross-flow filter (for example, a tubular cross-flow filter) or it can include two or more cross-flow filters.
[0008] In some examples, the system includes one or more additional TFF units disposed in the first conduit, the second conduit, or both. In some examples, the cross-flow filter(s) has an average pore size of about 0.2 micrometer.
[0009] In some examples, at least one pump is disposed in the first conduit or in the second conduit, or in both. In further examples, at least one pump is arranged in the system between any two TFF units. In some embodiments, at least one pump is disposed in the reservoir and proximate to the first or second fluid conduit. In some embodiments of all the methods described herein, at least one pump is a low turbulence (LTP) pump (e.g., a peristaltic pump). In some examples, the system includes a first and a second LTP, where the first LTP flows the cell culture in the first direction and the second LTP flows the cell culture in the second direction. In some embodiments, the system includes a single LTP, where the single LTP flows the cell culture in the first direction during the first and third time periods and the cell culture flows in the second direction during the second time period.
[00010] In some of the methods described in this document, the first, second and third time periods are about 30 seconds to about 15 minutes. In some embodiments, the cell culture is driven at one or more of (a), (b) and (c) at a rate of between about 0.5 l/minute (e.g., between about 3.0 l/minute). minute and about 80 l/minute and about 60 l/minute).
[00011] In some modalities, the single repetition of (b) and (c) results in an exchange fraction of more than 50%. In some examples, the filtered material does not contain a mammalian cell. In some embodiments, the cell culture contains a secreted recombinant protein and the filtered material contains the secreted recombinant protein. In some embodiments, the secreted recombinant protein is an antibody or antigen binding fragment thereof, a growth factor, a cytokine, or an enzyme, or a combination thereof. Some additional modalities include a step of isolating the secreted recombinant protein from the filtered material. For example, isolation can be performed using an integrated, continuous process that includes isolation through at least one multi-column chromatography system (MCCS). Some embodiments also include a step of formulating a therapeutic drug substance by mixing the isolated recombinant protein with a pharmaceutically acceptable excipient or buffer. In some embodiments, cell culture or filtered material, or both, are sterile. In some examples, the method runs continuously for a period of between about 14 days and about 80 days.
[00012] Open circuit filtration systems are also provided that include a reservoir, a tangential flow filtration unit (TFF) having first and second inlets, a first conduit in fluid communication between the reservoir and the first inlet of the unit. TFF, and a second conduit in fluid communication between the reservoir and the second inlet of the TFF unit, and at least one pump disposed within the system, wherein actuation of at least one pump reversibly propels fluid through the system to from the reservoir, through the first conduit, the TFF unit, the second conduit, and back to the reservoir. In some examples, the reservoir is a bioreactor or a refrigerated containment tank. In some embodiments, one or both of the first and second conduits comprise(s) a biocompatible tubing. In some embodiments, the TFF unit includes a single cross-flow filter (eg, a tubular cross-flow filter). In some embodiments, the TFF unit includes two or more cross-flow filters. In some examples, the system includes one or more additional TFF units disposed in the first conduit, the second conduit, or both. In some systems, the cross-flow filter(s) have an average pore size of about 0.2 micrometer.
[00013] In some embodiments of the systems described in this document, at least one pump is arranged in the first conduit or in the second conduit, or in both. In other embodiments, at least one pump is arranged in the system between any two TFF units. In other examples, at least one pump is disposed in the reservoir and close to the first or second fluid conduit. In some of the systems described in this document, at least one pump is a low turbulence (LTP) pump (e.g., a peristaltic pump). In some embodiments, the system includes first and second LTPs, wherein the first LTP is adapted to propel cell culture in a first flow direction and the second LTP is adapted to reverse the first flow direction and propel the culture of cells in a second flow direction. In other embodiments, the system includes a single LTP adapted to reversibly propel cell culture in a first and second flow direction. In some embodiments, the peristaltic pump has a pump column volume between about 20 ml and about 250 ml.
[00014] Some embodiments of the systems described in this document also include a filtered material containment tank and a filtered material conduit in fluid communication between the TFF unit and the filtered material containment tank. Some embodiments of the systems described herein also include a biological manufacturing system comprising at least a multiple column chromatography (MCCS) system and an inlet and an outlet and a filtered material conduit in fluid communication between the TFF unit and the input to the biological manufacturing system, where the device is configured in such a way that filtered material is passed to the input to the biological manufacturing system, through at least one MCCS, and leaves the device through the output of the manufacturing system biological. In any of the systems described in this document, the TFF unit is enclosed in an enclosure.
[00015] As used herein, the word "a" or "plurality" before a noun represents one or more of the particular noun. For example, the phrase "a mammalian cell" represents "one or more mammalian cells," and the phrase "plurality of microcarriers" means "one or more microcarriers".
[00016] The term "mammalian cell" refers to any cell of or derived from any mammal (for example, a human, a hamster, a mouse, a green monkey, a rat, a pig, a cow, or A rabbit).
[00017] In some embodiments, the mammalian cell can be, for example, an immortalized cell, a differentiated cell, or an undifferentiated cell.
[00018] The term "cell culture" refers to a plurality of mammalian cells (eg any of the mammalian cells described herein) suspended in a liquid culture medium (eg any of the media culture fluids described in this document). The cell culture can have a cell density of more than about 0.1 x 106 cells/ml (e.g., more than about 1 x 106 cells/ml, more than about 5 x 106 cells/ml. ml, more than about 10 x 106 cells/ml, more than about 15 x 106 cells/ml, more than about 20 x 106 cells/ml, more than about 25 x 106 cells/ml, more than about 30 x 106 cells/ml, more than about 35 x 106 cells/ml, more than about 40 x 106 cells/ml, more than about 45 x 106 cells/ml, more than than about 50 x 106 cells/ml, more than about 55 x 106 cells/ml, more than about 60 x106 cells/ml, more than about 65 x 106 cells/ml, more than about 70 x 106 cells/ml, more than about 75 x 106 cells/ml, more than about 80 x 106 cells/ml, more than about 85 x 106 cells/ml, more than about 90 x 106 cells/ml, more than about 95 x 106 cells/ml, or more than 100 x 106 cells/ml). In some examples, mammalian cells present in a cell culture are attached to microcarriers (for example, any of the microcarriers described herein or known in the art).
[00019] The term "bioreactor" is known in the prior art and refers to a vessel that can incubate a cell culture under a controlled set of physical conditions that allow the maintenance or growth of a mammalian cell in a culture medium liquid. For example, the bioreactor can incubate a cell culture under conditions that allow a mammalian cell in the cell culture to produce and secrete a recombinant protein. For example, a bioreactor typically includes an O2 and N2 sparge, a thermal sleeve, one or more fluid ports, and an agitation system. Non-limiting examples of bioreactors are described herein. Additional examples of bioreactors are known in the state of the art.
[00020] The term "open loop filtration system" refers to a reservoir (eg a bioreactor) and a continuous closed fluid circuit that both start and terminate in a reservoir, and includes a TFF unit through the which a fluid (eg a cell culture) in the closed fluid circuit can pass to and from the reservoir (in a first or second flow direction) through the TFF unit and back to the reservoir. The open loop filtration system also includes at least one pump suitable for pumping fluid (e.g. cell culture) to and/or from the reservoir through the TFF unit and back to the reservoir.
[00021] The terms "tangential flow filtration unit" or "TFF unit" are known in the prior art and refer to a device that includes at least one casing (such as a cylinder) and at least one flow filter cross positioned in the housing in such a way that a large portion of the filter surface is positioned parallel to the flow of a fluid (eg a cell culture) through the unit. TFF units are well known in the art and are commercially available. Exemplary commercially available TFF units include MinimateTM TFF capsules (Pall Corporation), Vivaflow® 50 and 200 systems (Sartorius), BioCap 25, E0170, E0340 and EI020 (3M) capsules, and ATF4 (Refine Technology). The housing may include a first inlet/outlet and a second inlet/outlet positioned, for example, to allow fluid to pass through the first inlet/outlet, cross at least one cross-flow filter, and through the second inlet/outlet. In some examples, an open circuit filtration system may include multiple TFF units, for example, connected in series and/or in parallel. For example, a system that includes two or more TFF units may include fluid conduits that fluidly connect neighboring pairs of TFF units in the system. In other examples, a system may include two or more sets of two or more TFF units fluidly connected by fluid conduits. Any of the TFF units described in this document or known in the state of the art can receive the fluid in a first flow direction and in a second flow direction.
[00022] The term "crossflow filter" is from the prior art and refers to a filter that is designed in such a way that it can be positioned in a TFF unit so that a large portion of the filter surface remains parallel to the flow (eg, first and second flow directions) of a fluid (eg, a cell culture). For example, a cross-flow filter can be any shape that allows tangential flow filtration, for example, a tubular or rectangular shape. Particularly useful cross-flow filters are designed to result in a low amount of fluid turbulence or shear stress in the fluid (eg cell culture) when the fluid is propelled in a first and second direction across the filter surface. of cross flow. Cross-flow filters are commercially available, for example, from Sartorius, MembraPure, Millipore and Pall Corporation.
[00023] The term "low turbulence pump" or "LTP" is known in the prior art and refers to a device that can move a fluid (for example, a cell culture) within the system in a single direction (by eg a first or second flow direction) or reversibly propelling a fluid (eg a cell culture) in two directions (a first and a second flow direction) within the system without inducing a substantial amount of stress shear or fluid turbulence in the fluid (eg cell culture). When an LTP is used to propel a fluid (eg, a cell culture) in alternating first and second flow directions, the second flow direction is more or less opposite to that first flow direction. An example of an LTP is a peristaltic pump. Other examples of LTPs are known in the prior art.
[00024] The terms "flow reversal" or "flow direction reversal" are well known to those skilled in the art. For example, those skilled in the art will appreciate that reversal of the flow of a fluid refers to changing the total flow direction of a fluid to a normally opposite total flow direction (eg, the flow direction of a crop. of cells in any of the methods or systems described in this document).
[00025] The term "exchange fraction" refers to the percentage of fluid (eg cell culture) that is returned to the reservoir after the fluid is propelled through the components of an open circuit filtration system out of the reservoir. (eg, through the first conduit, at least one TFF unit and the second conduit) in a first direction for a first period of time and by propelling the fluid in a second direction for a second period of time.
[00026] The term "substantially free" refers to a composition (eg a liquid or solid) that is at least or about 90% free (eg at least or about 95%, 96%, 97 %, 98%, or at least or about 99% free, or about 100% free) of a specific substance (e.g., a mammalian cell or a host mammalian cell protein or nucleic acid). For example, a filtered material generated using the methods described herein can be substantially free of a mammalian cell or a microcarrier. In another example, a recombinant protein isolated using any of the methods described herein may be substantially free of a host mammalian cell protein, nucleic acid, and/or contaminating virus.
[00027] The term "culture" or "cell culture" refers to the maintenance or growth of a mammalian cell in a liquid culture medium under a controlled set of physical conditions.
[00028] The term "liquid culture medium" refers to a fluid that contains sufficient nutrients to allow a mammalian cell to grow in the medium in vitro. For example, a liquid culture medium may contain one or more of: amino acids (eg 20 amino acids), a purine (eg hypoxanthine), a pyrimidine (eg thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, selenium, and other necessary trace metals, and sodium bicarbonate. A liquid culture medium can contain mammalian serum. In some examples, a liquid culture medium does not contain serum or another extract from a mammal (a defined liquid culture medium). A liquid culture medium may contain trace metals, a mammalian growth hormone, and/or a mammalian growth factor. Non-limiting examples of liquid culture medium are described herein and additional examples are known in the art and are commercially available.
[00029] The term "microcarrier" refers to a particle (eg an organic polymer) that has a size between 20 µm and about 1000 µm that contains a surface that is permissive or promotes the binding of a mammalian cell (for example any of the mammalian cells described herein or known in the art). A microcarrier may contain one or more pores (for example, pores with an average diameter of from about 10 µm to about 100 µm). Non-limiting examples of microcarriers are described in this document. Additional examples of microcarriers are known in the state of the art. A microcarrier can contain, for example, a polymer (for example, cellulose, polyethylene glycol, or poly(lactic-co-glycolic) acid).
[00030] The term "liquid culture medium free from animal-derived components" refers to a liquid culture medium that does not contain any components (eg, proteins or serum) derived from an animal.
[00031] The term "serum-free liquid culture medium" refers to a liquid culture medium that does not contain animal serum.
[00032] The term "liquid culture medium containing serum" refers to a liquid culture medium that contains animal serum.
[00033] The term "chemically defined liquid culture medium" refers to a liquid culture medium in which substantially all of the chemical components are known. For example, a chemically defined liquid culture medium does not contain fetal bovine serum, bovine serum albumin or human serum albumin, as such preparations typically contain a complex mixture of albumins and lipids.
[00034] The term "protein-free liquid culture medium" refers to a liquid culture medium that does not contain any protein (eg, any detectable protein).
[00035] The term "immunoglobulin" refers to a polypeptide that contains an amino acid sequence of at least 15 amino acids (for example, at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids) of an immunoglobulin protein (for example, a variable domain sequence, a framework sequence, or a constant domain sequence). The immunoglobulin can, for example, include at least 15 amino acids from a light chain immunoglobulin and/or at least 15 amino acids from a heavy chain immunoglobulin. The immunoglobulin can be an isolated antibody (for example an IgG, IgE, IgD, IgA or IgM). The immunoglobulin can be a subclass of IgG (for example, IgG1, IgG2, IgG3 or IgG4). The immunoglobulin can be an antibody fragment, for example a Fab fragment, an F(ab')2 fragment, or an scFv fragment. The immunoglobulin can also be a bispecific antibody or a trispecific antibody, or a dimer, trimer, or multimer antibody, or a diabody, an Affibody®, or a Nanobody®. The immunoglobulin can also be a engineered protein that contains at least one immunoglobulin domain (for example, a fusion protein). Non-limiting examples of immunoglobulins are described herein and additional examples of immunoglobulins are known in the art.
[00036] The term "protein fragment" or "polypeptide fragment" refers to a portion of a polypeptide sequence that has at least or about 4 amino acids, for example, at least or about 5 amino acids, at least or about 6 amino acids, at least or about 7 amino acids, at least or about 8 amino acids, at least or about 9 amino acids, at least or about 10 amino acids, at least or about 11 amino acids, at least or about of 12 amino acids, at least or about 13 amino acids, at least or about 14 amino acids, at least or about 15 amino acids, at least or about 16 amino acids, at least or about 17 amino acids, at least or about 18 amino acids, at least or about 19 amino acids, or at least or about 20 amino acids in length, or more than 20 amino acids in length. A recombinant protein fragment can be produced using any of the methods described herein.
[00037] The term "designed protein" refers to a polypeptide that is not naturally encoded by an endogenous nucleic acid present within an organism (eg a mammal). Examples of engineered proteins include enzymes (eg, with one or more amino acid substitutions, deletions, insertions, or additions that result in an increase in the stability and/or catalytic activity of the engineered enzyme), fusion proteins, antibodies (eg , divalent antibodies, trivalent antibodies, or a diabody), and antigen binding proteins that contain at least one recombinant scaffold sequence.
[00038] The term "isolate" or "isolation" in certain contexts refers to at least partial purification or purification (eg at least or about 5%, eg at least or about 10%, 15% , 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least or about 95 % pure by weight) of a recombinant protein from one or more other components present in the filtered material (eg a filtered material generated using the methods currently described), eg one or more DNA, RNA and/or other components proteins present in the filtered material. Non-limiting methods for isolating a protein from a filtered material are described herein and others are known in the art.
[00039] The term "secreted protein" or "secreted recombinant protein" refers to a protein or a recombinant protein that originally contains at least one secretion signal sequence when it is translated into a mammalian cell, and which through, at least in part, enzymatic cleavage of the secretion signal sequence in the mammalian cell, is released at least partially into the extracellular space (e.g., a liquid culture medium).
[00040] The phrase "perfusion gradient" is known in the prior art and refers to the incremental change (eg increase or decrease) in the volume of culture medium removed and added to an initial culture volume in incremental periods (by example, a period of about 24 hours, a period between about 1 minute and about 24 hours, or a period of more than 24 hours) during the culture period (e.g., the feedback rate of the culture medium in on a daily basis). The fraction of media removed and replaced each day may vary depending on the particular cells being grown, the density of initial seeding and the density of the cell at a particular time.
[00041] "Specific productivity rate" or "SPR" as used herein refers to the mass or enzymatic activity of a recombinant protein produced per mammalian cell per day. The SPR for a recombinant antibody is usually measured as mass/cell/day. The SPR for a recombinant enzyme is usually measured as units/cell/day or (units/mass)/cell/day.
[00042] "Volometric productivity rate" or "VPR", as used herein, refers to the mass or enzymatic activity of the recombinant protein produced per culture volume (e.g., per liter of bioreactor volume, of the pot or tube) per day. The VPR for a recombinant antibody is usually measured as mass/liter/day. The VPR for a recombinant enzyme is usually measured as units/liter/day or mass/liter/day.
[00043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. Materials, methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, this descriptive report, including definitions, will prevail.
[00044] Other detailed features and advantages of the invention will be apparent from the following description and figures, and from the claims. DESCRIPTION OF DRAWINGS
[00045] Figure 1 is a schematic diagram showing an exemplary open circuit filtration system that can be used to process a cell culture. The system shown includes a single pump 8 disposed in a first conduit 6.
[00046] Figure 2 is a schematic diagram showing an exemplary open circuit filtration system that includes a single pump 8 disposed in a second conduit 7.
[00047] Figure 3 is a schematic diagram showing an exemplary open circuit filtration system that includes a single pump 8 disposed in a reservoir 2 (for example, a bioreactor) and next to a first conduit 6.
[00048] Figure 4 is a schematic diagram showing an exemplary open circuit filtration system that includes two TFF 3 units, each of which includes two cross-flow filters 12, wherein the two TFF 3 units are connected fluidly by a third conduit 14, and a single pump 8 is disposed in the third conduit 14.
[00049] Figure 5 is a schematic diagram showing an exemplary open circuit filtration system that includes a single pump 8 disposed in a second conduit 7, and includes multiple pressure sensors 14, and a flow meter 15.
[00050] Figure 6 is a schematic diagram showing an exemplary open circuit filtration system that includes a pump 8 disposed in the first conduit 6 and a pump 8 disposed in a second conduit 7.
[00051] Figure 7 is a schematic diagram showing an exemplary open circuit filtration system that includes a reservoir 2 and first and second subsystems 19.
[00052] Figure 8 is a schematic diagram showing the first flow direction in an exemplary system.
[00053] Figure 9 is a diagram showing the flow of a cell culture for a first period of time in a first direction of flow, a reversal of the first direction of flow for a period of time (tr1), the flow of culturing cells for a second period of time in a second direction of flow (t2), reversing the second direction of flow for a period of time (tr2), and propelling cell culture for a third period of time in the first flow direction (t3). In the diagram, F represents the cell culture flow rate (l/minute).
[00054] Figure 10 is a graph of viable cell density in a cell culture processed using the methods provided in this document (GC2008 Set6 TFF V24; grey) or using ATFTM (Refine Technology) filtration (GC2008 Set5 ATFTM V21 ; black).
[00055] Figure 11 is a graph of the viable cell percentage in a cell culture processed using the methods provided herein (grey) or using ATFTM (Refine Technologie) filtration (black).
[00056] Figure 12 is a graph of the capacitance (pF) of the cell culture processed when using the methods provided in this document (grey) or when using ATFTM (Refine Technology) filtration (black).
[00057] Figure 13 is a graph of the mean viable cell diameter processed when using the methods provided in this document (gray) or when using ATFTM (Refine Technology) filtration (black).
[00058] Figure 14 is a graph of secreted immunoglobulin (IgG) detected in cell culture processed using the methods provided herein (grey) and using ATFTM (Refine Technology) filtration (black).
[00059] Figure 15 is a graph of the volumetric productivity (g/l/d) of the cell culture processed using the methods provided in this document (grey) and using ATFTM (Refine Technology) filtration (black).
[00060] Figure 16 is a graph of the specific productivity (pg/cell/day) of the cell culture processed using the methods provided in this document (grey) and using ATFTM (Refine Technology) filtration (black).
[00061] Figure 17 is a graph of the sieve percentage coefficient of the cell culture processed when using the methods provided in this document (gray) or when using ATFTM (Refine Technology) filtration (black).
[00062] Figure 18 is a graph of the specific glucose consumption (ng/cell/day) of the cell culture processed when using the methods provided in this document (GC2008 Set6 TFF V24) (grey) or when using ATFTM filtration (Refine Technology) (black).
[00063] Figure 19 is a graph of the specific lactate production (ng/cell/day) of the cell culture processed when using the methods provided herein (grey) or when using ATFTM (Refine Technology) filtration (black).
[00064] Figure 20 is a graph of the specific aerobic glucose consumption (cpmol/cell/hour) of the cell culture processed when using the methods described in this document (grey) or when using ATFTM (Refine Technology) filtration (black) .
[00065] Figure 21 is a graph of the glucose lactate yield (mol/mol) of the cell culture processed when using the methods described herein (grey) or when using ATFTM (Refine Technology) filtration (black). DETAILED DESCRIPTION
[00066] In the present document, open circuit filtration systems are provided that include a reservoir, a TFF unit having first and second inlets, a first conduit in fluid communication between the reservoir and the first inlet of the TFF unit, and a second conduit in fluid communication between the reservoir and the second inlet of the TFF unit, and at least one pump disposed within the system, wherein actuation of at least one pump reversibly propels fluid through the system from the reservoir. , through the fluid conduit, the TFF unit, the second conduit, and back to the reservoir. Methods for processing a cell culture that include the use of an open loop filtration system (for example, any of the open loop filtration systems described herein) are also provided. The systems and methods described in this document provide, for example, a high cell viability and/or percentage of cell viability during cell culture processing. Additional benefits of the systems and methods provided in this document are described below. Open circuit filtration systems
[00067] This descriptive report provides exemplary open circuit filtration systems useful for performing the methods described in this document. These systems are designed such that actuation of at least one pump (in the system) reversibly propels fluid through the system from the reservoir, through the first conduit, the TFF unit, the second conduit, and back to the reservoir. Exemplifying Individual Pump Systems
[00068] A non-limiting example of a system 1 is provided in FIGURE 1. System 1 includes a reservoir 2, for example a bioreactor, a first conduit 6, a second conduit 7 and a TFF unit 3 that includes a housing 11 and a single tubular cross-flow filter 12, a first inlet 4 and a second inlet 5. The single tubular cross-flow filter 12 may have a pore size, for example, of about 0.2 µm. The first conduit 6 is in fluid communication between the reservoir 2 and the first inlet 4. The second conduit 7 is in fluid communication between the reservoir 2 and the second inlet 5. The fluid conduit 6 and the fluid conduit 7 can be any type of biocompatible tubing, eg a silicone tubing. The TFF unit 3 may include a single tubular cross-flow filter 2, as shown in FIGURE 1, or two or more cross-flow filters.
[00069] The system 1 in FIGURE 1 also includes a pump 8, for example a low turbulence pump (LTP), such as a peristaltic pump, which is disposed in the first conduit 6. When actuated, the pump 8 impels the fluid reversibly through the system from reservoir 2, through first conduit 6, TFF unit 3, second conduit 7, and back to reservoir 2. Housing 11 of TFF unit 3 includes a material outlet filtered 13. System 1 also includes a filtered material holding tank 10 and a filtered material conduit 9 in fluid communication between the filtered material outlet 13 and the filtered material holding tank 10. The filtered material holding tank 10 can be, for example, a refrigerated containment tank. The filtered material conduit 9 can be any type of biocompatible tubing, for example a silicone tubing.
[00070] Another exemplary system 1 is shown in FIGURE 2, which is similar to that shown in FIGURE 1, except at least for the fact that the LTP is located in a different portion of the system. System 1 includes a reservoir 2, for example a bioreactor, a first conduit 6, a second conduit 7 and a TFF unit 3 which includes a housing 11 and a single tubular cross-flow filter 12, a first inlet 4 and a second inlet 5. The single tubular cross-flow filter 12 may have a pore size, for example, of about 0.2 µm. The first conduit 6 is in fluid communication between the reservoir 2 and the first inlet 4. The second conduit 7 is in fluid communication between the reservoir 2 and the second inlet 5. The fluid conduit 6 and the fluid conduit 7 can be any type of biocompatible tubing, eg a silicone tubing. The TFF unit 3 can include a single tubular cross-flow filter 12, as shown in FIGURE 2, or it can include a set of two or more cross-flow filters.
[00071] The system 1 in FIGURE 2 also includes a pump 8, for example a low turbulence pump (LTP), such as a peristaltic pump, which is arranged in the second conduit 7. When actuated, the pump 8 impels the fluid reversibly through the system from reservoir 2, through first conduit 6, TFF unit 3, second conduit 7, and back to reservoir 2. Housing 11 of TFF unit 3 includes a material outlet filtrate 13. System 1 also includes a filtered material holding tank 10 and a filtered material conduit 9 in fluid communication between the filtered material outlet 13 and the filtered material holding tank 10. The filtered material holding tank 10 can be, for example, a refrigerated containment tank. The filtered material conduit 9 can be any type of biocompatible tubing, for example a silicone tubing.
[00072] A further exemplary system 1 is shown in FIGURE 3. System 1 includes a reservoir 2, for example a bioreactor, a first conduit 6, a second conduit 7 and a TFF unit 3 which includes a housing 11 and a a single tubular cross-flow filter 12, a first inlet 4 and a second inlet 5. The single tubular cross-flow filter 12 may have a pore size, for example, of about 0.2 µm. The first conduit 6 is in fluid communication between the reservoir 2 and the first inlet 4. The second conduit 7 is in fluid communication between the reservoir 2 and the second inlet 5. The fluid conduit 6 and the fluid conduit 7 can be any type of biocompatible tubing, eg a silicone tubing. The TFF unit 3 can include a single tubular cross-flow filter 12, as shown in FIGURE 3, or it can contain a set of two or more cross-flow filters.
[00073] System 1 in FIGURE 3 also includes a single pump 8, for example a low turbulence pump (LTP), such as a peristaltic pump, which is disposed in reservoir 2, for example, bioreactor, and close to the first conduit 6. When actuated, pump 8 reversibly propels fluid through the system from reservoir 2, through first conduit 6, TFF unit 3, second conduit 7, and back to reservoir 2. housing 11 of the TFF unit 3 includes a filtered material outlet 13. System 1 also includes a filtered material holding tank 10 and a filtered material conduit 9 in fluid communication between the filtered material outlet 13 and the holding tank of filtered material 10. The filtered material holding tank 10 may be, for example, a refrigerated holding tank. The filtered material conduit 9 can be any type of biocompatible tubing, for example a silicone tubing.
[00074] The exemplary system 1 is shown in FIGURE 4, which is similar to those illustrated in FIGURES 1 to 3, except at least for the fact that the system includes multiple TFF units. System 1 includes a reservoir 2, for example a bioreactor, a first conduit 6, a second conduit 7 and two TFF units 3, each of which includes: a housing 11, a first inlet 4, a second inlet 5, and two cross-flow filters 12. The two TFF units 3 are fluidly connected by a third conduit 14. Each of the cross-flow filters 12 may have a pore size, for example, of about 0.2 µm . The first conduit 6 is in fluid communication between the reservoir 2 and the first inlet 4 of one of the two TFF units 3, and the second conduit 7 is in fluid communication between the reservoir 2 and the second inlet 5 of the other of the two TFF units. TFF 3. The third conduit is in fluid communication between the second inlet 5 of one TFF unit 3 and the first inlet 4 of the other TFF unit 3, as shown, for example, in FIGURE 4. The fluid conduits 6, 7, and 14 can be any type of biocompatible tubing, for example, silicone tubing. As will be appreciated by those skilled in the art, the TFF 3 units may alternatively contain a single cross-flow filter, for example a tubular cross-flow filter.
[00075] The system 1 in FIGURE 4 also includes a single pump 8, for example a low turbulence pump (LTP), such as a peristaltic pump, which is disposed in the third conduit 14. When actuated, the pump 8 impels the fluid reversibly through the system from reservoir 2, through first conduit 6, one TFF unit 3, third conduit 14, the other TFF unit 3, second conduit 7, and back to reservoir 2 The housing 11 of each of the two TFF units 3 includes a filtered material outlet 13. System 1 also includes two filtered material holding tanks 10 and two filtered material conduits 9. Each filtered material holding tank 10 The individual unit is fluidly connected to a filtered material outlet 13 in a TFF unit 3 by a filtered material conduit 9. The filtered material holding tank 10 can be, for example, a refrigerated holding tank. The filtered material conduits 9 can be any type of biocompatible tubing, for example a silicone tubing.
[00076] A further exemplary system 1 is shown in FIGURE 5. System 1 includes a reservoir 2, for example a bioreactor, a first conduit 6, a second conduit 7 and a TFF unit 3 which includes a housing 11 and a a single tubular crossflow filter 12, a first inlet 4 and a second inlet 5. The crossflow filter 12 may have, for example, a pore size of about 0.2 µm, a fiber count of about 830 fibres/filter, including fibers with an internal diameter of 1 mm and a length of 30 cm, and has a filtration area of 0.77 m2. The first conduit 6 is in fluid communication between the reservoir 2 and the first inlet 4. The second conduit 7 is in fluid communication between the reservoir 2 and the second inlet 5. The fluid conduits 6 and 7 can be any type of biocompatible tubing , for example, a silicone tubing. Fluid conduit 6 and 7 can be transfer tubing with an inside diameter (ID) of 0.5 inch.
[00077] System 1 in FIGURE 5 also includes a single pump 8, for example a low turbulence pump (LTP), such as a peristaltic pump, which is disposed in the second conduit 7. Pump 8 can be a pump Watson-Marlow 620 Du peristaltic tube equipped with a dual-channel GORE Sta-Pure tubing (16mm internal diameter, 4mm wall). When actuated, pump 8 reversibly propels fluid through the system from reservoir 2, through first conduit 6, TFF unit 3, second conduit 7, and back to reservoir 2. Unit housing 11 of TFF 3 includes a filtered material outlet 13. System 1 also includes a filtered material holding tank 10 and a filtered material conduit 9 in fluid communication between the filtered material outlet 13 and the filtered material holding tank. The filtered material containment tank 10 can be, for example, a refrigerated containment tank. The filtered material conduit 9 can be any type of biocompatible tubing, for example a silicone tubing. System 1 also includes pressure sensors 14 disposed in each of first conduit 6, filtered material conduit 9 and second conduit 7. Pressure sensors 14 may be PendoTECH PressureMAT™ pressure sensors. System 1 also includes a flowmeter 15 disposed in second conduit 7. Flowmeter 15 may be a non-invasive EM-TEC BioProTT real-time flowmeter.
[00078] System 1 in FIGURE 5 also includes port conduit 16 and a port 17, wherein port conduit 16 is in fluid communication between first conduit 6 and port 17. System 1 may also include a clamp 18 disposed in port duct 16. Port 17 and port duct 16 can be used for adding fluids to system 1 through first duct 6. Multiple Pump Systems Exemplifying
[00079] A non-limiting example of a system 1 that includes two pumps 8 is shown in FIGURE 6. System 1 includes a reservoir 2, for example a bioreactor, a first conduit 6, a second conduit 7 and a TFF unit 3 which includes a housing 11 and a single tubular cross-flow filter 12, a first inlet 4 and a second inlet 5. The single tubular cross-flow filter 12 may have a pore size of about 0.2 µm. The first conduit 6 is in fluid communication between the reservoir 2 and the first inlet 4. The second conduit 7 is in fluid communication between the reservoir 2 and the second inlet 5. The fluid conduits 6 and 7 can be any type of biocompatible tubing , for example, a silicone tubing. The TFF unit 3 can include a single tubular cross-flow filter 12, as shown in FIGURE 6, or it can include a set of two or more cross-flow filters.
[00080] The system 1 in FIGURE 6 also includes a pump 8, for example a low turbulence pump (LTP), such as a peristaltic pump, which is arranged in the first conduit 6, and a pump 8, a pump low turbulence (LTP), such as a peristaltic pump, which is disposed in second conduit 7. When actuated, pump 8 disposed in first conduit 6 propels fluid in a first direction through the system from reservoir 2, through first conduit 6, from the TFF unit 3, from the second conduit 7, and back to the reservoir 2. When actuated, the pump 8 disposed in the second conduit 7 propels fluid in a second direction (opposite to the first direction) through the system a from reservoir 2, through second conduit 7, TFF unit 3, first conduit 6, and back to reservoir 2. Housing 11 of TFF unit 3 includes a filtered material outlet 13. The system also includes a 10 filtered material holding tank and a mat flue. filtered material 9 in fluid communication between the filtered material outlet 13 and the filtered material holding tank 10. The filtered material holding tank 10 can be, for example, a refrigerated holding tank. The filtered material conduit 9 can be any type of biocompatible tubing, for example a silicone tubing. Exemplary systems that include two or more subsystems
[00081] Those skilled in the art will appreciate that multiple subsystems can be added to the system. An exemplary system 1 that includes two or more subsystems 19 is shown in FIGURE 7. System 1 includes a reservoir 2; and first and second subsystems 19, wherein each subsystem 19 includes a first conduit 6, a second conduit 7 and a TFF unit 3 which includes a housing 11 and a single tubular cross-flow filter 12, a first inlet 4 and a second inlet 5, as shown in FIGURE 7. The individual cross-flow tubular filters 12 may have a pore size of about 0.2 µm. In each subsystem, the first conduit 6 is in fluid communication between the reservoir 2 and the first inlet 4. The second conduit 7, in each subsystem, is in fluid communication between the reservoir 2 and the second inlet 5. The fluid conduits 6 and 7 can be any type of biocompatible tubing, eg silicone tubing. The TFF units 3 can include a single tubular cross-flow filter 12, respectively, as shown in FIGURE 7, or each can include a set of two or more cross-flow filters. The individual cross-flow tubular filters 12 can have a pore size of about 0.2 µm.
[00082] Each subsystem 19 in FIGURE 7 also includes a single pump 8, for example a low turbulence pump (LTP), such as a peristaltic pump, which is disposed in the first conduit 6. When actuated, the single pump 8 in each subsystem 19 propels fluid reversibly through the system from reservoir 2, through first conduit 6, TFF unit 3, second conduit 7, and back to reservoir 2. two TFF units 3 include a filtered material outlet 13. Each subsystem 19 also includes a filtered material holding tank 10 and a filtered material conduit 9 in fluid communication between the TFF unit 3 and the filtered material holding tank 10. The filtered material containment tank 10 may be, for example, a refrigerated containment tank. The filtered material conduits 9 can be any type of biocompatible tubing, for example a silicone tubing. Structures and Characteristics of Additional Exemplary Systems
[00083] The non-limiting exemplary structures that can be used for the reservoir, the conduits, the TFF unit(s), the pump(s), the containment tank(s) filtered material, flow meter(s), pressure sensor(s), clamp(s), port(s), and biological manufacturing system(s) are described below. Reservoirs
[00084] A reservoir can be a bioreactor. The bioreactor can have a volume, for example, between about 1 liter to about 10,000 liters (for example, between about 1 liter to about 50 liters, between about 50 liters to about 500 liters, between about 500 liters to about 1000 liters, between 500 liters to about 5000 liters, between about 500 liters to about 10,000 liters, between about 5000 liters to about 10,000 liters, between about 1 liter and about 10,000 liters, between about 1 liter and about 8,000 liters, between about 1 liter and about 6,000 liters, between about 1 liter and about 5,000 liters, between about 100 liters and about 5,000 liters, between about 10 liters and about 100 liters, between about 10 liters and about 4,000 liters, between about 10 liters and about 3,000 liters, between about 10 liters and about 2,000 liters, or between about 10 liters and about 1,000 liters). Any of the bioreactors described herein can be a perfusion bioreactor. Exemplary bioreactors can be purchased from a number of different commercial vendors (eg, Xcellerex (Marlborough, MA) and Holland Applied Technologies (Burr Ridge, IL)).
[00085] Alternatively or additionally, a reservoir can be a containment tank. For example, such a refrigerated holding tank can hold the cell culture containing a recombinant protein for a period of between about 5 minutes and about a week (for example, between about 5 minutes and about 6 days, between about 5 minutes and about 5 days, between about 5 minutes and about 4 days, between about 5 minutes and about 3 days, between about 5 minutes and about 2 days, between about 5 minutes and about 36 hours , between about 5 minutes and about 24 hours, between about 5 minutes and about 12 hours). The cell culture in the holding tank can be maintained at a temperature between about 15°C and about 37°C, between about 20°C and about 37°C, between about 25°C and about 37°C , between about 30°C and about 37°C, or between about 20°C and about 30°C. Conduits
[00086] A conduit described in this document can be a simple pipe, for example, a biocompatible pipe. Non-limiting examples of useful tubing include silicone rubber, polyurethane, polydioxanone (PDO), polyhydroxy alkanoate, polyhydroxy butyrate, poly(glycerol sebacate), polyglycolide, polylactide, polycaprolactone, or polyanhydride , or copolymers or derivatives including these and/or other polymers. Alternatively or additionally, any of the conduits described herein may include polyvinyl chloride. Any of the ducts can have, for example, an internal diameter (ID) between about 5 mm and about 50 mm (for example, between about 10 mm and about 40 mm, between about 10 mm and about 35 mm. mm, or between about 10 mm and about 30 mm, between about 10 mm and about 20 mm). A conduit can be weldable transfer tubing. Additional examples of conduits and conduit properties that can be used in the present devices and methods are well known to those skilled in the art. TFF Units and Cross Flow Filters
[00087] TFF units used in any of the systems or subsystems or methods described in this document may include one or more cross-flow filters. For example, a TFF unit described herein may include a single cross-flow filter (e.g., a tubular cross-flow filter). In other examples, a TFF unit may include two or more (eg, three, four, five, or six) cross-flow filters (eg, tubular cross-flow filters). The two or more crossflow filters in the TFF unit can be identical or they can be different (for example, different in number, type, shape, surface area, or pore size). In a specific example, the TFF unit may include two tubular cross-flow filters. The two or more crossflow filters present in a TFF unit can be curved rectangular in shape.
[00088] The cross-flow filter(s) may have an average pore size between about 0.1 µm to about 0.45 µm (for example, between about 0.15 µm to about 0.40 µm, between about 0.15 µm to about 0.35 µm, between about 0.15 µm to about 0.30 µm, between about 0.15 µm to about 0.25 µm), or about 0.20 µm. The cross-flow filter(s) may be a polyether sulfone (PES) composite spectrum filter.
[00089] The cross-flow filter(s) may have a surface area (filtering area) between about 0.1 m2 to about 5 m2 (for example, between about 0.5 m2 to about 4.5 m2, between about 0.5 m2 to about 4.0 m2, between about 0.5 m2 to about 3.5 m2, between about 0.5 m2 to about 3 ,0 m2, between about 0.5 m2 to about 2.5 m2, between about 0.5 m2 to about 2.0 m2, between about 0.5 m2 to about 1.5 m2, or between about 0.5 m2 to about 1.0 m2). Crossflow filters can have total fiber numbers per filter between about 500 fibers/filter to about 2,500 fibers/filter (e.g., between about 500 fibers/filter to about 2,400 fibers/filter, between about 500 fibers/filter to about 2300 fibers/filter, between about 500 fibers/filter to about 2200 fibers/filter, between about 500 fibers/filter to about 2,100 fibers/filter, between about 500 fibers/filter to about 2,000 fibers/filter, between about 500 fibers/filter to about 1,900 fibers/filter, between about 500 fibers/filter to about 1,800 fibers/filter, between about 500 fibers/filter to about 1,700 fibers/filter , between about 500 fibers/filter to about 1,600 fibers/filter, between about 500 fibers/filter to about 1,500 fibers/filter, between about 500 fibers/filter to about 1,400 fibers/filter, between about 500 fibers/filter to about 1,300 fibers/filter, between about 500 fibers/filter to about 1,200 fibers/filter, between and about 500 fibers/filter to about 1,100 fibers/filter, between about 500 fibers/filter to about 1,000 fibers/filter, between about 500 fibers/filter to about 900 fibers/filter, between about 600 fibers /filter to about 900 fibers/filter, between about 700 fibers/filter to about 900 fibers/filter, or between about 800 fibers/filter to about 900 fibers/filter). In some examples, the fibers within the cross-flow filter(s) have an internal diameter between about 0.05 mm to about 10 mm (for example, between about 0.1 mm to about 9 mm , between about 0.1 mm to about 8 mm, between about 0.1 mm to about 7 mm, between about 0.1 mm to about 6 mm, between about 0.1 mm to about 5 mm, between about 0.1 mm to about 4 mm, between about 0.1 mm to about 3 mm, between about 0.1 mm to about 2.5 mm, between about 0.1 mm to about 2.0 mm, between about 0.1 mm to about 1.5 mm, between about 0.5 mm to about 1.5 mm, or between about 0.75 mm to about 1.25 mm). The fibers present in the cross-flow filter(s) can have a length of between about 0.2 cm and about 200 cm (for example, between about 0.2 cm and about 190 cm, between about about 0.2 cm and about 180 cm, between about 0.2 cm and about 170 cm, between about 0.2 cm and about 160 cm, between about 0.2 cm and about 150 cm, between about 0.2 cm and about 140 cm, between about 0.2 cm and about 130 cm, between about 0.2 cm and about 120 cm, between about 0.2 cm and about 110 cm, between about 0.2 cm and about 100 cm, between about 0.2 cm and about 90 cm, between about 0.2 cm and about 80 cm, between about 0.2 cm and about 70 cm cm, between about 0.2 cm and about 60 cm, between about 0.2 cm and about 55 cm, between about 0.2 cm and about 50 cm, between about 1 cm and about 45 cm cm, between about 1 cm and about 40 cm, between about 1 cm and about 35 cm, between about 1 cm and about 35 cm, between about 1 cm and about 30 cm, between about 1 cm and about 25 cm, between about 1 cm and about 20 cm, between about 1 cm and about 15 cm, between about 1 cm and about 10 cm, between about 0.1 cm and about 5 cm, between about 20 cm and about 40 cm, or between about 25 cm and about 35 cm). The cross-flow filter(s) can be of any shape such that most of the surface area of the filter(s) is positioned parallel to the fluid flow (for example, the cell culture) in the system. For example, the cross-flow filter(s) can have a tubular shape or a curved rectangular or threaded shape. An example of a crossflow filter that can be used in the systems described in this document is the ATF4 (Refine Technology) filter. Additional cross-flow filters are described in this document and known in the state of the art.
[00090] As will be appreciated by those skilled in the art, the cross-flow filter(s) in the TFF unit can be housed in a casing (eg a hard plastic casing or metal). An enclosure can be of any shape, cylindrical or rectangular, and designed in such a way that it can contain one or more cross-flow filters. The housing may contain a surface that permits the insertion or removal of one or more cross-flow filters from the housing.
[00091] Some systems include two or more TFF units arranged in series or in parallel. For example, in systems where two or more TFF units are arranged in series, a fluid conduit can be used to fluidly connect two neighboring TFF units (e.g., any of the exemplary TFF units described herein. document or known in the state of the art). Such an exemplary arrangement of two TFF units in a system is shown in FIGURE 4. The two or more TFF units arranged in series can be designed in any manner, as long as the activation of at least one pump in the system results in reversible flow of the cell culture from the reservoir, for example, the bioreactor, through the first conduit, two or more TFF units, one or more conduits positioned between neighboring TFF units), from the second conduit, and back to the reservoir, for example , the bioreactor). The two or more TFF units can be identical (eg the same number and type of crossflow filters) or different (eg different number and type of crossflow filters). In some examples, two or more TFF units each contain a single tubular cross-flow filter. Each TFF unit can be fluidly connected to a filtered material conduit which allows the filtered material exiting the TFF unit to be propelled into a filtered material holding tank (eg any of the material holding tanks filtered described in this document). In some embodiments, the two or more TFF units may be arranged in a single housing (for example, any of the exemplary types of housing described herein or known in the prior art). bombs
[00092] The systems described in this document may include one or more pumps. In some examples, one or more pumps are low turbulence pumps (LTPs). LTPs are pumps that can move a fluid (eg cell culture) in a single direction (eg a first or second flow direction) or reversibly move a fluid (eg a cell culture ) in two directions (a first and a second flow directions) without inducing a substantial amount of shear stress and/or fluid turbulence in the fluid (e.g., cell culture). When an LTP is used to propel a fluid (eg, a cell culture) in alternate first and second flow directions, the second flow direction is more or less opposite to the first flow direction.
[00093] An example of an LTP pump is a peristaltic pump. A peristaltic pump can have the pump column having a volume of between about 20 ml to about 250 ml (for example, between about 20 ml and about 240 ml, between about 20 ml and about 220 ml, between about of 20 ml and about 200 ml, between about 20 ml and about 180 ml, between about 20 ml and about 160 ml, between about 20 ml and about 140 ml, between about 20 ml and about 120 ml, between about 20 ml and about 100 ml, between about 20 ml and about 80 ml, between about 20 ml and about 60 ml, between about 20 ml and about 50 ml, between about 20 ml and about 40 ml, between about 20 ml and about 30 ml, between about 30 ml and about 240 ml, between about 30 ml and about 220 ml, between about 30 ml and about 200 ml, between about 30 ml and about 180 ml, between about 30 ml and about 160 ml, between about 30 ml and about 140 ml, between about 30 ml and about 120 ml, between about 30 ml and about 100 ml, between about 30 ml and about 80 ml, between cer from about 30 ml to about 60 ml, between about 40 ml and about 250 ml, between about 40 ml and about 240 ml, between about 40 ml and about 220 ml, between about 40 ml and about 200 ml, between about 40 ml and about 180 ml, between about 40 ml and about 160 ml, between about 40 ml and about 140 ml, between about 40 ml and about 120 ml, between about 40 ml and about 100 ml, between about 40 ml and about 80 ml, between about 40 ml and about 60 ml, between about 50 ml and about 250 ml, between about 50 ml and about 240 ml, between about 50 ml and about 220 ml, between about 50 ml and about 200 ml, between about 50 ml and about 180 ml, between about 50 ml and about 160 ml, between about 50 ml and about 140 ml, between about 50 ml and about 120 ml, between about 50 ml and about 100 ml, between about 50 ml and about 80 ml, between about 50 ml and about 75 ml, between about 60 ml and about 250 ml, between about 60 ml and about 240 ml, between about 60 ml and about 220 ml, between about 60 ml and about 200 ml, between about 60 ml and about 180 ml, between about 60 ml and about 160 ml, between about 60 ml and about 140 ml between about 60 ml and about 120 ml, between about 60 ml and about 100 ml, between about 60 ml and about 80 ml, between about 70 ml and about 250 ml, between about 70 ml and about 240 ml, between about 70 ml and about 220 ml, between about 70 ml and about 200 ml, between about 70 ml and about 180 ml, between about 70 ml and about 160 ml , between about 70 ml and about 140 ml, between about 70 ml and about 120 ml, between about 70 ml and about 100 ml, between about 80 ml and about 250 ml, between about 80 ml and about 240 ml, between about 80 ml and about 220 ml, between about 80 ml and about 200 ml, between about 80 ml and about 180 ml, between about 80 ml and about 160 ml, between about 80 ml and about 140 ml, between about 80 ml and about 120 ml, between about 80 ml and about 100 ml, between about 90 ml and about 250 ml, between about 90 ml and about 240 ml, between about 90 ml and about 220 ml, between about 90 ml and about 200 ml, between about 90 ml and about 180 ml, between about 90 ml and about 160 ml, between about 90 ml and about 140 ml, between about 90 ml and about 120 ml, between about 90 between about 100 ml and about 100 ml, between about 100 ml and about 250 ml, between about 100 ml and about 240 ml, between about 100 ml and about 220 ml, between about 100 ml and about 200 ml , between about 100 ml and about 180 ml, between about 100 ml and about 160 ml, between about 100 ml and about 140 ml, or between about 100 ml and about 120 ml). The peristaltic pump can have tubing with an internal diameter between about 5 mm and about 400 mm (for example, between about 5 mm and about 380 mm, between about 5 mm and about 360 mm, between about 5mm and about 340mm, between about 5mm and about 320mm, between about 5mm and about 300mm, between about 5mm and about 280mm, between about 5mm and about 260mm mm, between about 5 mm and about 240 mm, between about 5 mm and about 220 mm, between about 5 mm and about 200 mm, between about 5 mm and about 180 mm, between about 5 mm and about 160 mm, between about 5 mm and about 140 mm, between about 5 mm and about 120 mm, between about 5 mm and about 100 mm, between about 5 mm and about 80 mm , between about 5 mm and about 60 mm, between about 5 mm and about 55 mm, between about 5 mm and about 50 mm, between about 5 mm and about 45 mm, between about 5 mm and about 40 mm, between about 5 mm and about 35 mm, between about 5 mm and about 30 mm, between about 5 mm and about 25 mm, between about 5 mm and about 20 mm, between about 5 mm and about 15 mm, between about 5 mm and about 10 mm, between about 1 mm and about 10 mm, between about 10 mm and about 60 mm, between about 10 mm and about 35 mm, between about 10 mm and about 25 mm, between about 10 mm and about 20 mm, between about 20 mm and about 60 mm, between about 20 mm and about 50 mm, or between about 30 mm and about 50 mm). The tubing inside a peristaltic pump can have a wall diameter between about 1 mm to about 30 mm (for example, between about 1 mm to about 25 mm, between about 1 mm to about 20 mm, between about 1 mm to about 18 mm, between about 1 mm to about 16 mm, between about 1 mm to about 14 mm, between about 1 mm to about 12 mm, between about 1 mm to about 10 mm, between about 1 mm to about 8 mm, between about 1 mm to about 6 mm, or between about 1 mm to about 5 mm). Examples of peristaltic pump(s) that can be used in the present systems and methods are the Watson Marlow 620 and Watson Marlow 800 pumps. Any of the peristaltic pumps described in this document may have a dual channel and/or contain the GORE Sta-Pure tubing (eg tubing with an inner diameter of 16 mm and a wall of 4 mm).
[00094] Additional examples of LTP pumps are described in U.S. Patents no. 4,037,984; 5,033,943; and 5,458,459; in U.S. Patent Application Publication no. 2009/0199904, and in International Patent Application number WO 06/021873. Other examples of LTP pumps include rotary positive displacement pumps, lobe pumps, internal gear pumps, and progressive cavity pumps. Those skilled in the art will appreciate that other types of LTPs are commercially available and can be used in any of the systems and methods described herein.
[00095] In some examples, at least one pump is arranged in the first or second conduit, or both. In other examples, at least one pump is disposed in the reservoir and close to the first or second fluid conduits. In systems that include two or more TFF units, at least one pump may be disposed in a conduit placed between two neighboring TFF units (for example, conduit 14 shown in FIGURE 4). At least one pump may be disposed anywhere in the systems provided herein provided that, the actuation of at least one pump results in the fluid flowing reversibly through the system from the reservoir, through the first conduit, of the pump unit. TFF, from the second conduit, and back to the reservoir, or in systems containing two or more TFF units, the fluid flow reversibly through the system from the reservoir, through the first conduit, of the two or more units of TFF, from one or more conduits between neighboring TFF units, from the second conduit, and back to the reservoir. Filtered material containment tank
[00096] A filtered material containment tank can be optionally included in the system, for example to store the filtered material. For example, the filtered material can be stored for a period of between about 1 hour and about a week (for example, between about 1 hour and about 6 days, between about 1 hour and about 5 days, between about between about 1 hour and about 4 days, between about 1 hour and about 3 days, between about 1 hour and about 2 days, between about 1 hour and about 36 hours, between about 1 hour and about 24 hours, between about 1 hour and about 20 hours, between about 1 hour and about 16 hours, between about 1 hour and about 12 hours, or between about 1 hour and about 6 hours). The filtered material holding tank can have an internal volume of between about 50 ml and about 50 liters (for example, between about 50 ml and about 45 liters, between about 50 ml and about 40 liters, between about of 50 ml and about 35 liters, between about 50 ml and about 30 liters, between about 50 ml and about 25 liters, between about 50 ml and about 20 liters, between about 50 ml and about 18 liters, between about 50 ml and about 16 liters, between about 50 ml and about 14 liters, between about 50 ml and about 12 liters, between about 50 ml and about 10 liters, between about 50 ml and about 9 liters, between about 50 ml and about 8 liters, between about 50 ml and about 7 liters, between about 50 ml and about 6 liters, between about 50 ml and about 5 liters, between about 50 ml and about 4.5 liters, between about 50 ml and about 4.0 liters, between about 50 ml and about 3.5 liters, between about 50 ml and about 3 ,0 liters, between about 50 ml and about 2.5 liters, between about 50 ml and about 2.0 liters, between about 50 ml and about 1.5 liter, between about 50 ml and about 1.0 liter, between about 100 ml and about 1.0 liter, or between about 500 ml and about 1.0 liter). The inner surface of the filtered material holding tank can contain a biocompatible material (for example, any biocompatible material known in the prior art). The filtered material holding tank can be a refrigerated holding tank that is capable of storing the filtered material at a temperature between about 10°C and about 35°C (e.g., between about 10°C and about 30°C, between about 10°C and about 25°C, between about 10°C and about 20°C, between about 10°C and about 15°C, or between about 15°C and about 25°C). As one skilled in the art can appreciate, a number of different commercially available containment tanks can be used as a containment tank for filtered material in the systems and methods described herein. flow meters
[00097] Some examples of the systems described in this document may include one or more (eg two, three, four or five) flow meters. For example, one or more flow meters may be disposed in one or more of any of the conduits in the system (eg, the first conduit, the second conduit, one or more conduits between neighboring TFF units, and/or the conduit of filtered material). For example, a flowmeter can be placed between two neighboring TFF units. In some examples, the flow meter(s) is/are non-invasive. Those skilled in the art should understand the wide variety of commercially available flow meters that can be used in the present systems and methods. For example, a TEC BioProTT non-invasive real-time flowmeter, a PT878 (Rshydro) ultrasonic flowmeter, and a Sono-Trak (EMCO) noninvasive ultrasonic flowmeter are commercially available flowmeters that can be used in the present systems and methods. Pressure Sensors
[00098] The systems described in this document may include one or more pressure sensors. For example, one or more pressure sensors may be disposed in any of the conduits in the system (eg, the first conduit, the second conduit, one or more conduits between neighboring TFF units, and/or the filtered material conduit ). For example, a pressure sensor can be placed between two neighboring TFF units in a system. Those skilled in the art should understand the wide variety of commercially available pressure sensors that can be used in the present systems and methods. A non-limiting example of a pressure sensor that can be used in the systems and methods described in this document is a PendoTECH PressureMAT pressure sensor. Clamps/Doors
[00099] Any of the systems described in this document may optionally include a door conduit between the first or second conduits and a door that fluidly connects the first or second conduits, respectively, to the door. The port can be used to apply or remove a fluid (eg cell culture or wash solution) from the system (through the first or second conduit, respectively). A clamp can be arranged on the door flue. A wide variety of suitable clamps are known in the prior art (for example a screw clamp). The door duct can have any combination of the characteristics described above for ducts. The door can be any type of door commonly known in the prior art. For example, a port can be an injection port or it can have a ribbed thread. Biological Manufacturing Systems
[000100] Any of the devices described in this document may include a biological manufacturing system that includes at least one (for example, two, three, or four) multiple column chromatography (MCCS) systems that have an input and an output , and a filtered material conduit between the TFF unit or the filtered material containment tank, wherein the device is configured in such a way that the filtered material is passed to the inlet of the biological manufacturing system, through at least one MCCS, and leaves the device through the output of the biological manufacturing system. An MCCS can include two or more chromatography columns, two or more chromatographic membranes, or a combination of at least one chromatography column and at least one chromatographic membrane. In non-limiting examples, an MCCS can include four chromatographic columns, three chromatographic columns and one chromatographic membrane, three chromatographic columns, two chromatographic columns, two chromatographic membranes, and two chromatographic columns and one chromatographic membrane. Additional examples of combinations of chromatography columns and/or chromatographic membranes can be employed for use in an MCCS by the skilled artisan without limitation. The individual chromatography columns and/or chromatographic membranes present in an MCCS may be identical (eg have the same shape, volume, resin, capture mechanism and unit operation), or they may be different (eg have one or more than one different shape, volume, resin, capture mechanism and unit operation). The individual chromatography column(s) and/or chromatographic membrane(s) present in an MCCS can perform the same unit operation (for example, the capture unit operation , purification, polishing, virus inactivation, adjustment of the ionic concentration and/or pH of a fluid containing the recombinant therapeutic protein, and filtration) or different unit operations (eg different unit operations selected from, for example, group capture, purification, polishing, virus inactivation, adjustment of the ionic concentration and/or pH of a fluid containing the recombinant therapeutic protein, and filtration).
[000101] One or more (for example, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, or twenty-four) different types of plug may be employed while using one or more MCCS in any of the biological manufacturing devices described herein. As is known in the prior art, one or more types of buffer used in one or more MCCS used in the biological manufacturing systems described herein will depend on the resin present in the chromatography column(s) and/or in the chromatographic membrane(s) of one or more MCCS (e.g., first and second MCCS), recombinant therapeutic protein, and unit operation (e.g., any of the exemplary unit operations described herein document) performed on chromatography column(s) and/or specific chromatography membranes of one or more MCCS. The volume and type of buffer employed when using one or more MCCS in any of the biological processing devices described herein can also be determined by one of ordinary skill in the art. For example, the volume and type(s) of buffer employed when using one or more MCCS in any of the processes described herein can be chosen in order to optimize one or more of the following in the isolated recombinant protein resulting (eg, drug product): the total yield of the recombinant therapeutic protein, the activity of the recombinant therapeutic protein, the level of purity of the recombinant therapeutic protein, and the removal of biological contaminants from a fluid containing the recombinant therapeutic protein ( for example, the absence of active viruses, microbacteria, yeast, bacteria, or mammalian cells).
[000102] One or more MCCS can be a periodic countercurrent chromatography system (PCCS). A PCCS can, for example, include two or more chromatography columns (eg, three columns or four columns) that are switched to allow continuous elution of the recombinant therapeutic protein from the two or more chromatography columns. A PCCS can include two or more chromatography columns, two or more chromatographic membranes, or at least one chromatography column and at least one chromatographic membrane. A column operation generally consists of loading, washing, elution and regeneration steps. In PCCSs, multiple columns are used to distinctly and continuously perform the same steps in a cyclic fashion. Since the columns are run in series, the flow through one column and the wash is captured by another column. This unique feature of PCCS allows the resin to charge close to its static binding capacity rather than the dynamic binding capacity, as is typical during batch mode chromatography. As a result of continuous cycling and elution, the fluid entering a PCCS is continuously processed, and the eluate containing the recombinant therapeutic protein is continuously produced.
[000103] One or more unit operations that can be performed by at least one MCCS in biological manufacturing systems include, for example, capture of recombinant therapeutic protein, inactivation of viruses present in a fluid containing the recombinant therapeutic protein, purification of recombinant therapeutic protein, polishing of recombinant therapeutic protein, containment of a fluid containing the recombinant therapeutic protein (eg, when using a decomposition tank), filtering or removing particulate matter from a fluid containing the recombinant therapeutic protein, and adjusting the ionic concentration and/or pH of a fluid containing the recombinant therapeutic protein.
[000104] The operation of the capture unit can be performed by using one or more MCCS that includes(s) at least one chromatography column and/or chromatography resin, for example, that uses a capture mechanism. Non-limiting examples of capture mechanisms include a protein A binding capture mechanism, an antibody or antibody fragment binding capture mechanism, a substrate binding capture mechanism, an aptamer binding capture mechanism , a tag-binding capture mechanism (e.g., a poly-His tag-based capture mechanism), and a cofactor-binding capture mechanism. Capture can also be performed using a resin that can be used to perform cation exchange or anion exchange chromatography, or molecular sieve chromatography. Examples of resins that can be used to capture a therapeutic recombinant protein are known in the art.
[000105] The operation of the virus inactivation unit present in a fluid containing the recombinant therapeutic protein can be performed by using one or more MCCSs that include, for example, a chromatography column, a chromatography membrane, or a holding tank that is capable of incubating a fluid containing the recombinant therapeutic protein at a pH of between about 3.0 to 5.0 (e.g., between about 3.5 to about 4.5, between about 3.5 to about 4.25, between about 3.5 to about 4.0, between about 3.5 to about 3.8, or about 3.75 for a period of at least 30 minutes (for example, a period between about 30 minutes to 1.5 hours, a period between about 30 minutes to 1.25 hours, a period between about 0.75 hours to 1.25 hours, or a period of about 1 hour).
[000106] The operation of the purification unit of a recombinant protein can be performed using one or more MCCSs that include, for example, a chromatography column or a chromatographic membrane that contains a resin, for example, that uses a capture system. Non-limiting examples of capture mechanisms include a protein A binding capture mechanism, antibody or antibody fragment binding capture mechanism, a substrate binding capture mechanism, an aptamer binding capture mechanism, an tag-binding capture mechanism (eg, a poly-His tag-based capture mechanism), and a cofactor-binding capture mechanism. Purification can also be performed using a resin that can be used to perform cation exchange or anion exchange chromatography, or molecular sieve chromatography. Examples of resins that can be used in the purification of a therapeutic recombinant protein are known in the art.
[000107] The operation of the polishing unit of a recombinant protein can be performed by using one or more MCCSs that include, for example, a chromatography column or a chromatographic membrane that contains a resin, for example, which can be used to perform cation exchange, anion exchange, or molecular sieve chromatography. Examples of resins that can be used in polishing a therapeutic recombinant protein are known in the art.
[000108] The operation of the containment unit of a fluid containing the recombinant therapeutic protein can be performed using an MCCS that includes at least one reservoir (for example, a decomposition tank) or a maximum of 1, 2, 3, 4 or 5 reservoirs (eg decomposition tank(s)) in one or more MCCS in the biological manufacturing system. For example, each of the reservoir(s) (eg, decomposition tank(s)) that can be used to perform this unit operation may have a volume between about 1 ml to about 1 liter (for example, between about 1 ml to about 800 ml, between about 1 ml to about 600 ml, between about 1 ml to about 500 ml, between about 1 ml to about 400 ml, between about 1 ml to about 350 ml, between about 1 ml to about 300 ml, between about 10 ml and about 250 ml, between about 10 ml and about 200 ml, between about 10 ml and about 150 ml, and between about 10 ml to about 100 ml). The reservoir(s) (eg decomposition tank(s)) used in the biological manufacturing systems described in this document may have a capacity which is, for example, between 1 ml and about 300 ml, including, for example, between 1 ml and about 280 ml, about 260 ml, about 240 ml, about 220 ml, about 200 ml, about 180 ml, about 160 ml, about 140ml, about 120ml, about 100ml, about 80ml, about 60ml, about 40ml, about 20ml, or about 10ml inclusive. Each of the reservoir(s) (eg, decomposition tank(s)) in the biological manufacturing system can hold the fluid containing the recombinant therapeutic protein for at least 10 minutes (eg, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, or at least 6 hours). In other examples, the reservoir(s) (eg decomposition tank(s)) in the biological manufacturing system only contain a therapeutic protein for a total period of time, eg between about 5 minutes and less than about 6 hours, inclusive, for example, between about 5 minutes and about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, or about 30 minutes, inclusive . Reservoir(s) (eg decomposition tank(s)) in the biological manufacturing system can be used to contain and refrigerate (eg at a temperature of less than 25°C, less than 15°C , or less than 10°C) the fluid containing the recombinant therapeutic protein. The reservoir can be any shape, including a circular cylinder, an oval cylinder, or a more or less rectangular sealed non-permeable bag.
[000109] The operations of the filtration unit of a fluid that contains the recombinant therapeutic protein can be performed using an MCCS that includes, for example, a filter, or a chromatography column or a chromatographic membrane that contains a molecular sieve resin . As is known in the prior art, a wide variety of submicron filters (eg a filter with a pore size of less than 1 µm, less than 0.5 µm, less than 0.3 µm, about 0, 2 μm, less than 0.2 μm, less than 100 nm, less than 80 nm, less than 60 nm, less than 40 nm, less than 20 nm, or less than 10 nm) is available in the prior art, which are capable of removing any precipitated material and/or cell (e.g., unbroken protein; precipitated unwanted host cell proteins; precipitated lipids; bacteria; yeast cells; fungal cells; and/or microbacteria). It is known that filters having a pore size of about 0.2 µm or less than 0.2 µm effectively remove bacteria from the fluid containing the recombinant therapeutic protein. As is known in the prior art, a chromatography column or a chromatographic membrane containing a molecular sieve resin can also be used in an MCCS to carry out the operation of the filtration unit on a fluid containing a therapeutic recombinant protein.
[000110] Unit operations for adjusting the ionic concentration and/or pH of a fluid containing the recombinant therapeutic protein can be performed using an MCCS that includes and utilizes a buffer adjustment reservoir (for example, a reservoir of in-line buffer adjustment) that adds a new buffer solution to a fluid containing the recombinant therapeutic protein (eg, between columns within a single MCCS, or after the last column in a penultimate MCCS and before the fluid that contains the recombinant therapeutic protein is fed into the first column of the next MCCS (eg the second MCCS) As can be appreciated in the prior art, the in-line buffer fitting reservoir can be of any size (eg more 100 ml) and can contain any protected solution (eg a protected solution that has one or more of: an increased or decreased pH compared to fluid containing the recombinant therapeutic protein, u an increased or decreased ionic concentration (eg, salt) compared to the fluid containing the recombinant therapeutic protein, and/or an increased or decreased concentration of an agent that competes with the recombinant therapeutic protein to bind to the resin present in at least a chromatographic column or on at least one chromatographic membrane in an MCCS (eg, the first or second MCCS)).
[000111] An MCCS can perform two or more unit operations. For example, an MCCS can perform at least the following unit operations: capturing the recombinant therapeutic protein and inactivating the viruses present in the fluid containing the recombinant therapeutic protein; capturing the recombinant therapeutic protein, inactivating the viruses present in the fluid containing the recombinant therapeutic protein, and adjusting the ionic concentration and/or pH of a fluid containing the recombinant therapeutic protein; purification of the recombinant therapeutic protein and polishing of the recombinant therapeutic protein; purifying the recombinant therapeutic protein, polishing the recombinant therapeutic protein, and filtering a fluid containing the recombinant therapeutic protein or removing precipitates and/or particulate matter from a fluid containing the recombinant therapeutic protein; and purifying the recombinant therapeutic protein, polishing the recombinant therapeutic protein, filtering a fluid containing the recombinant therapeutic protein or removing precipitates and/or particulate matter from a fluid containing the recombinant therapeutic protein, and adjusting the ionic concentration and/ or the pH of a liquid containing the recombinant therapeutic protein.
[000112] Additional exemplary features of biological manufacturing systems that can be used in the present devices and methods are described in U.S. Patent Application no. Serial 61/775,060, filed March 8, 2013, and in U.S. Patent Application no. series 61/856,390, filed on July 19, 2013. Benefits conferred by these systems
[000113] The systems described in this document provide for continuous filtration of cell culture that has one or more (for example, two, three, four, five, six, or seven) of the following benefits: decreased external volume of the cell culture (outside the reservoir), increased exchange fraction (inside the first conduit, the TFF unit and the second conduit), decreased cell culture external residence time (outside the reservoir), decreased shear stress during culture filtration of cells, improved cell viability in cell culture, high viable cell density in cell culture, and decreased filter fouling compared to other unidirectional open circuit filtration systems (eg unidirectional TFF systems) or filtration systems. bidirectional closed-loop filtration (closed-loop ATF™ systems).
[000114] The exchange fraction and the external residence time of a system described in this document can be calculated using equations 1 and 2 below.

[000115] For example, the present systems can only have a total external volume of the cell culture that is between about 1% and about 7% (for example, between about 1.0% and about 6.5% , between about 1% and about 6.0%, between about 1% and about 5.5%, or between about 1% and about 5.0%) of the total volume of cell culture in the reservoir , in the first conduit, in the second conduit and in the TFF unit. The systems provided herein can also provide a reduced residence time of the cell culture outside the reservoir (reduced external residence time) between about 1 second and about 60 seconds (for example, between about 1 second and about 55 seconds, between about 1 second and about 50 seconds, between about 1 second and 45 seconds, between about 1 second and about 30 seconds, between about 1 second and about 25 seconds, between about 1 second and about 20 seconds, between about 1 second and about 15 seconds, between about 1 second and 13 seconds, between about 1 second and 10 seconds, between about 1 second and about 8 seconds, between about 1 second and about 5 seconds, or between about 10 seconds and 14 seconds). Table 1 below compares the residence time of the exemplary system described in the example and a closed-loop alternate tangential filtration system (ATF4). Table 1. Comparison of external residence time and external fraction of the exemplary system provided in this document and the closed system ATF4

[000116] The present systems can provide an improved exchange fraction of more than about 50% (eg more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, or more than about 85%). The systems described herein can provide high viable cell densities in cell culture, for example, a viable cell density of more than about 30 x 106 cells, more than about 32 x 106 cells, more than about 34 x 106 cells, more than about 36 x 106 cells, more than about 38 x 106 cells, more than about 40 x 106 cells, more than about 42 x 106 cells, more than about 44 x 106 cells, more than about 46 x 106 cells, more than about 48 x 106 cells, more than about 50 x 106 cells, more than about 52 x 106 cells, more than about 54 x 106 cells, more than about 56 x 106 cells, more than about 58 x 106 cells, or more than about 60 x 106 cells. The systems described herein can provide a viable cell density of more than about 65 x 106 cells, more than about 70 x 106 cells, more than about 75 x 106 cells, more than about 80 x 106 cells, more than about 85 x 106 cells, more than about 90 x 106 cells, more than about 95 x 106 cells, more than about 100 x 106 cells, more than about 105 x 106 cells, more than about 110 x 106 cells, more than about 115 x 106 cells, more than about 120 x 106 cells, more than about 125 x 106 cells, more than about 130x 106 cells, more than about 135 x 106 cells, more than about 140 x 106 cells, more than about 145 x 106 cells, more than about 150 x 106 cells, more than about 155 x 106 cells, more than about 160 x 106 cells, more than about 165 x 106 cells, more than about 170 x 106 cells, more than about 175 x 106 cells s, more than about 180 x 106 cells, more than about 185 x 106 cells, more than about 190 x 106 cells, more than about 200 x 106 cells, more than about 210 x 106 cells, more than about 220 x 106 cells, more than about 230 x 106 cells, more than about 240 x 106 cells, or more than about 250 x 106 cells.
[000117] The systems provided in this document also provide an optimized exchange rate (also called throughput in this document). As can be appreciated by those of skill in the art, an exchange rate that is too high can result in a level of shear stress that negatively impacts cell growth and cell culture performance, and an exchange rate that is Too low can result in filter fouling and a longer external residence time of the cell culture. The systems provided in this document provide for obtaining any of the exemplary flows described in this document.
[000118] The systems provided in this document also provide an optimized ratio between the exchange rate (XR) and the perfusion rate (PR). As a person skilled in the art can appreciate, systems and methods that provide higher XR:PR ratios result in more efficient cell culture production methods (e.g., use less cell culture media during the perfusion process ). In some examples, the exemplary devices and methods herein provide an XR:PR ratio of more than about 2 (e.g., more than about 3, more than about 4, more than about of 5, more than about 6, more than about 7, more than about 8, more than about 9, more than about 10, more than about 11, more than about 12, more than about 13, more than about 14, more than about 15, more than about 16, more than about 17, more than about 18, more than about 19 , more than about 20, more than about 21, more than about 22, more than about 23, more than about 24, more than about 25, more than about 50, more than about 75, more than about 100, more than about 125, more than about 150, more than about 175, more than about 200, more than about 225, more than about 250, more than about 275, more than about 300, more than about 325, more than about 350, more than about 375, more than about 400, more than about 425, more than about 450, more than about 475, more than about 500, more than about 525, greater than about 550, more than about 575, or more than about 600), or between about 5 and about 600 (per for example, between about 10 and about 550, between about 10 and about 500, between about 10 and about 450, between about 10 and about 400, between about 10 and about 350, between about 10 and about 300, between about 10 and about 250, between about 10 and about 200, between about 10 and about 150, between about 10 and about 100, or between about 10 and about 50). Cell Culture Processing Methods
[000119] Methods for processing a cell culture are also provided which include (a) the provision of an open loop filtration system (e.g. any of the open loop filtration systems described in this document), (b ) the flow of the cell culture from the reservoir through the TFF unit in a first flow direction for a first period of time, (c) the reversal of the first flow direction and the flow of the cell culture through the TFF in a second flow direction for a second period of time, (d) reversing the second flow direction and crop flow through the TFF unit in the first flow direction for a third period of time, (e) to repetition of steps (c) - (d) at least two (eg at least three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, fifty, sixty, seventy, eighty , ninety or a hundred, or more than a hundred) times, and (f) the collection of the filtered material. Several exemplary aspects of these methods are described below. cell culture
[000120] The cell culture to be processed in the methods provided herein may contain a plurality of any type of mammalian cell in a liquid culture medium. In some examples of all the methods described herein, the mammal is a cell that grows in suspension culture. In other examples, the mammalian cell is an adherent cell (eg, a cell that requires a solid substrate, such as microcarriers, for growth in a perfusion bioreactor). Non-limiting examples of mammalian cells that may be present in a cell culture include: Chinese hamster ovary (CHO) cells (eg, CHO DG44 cells, CHO H1s cells, Sp2.0, myeloma cells ( eg NS/0), B cells, hybridoma cell, T cells, human embryonic kidney (HEK) cells (eg HEK 293E and HEK 293F), African green monkey kidney epithelial cells (Vero), cells Madin-Darby Canine Kidney Epithelial (Cocker Spaniel) (MDCK) Additional mammalian cells that may be present in a cell culture are known in the art.
[000121] A cell culture processed using any of the methods described herein may contain a viable cell density of more than about 0.5 x 106 cells, more than about 1.0 x 106 cells, more than about 5.0 x 106 cells, more than about 10.0 x 106 cells, more than about 15.0 x 106 cells, more than about 20.0 x 106 cells, more than than about 25.0 x 106 cells, more than about 30.0 x 106 cells, more than about 35.0 x 106 cells, more than about 40.0 x 106 cells, more than about 45.0 x 106 cells, more than about 50.0 x 106 cells, more than about 55.0 x 106 cells, more than about 60.0 x 106 cells, more than about 65 .0 x 106 cells, more than about 70.0 x 106 cells, more than about 75.0 x 106 cells, more than about 80.0 x 106 cells, more than about 85.0 x 106 cells, more than about 90.0 x 106 cells, more than about 95.0 x 10 6 cells, more than about 100.0 x 106 cells, more than about 105.0 x 106 cells, more than about 110.0 x 106 cells, more than about 120.0 x 106 cells , more than about 125.0 x 106 cells, more than about 130.0 x 106 cells, more than about 135.0 x 106 cells, more than about 140.0 x 106 cells, more than about 145.0 x 106 cells, more than about 150.0 x 106 cells, more than about 155.0 x 106 cells, more than about 160.0 x 106 cells, more than about 170.0 x 106 cells, more than about 175.0 x 106 cells, more than about 180.0 x 106 cells, more than about 185.0 x 106 cells, more than about 190.0 x 106 cells, more than about 195.0 x 106 cells, more than about 200.0 x 106 cells, more than about 205.0 x 106 cells, more than about 210, 0 x 106 cells, more than about 215.0 x 106 cells, more than about 220.0 x 106 cells, more than about 22 5.0 x 106 cells, more than about 230.0 x 106 cells, more than about 235.0 x 106 cells, more than about 240.0 x 106 cells, more than about 245, 0 x 106 cells, or more than about 250.0 x 106 cells). In some examples, the cell culture has a viable cell concentration of between about 30 x 106 cells and about 100 x 106 cells (for example, between about 30 x 106 cells and about 95 x 106 cells, between about about 30 x 106 cells and about 90 x 106 cells, between about 30 x 106 cells and about 85 x 106 cells, between about 35 x 106 cells and about 80 x 106 cells, between about 40 x 106 cells and about 80 x 106 cells, between about 40 x 106 cells and about 60 x 106 cells, or between about 60 x 106 cells and about 80 x 106 cells). In some examples, the cell culture has a viable cell concentration of between about 110 x 106 cells and about 250 x 106 cells (for example, between about 110 x 106 cells and about 240 x 106 cells, between about about 110 x 106 cells and about 230 x 106 cells, between about 110 x 106 cells and about 220 x 106 cells, between about 110 x 106 cells and about 210 x 106 cells, between about 110 x 106 cells and about 200 x 106 cells, between about 110 x 106 cells and about 190 x 106 cells, between about 110 x 106 cells and about 180 x 106 cells, between about 110 x 106 cells and about 170 x 106 cells, between about 110 x 106 cells and about 160 x 106 cells, between about 110 x 106 cells and about 150 x 106 cells, between about 110 x 106 cells and about 140 x 106 cells, between about 110 x 106 cells and about 130 x 106 cells, between about 120 x 106 cells and about 250 x 106 cells, between about 120 x 106 c cells and about 240 x 106 cells, between about 120 x 106 cells and about 230 x 106 cells, between about 120 x 106 cells and about 220 x 106 cells, between about 120 x 106 cells and about 210 x 106 cells, between about 120 x 106 cells and about 200 x 106 cells, between about 120 x 106 cells and about 190 x 106 cells, between about 120 x 106 cells and about 180 x 106 cells, between about 120 x 106 cells and about 170 x 106 cells, between about 120 x 106 cells and about 160 x 106 cells, between about 120 x 106 cells and about 150 x 106 cells, between about 120 x 106 cells and about 140 x 106 cells, between about 130 x 106 cells and about 250 x 106 cells, between about 130 x 106 cells and about 240 x 106 cells, between about 130 x 106 cells and about 230 x 106 cells, between about 130 x 106 cells and about 220 x 106 cells, between about 130 x 106 cells and about 210 x 106 cells, between about 130 x 106 cells and about 200 x 106 cells, between about 130 x 106 cells and about 190 x 106 cells, between about 130 x 106 cells and about 180 x 106 cells, between about 130 x 106 cells and about 170 x 106 cells, between about 130 x 106 cells and about 160 x 106 cells, between about 130 x 106 cells and about 150 x 106 cells, between about 140 x 106 cells and about 250 x 106 cells , between about 140 x 106 cells and about 240 x 106 cells, between about 140 x 106 cells and about 230 x 106 cells, between about 140 x 106 cells and about 220 x 106 cells, between about 140 x 106 cells and about 210 x 106 cells, between about 140 x 106 cells and about 200 x 106 cells, between about 140 x 106 cells and about 190 x 106 cells, between about 140 x 106 cells and about 180 x 106 cells, between about 140 x 106 cells and about 170 x 106 cells, between about 140 x 106 cells and about 160 x 106 cells, between approx. that of 150 x 106 cells and about 250 x 106 cells, between about 150 x 106 cells and about 240 x 106 cells, between about 150 x 106 cells and about 230 x 106 cells, between about 150 x 106 and about 220 x 106 cells, between about 150 x 106 cells and about 210 x 106 cells, between about 150 x 106 cells and about 200 x 106 cells, between about 150 x 106 cells and about 190 x 106 cells, between about 150 x 106 cells and about 180 x 106 cells, between about 150 x 106 cells and about 170 x 106 cells, between about 160 x 106 cells and about 250 x 106 cells, between about 160 x 106 cells and about 240 x 106 cells, between about 160 x 106 cells and about 230 x 106 cells, between about 160 x 106 cells and about 220 x 106 cells, between about 160 x 106 cells and about 210 x 106 cells, between about 160 x 106 cells and about 200 x 106 cells, between about 160 x 106 cells and about 190 x 106 cells, between about that of 160 x 106 cells and about 180 x 106 cells, between about 170 x 106 cells and about 250 x 106 cells, between about 170 x 106 cells and about 240 x 106 cells, between about 170 x 106 cells and about 230 x 106 cells, between about 170 x 106 cells and about 220 x 106 cells, between about 170 x 106 cells and about 210 x 106 cells, between about 170 x 106 cells and about 200 x 106 cells, between about 170 x 106 cells and about 190 x 106 cells, between about 180 x 106 cells and about 250 x 106 cells, between about 180 x 106 cells and about 240 x 106 cells, between about 180 x 106 cells and about 230 x 106 cells, between about 180 x 106 cells and about 220 x 106 cells, between about 180 x 106 cells and about 210 x 106 cells, between about 180 x 106 cells and about 200 x 106 cells, between about 190 x 106 cells and about 250 x 106 cells, between about 190 x 106 cells and about 240 x 106 cells, en between about 190 x 106 cells and about 230 x 106 cells, between about 190 x 106 cells and about 220 x 106 cells, between about 190 x 106 cells and about 210 x 106 cells, between about 200 x 106 cells and about 250 x 106 cells, between about 200 x 106 cells and about 240 x 106 cells, between about 200 x 106 cells and about 230 x 106 cells, between about 200 x 106 cells and about 220 x 106 cells, between about 210 x 106 cells and about 250 x 106 cells, between about 210 x 106 cells and about 240 x 106 cells, between about 220 x 106 cells and about 240 x 106 cells, or between about 230 x 106 cells and about 250 x 106 cells).
[000122] The total amount of cell culture in the system (with the exception of the filtered material conduit and the filtered material containment tank) can be between 0.2 liter and about 10,000 liters (for example, between about 0, 2 liter and about 9,500 liters, between about 0.2 liter and about 9,000 liters, between about 0.2 liter and about 8,500 liters, between about 0.2 liter and about 8,000 liters, between about 0.2 liter and about 7,500 liters, between about 0.2 liter and about 7,000 liters, between about 0.2 liter and about 6,500 liters, between about 0.2 liter and about 6,500 liters, between about 0.2 liter and about 6,000 liters, between about 0.2 liter and 5,500 liters, between about 0.2 liter and about 5,000 liters, between about 0.2 liter and about 4,500 liters, between about 0.2 liter and about 4,000 liters, between about 0.2 liter and about 3,500 liters, between about 0.2 liter and about 3,000 liters, between about 0.2 liter and about 2.50 0 liters, between about 0.2 liter and about 2,000 liters, between about 0.2 liter and about 1,500 liters, between about 0.2 liter and about 1,000 liters, between about 0.2 liter and about 500 liters, between about 0.2 liter and about 400 liters, between about 0.2 liter and about 300 liters, between about 0.2 liter and about 200 liters, between about 0.2 liter and about 150 liters, between about 0.2 liter and about 100 liters, between about 0.2 liter and about 50 liters, or between about 0.2 liter and about 10 liters).
[000123] Mammalian cells present in a cell culture may contain a recombinant nucleic acid (for example, a nucleic acid stably integrated into the genome of the mammalian cell) that encodes a recombinant protein (for example, a recombinant protein that is secreted by the mammalian cell). A nucleic acid encoding a recombinant protein can be introduced into a mammalian cell using a wide variety of methods known in molecular biology and molecular genetics. Non-limiting examples include transfection (eg lipofection), transduction (eg lentivirus, adenovirus or retrovirus infection), and electroporation. In some examples, nucleic acid encoding a recombinant protein is not stably integrated into a mammalian cell chromosome (transient transfection), whereas in others the nucleic acid is integrated. Alternatively or additionally, nucleic acid encoding a recombinant protein may be present on a plasmid and/or a mammalian artificial chromosome (e.g., a human artificial chromosome). Alternatively or additionally, the nucleic acid can be introduced into the cell using a viral vector (for example, a lentivirus, retrovirus or adenovirus vector). Nucleic acid can be operably linked to a promoter sequence (for example, a strong promoter, such as a β-actin promoter and a CMV promoter, or an inducible promoter). A nucleic acid sequence encoding a soluble recombinant protein may contain a sequence encoding a secretion signal peptide at the N- or C-terminus of the recombinant protein, which is cleaved by an enzyme present in the mammalian cell, and subsequently released into the medium of culture. A vector that contains the nucleic acid can also, if desired, contain a selectable marker (for example, a gene that confers resistance to hygromycin, puromycin or neomycin to the mammalian cell).
[000124] Non-limiting examples of recombinant proteins that can be secreted by mammalian cells in cell culture include immunoglobulins (including heavy and light chain immunoglobulins, antibodies, or antibody fragments (for example, any of the antibody fragments). described herein), enzymes (for example, a galactosidase (for example, an alpha-galactosidase), Miozyme, or Cerezyme), proteins (for example, human erythropoietin, tumor necrosis factor (TNF), or an alpha or interferon beta), or immunogenic or antigenic proteins or protein fragments (eg, proteins for use in a vaccine). multifunctional recombinant protein (see, for example, the recombinant antigen binding proteins described in Gebauer et al., Current Opin. Chem. Biol. 13:245 -255, 2009; and U.S. Patent Application Publication no. 201210164066 (incorporated by way of reference in this document in its entirety)). Non-limiting examples of the recombinant proteins that are antibodies include: panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab, alirocumabumabumabuma, altumomab, alirocumabumabumabumab basilizimab, bectumomab, belimumab, bevacizumab, biciromab, canakinumab, cetuximab, daclizumab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab, etaracizumab, daclizumab, stubumabximab, golimumabximab, golimumabximab, golimumabuma, . Additional examples of therapeutic antibodies that can be produced by the methods described herein are known in the art. Additional non-limiting examples of recombinant proteins that can be secreted by mammalian cells in cell culture include: alpha alglucosidase, laronidase, abatacept, galsulfase, alpha lutropin, anti-hemophilic factor, beta agalsidase, interferon beta-1a, darbepoetin alpha, tenecteplase, etanercept, coagulation factor IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alpha, epoetin alpha, and alteplase.
[000125] Liquid culture media are known in the state of the art. The liquid culture medium can be supplemented with a mammalian serum (eg, calf serum and fetal bovine serum), and/or a growth hormone or growth factor (eg, insulin, transferrin and factor of epidermal growth). Alternatively or additionally, the liquid culture medium may be a chemically defined liquid culture medium, an animal-derived component-free liquid culture medium, a serum-free liquid culture medium, or a serum-containing liquid culture medium. Examples of chemically defined liquid culture media, liquid culture media free from animal-derived components, liquid culture media free from serum, and liquid culture media containing serum are commercially available.
[000126] A liquid culture medium typically contains a source of energy (eg a carbohydrate such as glucose), essential amino acids (eg the basic set of twenty amino acids plus cysteine), vitamins and/or other compounds organics required at low concentrations, free fatty acids, and/or trace elements. The liquid culture medium may, if desired, for example be supplemented with a hormone or mammalian growth factor (for example, insulin, transferrin, or epidermal growth factor), salts and buffers (for example, salts of calcium, magnesium and phosphate), nucleosides and bases (eg adenosine, thymidine and hypoxanthine), protein and tissue hydrolysates, and/or any combination of these or other additives.
Non-limiting examples of liquid culture media include, for example, CD CHO, Opti CHO, and Forti CHO (all available from Life Technologies; Grand Island, NY), Hycell CHO medium (Thermo Fisher Scientific, Inc.; Waltham, MA), Ex-cell CD CHO Fusion medium (Sigma-Aldrich Co.; St. Louis, MO), and PowerCHO medium (Lonza Group, Ltd.; Basel, Switzerland). Media components that may also be present in a liquid culture medium include, but are not limited to, chemically defined hydrolysates (CD), eg, CD peptone, CD polypeptides (two or more amino acids), and growth factors CD. Additional examples of liquid tissue culture medium and medium components are known in the prior art.
[000128] A cell culture containing adherent mammalian cells can be grown in a perfusion bioreactor using, for example, microcarriers. Non-limiting exemplary microcarriers that can be used include CytoPoreTM 1 and CytoPoreTM 2 (available from GE Healthcare, Life Sciences, Piscataway, New Jersey). Additional examples of microcarriers that can be used are publicly available and known in the art. Use of Exemplary Open Circuit Filtration Systems
[000129] Any of the open circuit filtration systems described in this document can be used in the methods provided to process a cell culture. For example, the bioreactor in the open loop filtration system used in the methods described herein can be a bioreactor (for example any perfusion bioreactor known in the art) or a refrigerated containment tank. The open loop filtration system used in the methods may include one or more conduits (e.g., the first conduit, the second conduit, one or more conduits between neighboring TFF units, and/or the filtered material conduit) that is / are a biocompatible pipe. In some examples, the open loop filtration system contains a reservoir and two or more subsystems (as described in this document).
[000130] The open circuit filtration systems used in the methods may include a TFF unit with a single crossflow filter (eg a tubular crossflow filter) or two or more (eg two, three, four or five) cross-flow filters (e.g. tubular cross-flow filters) as described herein. In other examples, the open circuit filtration systems used may include two or more (e.g. two, three, or four) TFF units, where each pair of neighboring TFF units is fluidly connected by a duct of fluid. TFF units can provide a total filtration area of between about 0.1 m2 to about 150 m2 (for example, between about 0.1 m2 to about 145 m2, between about 0.1 m2 and 140 m2 , between about 0.1 m2 and about 135 m2, between about 0.1 m2 and about 130 m2, between about 0.1 m2 and about 125 m2, between about 0.1 m2 and about 120 m2, between about 0.1 m2 and about 115 m2, between about 0.1 m2 and about 110 m2, between about 0.1 m2 and about 105 m2, between about 0.1 m2 and about 100 m2, between about 0.1 m2 and about 95 m2, between about 0.1 m2 and about 90 m2, between about 0.1 m2 and about 85 m2, between about 0.1 m2 and 80 m2, between about 0.1 m2 and 75 m2, between about 0.1 m2 and about 70 m2, between about 0.1 m2 and about 65 m2, between about 0.1 m2 and 60 m2, between about 0.1 m2 and about 55 m2, between about 0.1 m2 and about 50 m2, between about 0.1 m2 and about 45 m2, between about 0.1 m2 and about 40 m2, between about 0.1 m2 and about 35 m2, between about 0.1 m2 and about 30 m2, between about 0.1 m2 and about 25 m2, between about 0.1 m2 and about 20 m2, between about 0.1 m2 and about 15 m2, between about 0.1 m2 and about 10 m2, or between about 0.1 m2 and about 5 m2). The filter(s) present in a TFF unit can have any combination of pore sizes (eg about 0.2 µm), shapes, fiber inner diameters and/or fiber lengths. described in this document.
[000131] The open circuit filtration systems used herein may include at least one pump disposed in the first conduit or in the second conduit, or in both. At least one pump may also be disposed in one or more of the conduits in the system (e.g., in one or more of the first conduit, the second conduit, and/or one or more conduits between neighboring TFF units). The system used may include at least one pump disposed in the reservoir and close to the first or second conduits (for example, a distance between 0.01 cm to 5 cm (for example, between 0.01 cm and 4 cm, between 0, 01 cm and 3 cm, between 0.01 cm and 2 cm, or between 0.01 cm and 1 cm) from the pump to position where the first conduit or second conduit connects with the bioreactor). Some systems include only a single pump that drives cell culture in the first direction during the first and third time periods, and drives cell culture in the second direction during the second time period. Other systems include a first and a second pump, where the first pump drives the cell culture in the first direction and the second pump drives the cell culture in the second direction.
[000132] In any of the systems used in the methods, at least one pump (for example, one, two, three or four pumps) can be an LTP (for example, any of the LTPs described in this document, such as a pump peristaltic). At least one pump (eg, at least one LTP) present in the system used in the methods can have any combination of the uniqueness or characteristics of the pump (eg, LTPs) described herein (eg, pump column volume , the type and/or the piping). In either method, at least one pump is used at a pump speed (rpm) between about 10 rpm and about 100 rpm (for example, between about 10 rpm and about 95 rpm, between about 10 rpm and about 90 rpm, between about 10 rpm and about 85 rpm, between about 10 rpm and about 80 rpm, between about 10 rpm and about 75 rpm, between about 10 rpm and about 70 rpm, between about 10 rpm and about 65 rpm, between about 10 rpm and about 60 rpm, between about 10 rpm and about 55 rpm, between about 10 rpm and about 50 rpm, between about 10 rpm and about 45 rpm, between about 10 rpm and about 40 rpm, between about 10 rpm and about 35 rpm, between about 10 rpm and about 30 rpm, between about 10 rpm and about 25 rpm, or between about 10 rpm and about 20 rpm). In some examples, the methods result in a perfusion flow between about 0.5 l/m2/hr to about 40 l/m2/hr, between about 0.5 l/m2/hr to about 35 l/ m2/hour, between about 0.5 l/m2/hour to about 30 l/m2/hour, between about 0.5 l/m2/hour and about 25 l/m2/hour, between about 0 .5 l/m2/hour to about 20 l/m2/hour, between about 0.5 l/m2/hour to about 15 l/m2/hour, between about 0.5 l/m2/hour to about 10 l/m2/hour, between about 0.5 l/m2/hour to about 9 l/m2/hour, between about 0.5 l/m2/hour to about 8 l/m2/hour , between about 0.5 l/m2/hour to about 7 l/m2/hour, between about 0.5 l/m2/hour to about 6 l/m2/hour, between about 0.5 l /m2/hour to about 5 l/m2/hour, between about 0.5 l/m2/hour to about 4 l/m2/hour, between about 0.5 l/m2/hour to about 3 l/m2/hour, between about 0.5 l/m2/hour to about 2 l/m2/hour, or between about 0.8 l/m2/hour to about 1.2 l/m2/hour ). In some examples, the use of at least one pump results in a system shear rate of between about 50 s-1 to about 1000 s-1, for example, between about 50 s-1 to about 950 s- 1, between about 50 s-1 to about 900 s-1, between about 50 s-1 to about 850 s-1, between about 50 s-1 to about 800 s-1, between about 50 s-1 to about 750 s-1, between about 50 s-1 to about 700 s-1, between about 50 s-1 to about 650 s-1, between about 50 s-1 to about 600 s-1, between about 50 s-1 to about 550 s-1, between about 50 s-1 to about 500 s-1, between about s-1 to about 450 s-1 , between about 50 s-1 to about 400 s-1, between about 50 s-1 to about 350 s-1, between about 50 s-1 to about 300 s-1, between about 50 s-1 to about 250 s-1, between about 50 s-1 to about 200 s-1, between about 50 s-1 to about 150 s-1, or between about 50 s-1 to about 100 s-1). Specific examples of pumps that can be used in these methods include a Watson-Marlow 620 peristaltic pump with 16 mm tubing or a Watson-Marlow 800 peristaltic pump with 40 mm tubing.
[000133] As a person skilled in the art can appreciate, the total volume of cell culture in the system (excluding the volume of filtered material in the filtered material conduit and in the filtered material containment tank), of the total filtration area provided for at least one TFF unit, and the throughput (eg, in the second and third time periods) must be run at a reasonable rate (eg, the exemplary values and parameters described in this document) that provides for one or more benefits of the systems and methods presently provided. Flow Cycle
[000134] In the methods described herein, the first, second and/or third time periods can be between about 20 seconds and about 15 minutes (for example, between about 30 seconds and about 15 minutes, between about 20 seconds and about 14 minutes, between about 20 seconds and about 13 minutes, between about 20 seconds and about 12 minutes, between about 20 seconds and about 11 minutes, between about 20 seconds and about 10 minutes, between about 20 seconds and about 9 minutes, between about 20 seconds and about 8 minutes, between about 20 seconds and about 7 minutes, between about 20 seconds and about 6 minutes, between about 20 seconds and about 5 minutes, between about 20 seconds and about 4 minutes, between about 20 seconds and about 3 minutes, between about 20 seconds and about 2 minutes, between about 20 seconds and about 115 seconds, between about 20 seconds and about 110 seconds, between about 20 seconds and 105 seconds, between about 20 seconds and about 100 seconds, between about 20 seconds and about 95 seconds, between about 20 seconds and about 90 seconds, between about 20 seconds and about 85 seconds, between about 20 seconds and about 80 seconds, between about 20 seconds and about 75 seconds, between about 20 seconds and about 70 seconds, between about 20 seconds and about 65 seconds, between about 20 seconds and about 60 seconds, between about 20 seconds and about 55 seconds, between about 20 seconds and about 50 seconds, between about 20 seconds and about 45 seconds, between about 20 seconds and about 40 seconds, between about 20 seconds and about 35 seconds, between about 20 seconds and about 30 seconds, between about 20 seconds and about 25 seconds, between about 30 seconds and about 90 seconds, between about 35 seconds and about 85 seconds, between about 40 seconds and about 80 seconds, between about 45 seconds and about 75 seconds, between about 50 seconds and about 70 seconds, between about 55 seconds and about 65 seconds, between about 30 seconds and 14 minutes, between about 30 seconds and 13 minutes, between about 30 seconds and about 12 minutes, between about 30 seconds and about 11 minutes, between about 30 seconds and about 10 minutes, between about 30 seconds and about 9 minutes, between about 30 seconds and about 8 minutes, between about 30 seconds and about 7 minutes, between about 30 seconds and about 6 minutes, between about 30 seconds and about 5 minutes, between about 30 seconds and about 4 minutes, between about 30 seconds and about 3 minutes, between about 30 seconds and about 2 minutes, between about 30 seconds and about 90 seconds, between about 30 seconds and about 1 minute, between about 1 minute and about 15 minutes, between about 1 minute and about 14 minutes, between about 15 minutes and about 13 minutes between about 1 minute and about 12 minutes, between about 1 minute and about 11 minutes, between about 1 minute and about 10 minutes, between about 1 minute and about 9 minutes, between about 1 minute and about 8 minutes, between about 1 minute and about 7 minutes, between about 1 minute and about 6 minutes, between about 1 minute and about 5 minutes, between about 1 minute and about 4 minutes , between about 1 minute and about 3 minutes, between about 1 minute and about 2 minutes, or between about 1 minute and about 90 seconds). In some instances, the first, second, and third time periods are more or less identical. In other examples, the first, second, and third time periods are not the same.
[000135] In some examples, the first flow direction in the first period of time flows cell culture from the reservoir through the first or second conduit in which at least one pump is arranged (eg a single pump), then through of at least one TFF unit, and then back to the reservoir through the other conduit (for example, for a period of between about 30 seconds and about 60 minutes, between about 30 seconds and about 50 minutes, between about between about 30 seconds and about 40 minutes, between about 30 seconds and about 30 minutes, between about 30 seconds and about 20 minutes, between about 30 seconds and about 15 minutes, between about 30 seconds and about 10 minutes, or between about 30 seconds and about 5 minutes). In such examples, the flow during the first time period is used to balance at least one TFF unit of the system (and at least one cross-flow filter therein). FIGURE 8 is a schematic diagram showing cell culture flow in the first flow direction for the purpose of balancing at least one TFF unit in the system.
[000136] FIGURE 9 shows an example of reservoir cell culture flow through the TFF unit in a first flow direction for a first period of time (t1), the reversal of the first flow direction for a period of time (tr1) and the flow of the cell culture through the TFF unit in a second flow direction for a second period of time (t2), the inversion of the second flow direction for a period of time (tr2) and the flow of culture through the TFF unit in the first flow direction for a third period of time (t3). For example, tr1 and/or tr2 can be between about 1 second and about 1 minute (for example, between about 1 second and about 55 seconds, between about 1 second and about 50 seconds, between about 1 second and about 45 seconds, between about 1 second and about 40 seconds, between about 1 second and about 35 seconds, between about 1 second and about 30 seconds, between about 1 second and about 25 seconds , between about 1 second and about 20 seconds, between about 1 second and about 15 seconds, between about 1 second and about 10 seconds, between about 1 second and about 5 seconds, between about 5 seconds and about 60 seconds, between about 5 seconds and about 55 seconds, between about 5 seconds and about 50 seconds, between about 5 seconds and about 45 seconds, between about 5 seconds and about 40 seconds, between about 5 seconds and about 35 seconds, between about 5 seconds and about 30 seconds, between about 5 seconds and about 25 seconds, between about 5 seconds and about 20 seconds, between about 5 seconds and about 15 seconds, between about 5 seconds and about 10 seconds, or between about 2 seconds and about 10 seconds, between about 2 seconds and about 8 seconds, between about 2 seconds and about 6 seconds, or between about 2 seconds and about 4 seconds).
[000137] Flow in the first and/or second directions (for example any one of the first, second and/or third time periods) can result in a flow between about 0.5 l/minute to about 120 l/minute (for example, between about 0.5 l/minute to about 115 l/minute, between about 0.5 l/minute to about 110 l/minute, between about 0.5 l/minute, minute to about 105 l/minute, between about 0.5 l/minute to about 100 l/minute, between about 0.5 l/minute to about 95 l/minute, between about 0.5 l /minute to about 90 l/minute, between about 0.5 l/minute to about 85 l/minute, between about 0.5 l/minute to about 80 l/minute, between about 0.5 l/minute to about 75 l/minute, between about 0.5 l/minute to about 70 l/minute, between about 0.1 l/minute to about 65 l/minute, between about 0. 1 l/minute to about 60 l/minute, between about 0.1 l/minute to about 55 l/minute, between about 0.1 l/minute to about 50 l/minute, between about 0 .1 l/minute to approx. at 45 l/minute, between about 0.1 l/minute to about 40 l/minute, between about 0.1 l/minute to about 35 l/minute, between about 0.1 l/minute at about 30 l/minute, between about 0.1 l/minute to about 25 l/minute, between about 0.1 l/minute to about 20 l/minute, between about 0.1 l/minute, minute to about 15 l/minute, between about 0.1 l/minute to about 10 l/minute, or between about 0.1 l/minute to about 5 l/minute).
[000138] The simple iteration of (i) cell culture flow in the first flow direction for the first period of time and (ii) cell culture flow in the second flow direction for the second period of time can result in a fraction of exchange between about 40% to about 95% (for example, between about 40% to about 90%, between about 40% to about 85%, between about 40% to about 80%, between about 40% to about 75%, between about 45% to about 80%, between about 50% to about 80%, between about 55% to about 75%, between about 60% and about 85%, between about 70% and about 95%, or between about 70% and about 85%).
[000139] In the methods provided in this document, the volume of cell culture in the system (with the exception of the filtered material conduit, the filtered material containment tank and/or the biological manufacturing system) can be between about 0. 1 liter and about 50 liters (for example, between about 0.1 liter and about 45 liters, between about 0.1 liter and about 40 liters, between about 0.1 liter and about 35 liters, between about 0.1 liter and about 30 liters, between about 0.1 liter and about 25 liters, between about 0.1 liter and about 20 liters, between about 0.1 liter and about 18 liters, between about 0.1 liter and about 16 liters, between about 0.1 liter and about 14 liters, between about 0.1 liter and about 12 liters, between about 0.1 liter and about 10 liters, between about 0.1 liter and about 8 liters, between about 0.1 liter and about 6 liters, between about 0.1 liter and about 4 liters, between about 0.1 liter and about 3 liters, en between about 0.1 liter and about 2 liters, or between about 0.1 liter and about 1 liter). The amount of time the cell culture spends outside the reservoir (eg, in the perfusion bioreactor) in the methods described herein can be between 5 seconds to 45 seconds (eg, between about 5 seconds and about 40 seconds, between about 5 seconds and about 35 seconds, between about 5 seconds and about 30 seconds, between about 5 seconds and about 25 seconds, between about 5 seconds and about 20 seconds, between about 5 seconds and about 15 seconds, between about 5 seconds and about 10 seconds).
[000140] Some embodiments of the methods provided herein produce a filtered material that does not contain a mammalian cell. The methods provided herein can also produce a filtered material that contains a secreted recombinant protein (e.g., an antibody or antigen-binding fragment thereof, a growth factor, a cytokine, or an enzyme) from a culture of cells that contain the secreted recombinant protein. In some embodiments, the cell culture and/or filtered material is sterile.
[000141] The present methods can be scaled up or down to filter a larger volume of cell culture per unit of time. As will be appreciated by those skilled in the art, a larger volume of cell culture can be processed per unit of time by incorporating at least one pump with a larger pump column volume and larger tubing and/or a number greater number of cross-flow filters in the TFF unit(s) or a greater number of TFF units (eg a larger total filtration area). These changes can be implemented in the open circuit filtration system used to perform the methods described in this document and can be tested to ensure that the larger scale system has one or more (eg two, three, four, five, six or seven) of the following benefits: decreased cell culture external volume (outside the reservoir), increased exchange fraction (eg, within the first conduit, TFF unit and second conduit), external culture residence time cell count (outside the reservoir), decreased shear stress during cell culture filtration, improved cell viability in cell culture, high viable cell density in cell culture, and decreased filter fouling compared to other systems one-way open circuit filtration systems (eg, one-way TFF systems) or bidirectional closed-loop filtration systems (circuit ATFTM systems). I am closed). Examples of the physical and functional parameters of three different exemplifying methods and the open circuit filtration systems used to perform each method are shown in Table 2 (below).
[000142] Any of the methods described herein can be carried out continuously for a period between about 14 days and about 100 days (for example, between about 14 days and about 90 days, between about 14 days and about 100 days 80 days, between about 14 days and about 70 days, between about 14 days and about 60 days, between about 14 days and about 50 days, between about 14 days and about 40 days, between about between about 14 days and about 30 days, between about 14 days and about 20 days, between about 20 days and about 100 days, between about 20 days and about 90 days, between about 20 days and about 80 days, between about 20 days and about 70 days, between about 20 days and about 60 days, between about 20 days and about 50 days, between about 20 days and about 40 days, between about 20 days and about 30 days, between about 30 days and about 100 days, between about 30 days and about 90 days, between about 30 days and about 80 days, between about 30 days and about 70 days, between about 30 days and about 60 days, between about 30 days and about 50 days, between about 30 days and about 40 days, between about 40 days and about 100 days, between about 50 days and about 90 days, between about 50 days and about 80 days, between about 50 days and about 70 days, between about 50 days and about 60 days, between about 60 days and about 100 days, between about 60 days and about 90 days, between about 60 days and about 80 days, or between about 60 days and about 70 days). Table 2. Parameters of three different sample methods and the system used to execute each method

[000143] In some embodiments, the change in pressure across the filter fibers in one or more cross-flow filters in at least one TFF unit and/or the change in pressure across the filter membrane in one or more cross-flow filters cross flow in at least one TFF unit remain substantially the same (eg, within about ±20%, within about ±19%, within about ±18%, within about ±17%, within of about ± 16%, within about ± 15%, within about ± 14%, within about ± 13%, within about ± 12%, within about ± 11%, within about within about ±10%, within about ±9%, within about ±8%, within about ±7%, within about ±6%, within about ±5%, within about ±4 %, within about ± 3%, within about ± 2.5%, within about ± 2.0%, within about ± 1.5%, within about ± 1.0%, or within about ± 0.5% of the initial change in pressure through the filter fibers or through of the filter membrane at the beginning of the method) during the execution of the method for a period of about, for example, between about 1 hour and about 100 days (for example, between about 1 hour and about 95 days, between about 1 hour and about 90 days, between about 1 hour and about 90 days, between about 1 hour and about 85 days, between about 1 hour and about 80 days, between about 1 hour and about 75 days, between about 1 hour and about 70 days, between about 1 hour and about 65 days, between about 1 hour and about 60 days, between about 1 hour and about 55 days, between about between about 1 hour and about 50 days, between about 1 hour and about 45 days, between about 1 hour and about 40 days, between about 1 hour and about 35 days, between about 1 hour and about 30 days, between about 1 hour and about 25 days, between about 1 hour and about 20 days, between about 1 hour and about 15 days, between about 1 hour and about 10 days, between about 1 hour and about 5 days, between about 1 day and about 100 days, between about 1 day and about 90 days, between about 1 day and about 85 days, between about 1 day and about 80 days, between about 1 day and about 75 days, between about 1 day and about 70 days, between about 1 day and about 65 days, between about 1 day and about 60 days, between about 1 day and about 55 days, between about 1 day and about 50 days, between about 1 day and about 45 days, between about 1 day and about 40 days, between about 1 day and about 35 days, between about 1 day and about 30 days, between about 1 day and about 25 days, between about 1 day and about 20 days, between about 1 day and about 15 days, between about 1 day and about 10 days , between about 5 days and about 100 days, between about 5 days and about 95 days, between about 5 days and about 90 days, between about 5 days and about 85 days, between about 5 days and about 80 days, between about 5 days and about 75 days, between about 5 days and about 70 days, between about 5 days and about 65 days, between about 5 days and about 60 days, between about 5 days and about 55 days, between about 5 days and about 50 days, between about 5 days and about 45 days, between about 5 days and about 40 days, between about 5 days and about 35 days, between about 5 days and about 30 days, between about 5 days and about 25 days, between about 5 days and about 20 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 100 days, between about 10 days and about 95 days, between about 10 days and about 90 days, between about 10 days and about 85 days, between about 10 days and about 80 days, between about between about 10 days and about 75 days, between about 10 days and about 70 days, between about 10 days and about 65 days, between about 10 days and about 60 days, between about 10 days and about 55 days, between about 10 days and about 50 days, between about 10 days and about 45 days, between about 10 days and about 40 days, between about 10 days and about 35 days, between about 10 days and about 30 days, between about 10 days and about 25 days, between about 10 days and about 20 days, between about 15 days and about 100 days, between about 15 days and about 95 days, between about 15 days and about 90 days, between about 15 days and about 85 days, between about 15 days and about 80 days, between about 15 days and about 75 days, between about 15 days and about 70 days, between about 15 days and about 65 days, between about 15 days and about 60 days, between about 15 days and about 55 days, between about 15 days and about 50 days, between about 15 days and about 45 days, between about 15 days and about 40 days, between about 15 days and about 35 days, between about 15 days and about 30 days, between about 15 days and about 25 days, or between about 15 days and about 20 days). A significant increase in the change in pressure across the filter fiber or filter membrane indicates dirt in at least one cross-flow filter in at least one TFF unit in the system. Incubation of cell culture in reservoir
[000144] Some modalities also include incubating the cell culture in the reservoir (eg, in the perfusion bioreactor) under conditions that allow the mammalian cell to secrete a recombinant protein into the tissue culture medium. For example, the cell culture in the reservoir can be incubated at a temperature from about 32°C to about 39°C. Those skilled in the art will appreciate that the temperature can be changed at the specific time point(s) during incubation (eg, on an hourly or daily basis). For example, the temperature can be changed or changed (eg, raised or lowered) in about one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days , eleven days, twelve days, fourteen days, fifteen days, sixteen days, seventeen days, eighteen days, nineteen days, or approximately twenty days or more after placing the cell culture in the reservoir). For example, the temperature can be changed upwards (eg a change of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0 .8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 , 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0°C). For example, the temperature can be changed to low (eg a change of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0 .8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 , 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10°C). Incubation of cell culture in a reservoir can also be carried out in an atmosphere that contains at most or so about 1% to 15% CO2 (eg at most or so about 14% CO2, 12% CO2 , 10% CO2, 8% CO2, 6% CO2, 5%, 4% CO2, 3% CO2, 2% CO2, or at most or else about 1% CO2). In addition, any of the methods described herein may include incubating the cell culture in a humidified atmosphere (eg, at least or so about 20%, 30%, 40%, 50%, 60%, 70% , 85%, 80%, 85%, 90%, or at least or so about 95% humidity, or about 100% humidity).
[000145] The incubation of cell culture in a reservoir (for example, a perfusion bioreactor) during the reiteration of the first, second and third time periods, may include a step of adding a volume of liquid culture medium to the bioreactor . For example, adding a volume of liquid culture medium to the bioreactor can offset the loss of liquid culture medium leaving the system as filtered material. The addition of liquid culture medium to the reservoir can be performed continuously or periodically (eg, once every third day, once every other day, once a day, twice a day, three times a day, four times a day, five times a day, or more than five times a day), or any combination of these. The volume of liquid culture medium added to the reservoir can in some cases be executed in such a way that the initial volume of cell culture in the system (excluding the volume of filtered material present in the filtered material conduit and in the filtered material containment tank ) is more or less the same for every 24-hour period or for the entire period that the method is run. As is known in the prior art, the rate at which the liquid culture medium is removed from the system as filtered material (volume/time unit) and the rate at which the volume of liquid culture medium is added to the reservoir (volume /time unit) can be varied. The rate at which the liquid culture medium is removed from the system as filtered material (volume/time unit) and the rate at which the liquid culture medium volume is added (volume/time unit) can be more or less identical or they may be different.
[000146] Alternatively, the volume removed from the system as filtered material and the volume added to the reservoir may change (eg gradually increase) in each 24-hour period (or, alternatively, an incremental time period between 0.1 hour and about 24 hours or an incremental time period of more than 24 hours) during method execution. For example the volume of liquid culture medium removed from the system as filtered material and the volume of liquid culture medium added within each 24 hour period (or alternatively an incremental time period between about 1 hour and above 24 hours or an incremental time period of more than 24 hours) in the execution of the method can be increased (eg, gradually or through alternating increments), for example, from a volume that is between 0.5% to about 20% from the reservoir volume or total cell culture volume at the beginning of the method run to about 25% to about 150% of the reservoir volume or the total cell culture volume at the beginning of the method run. As can be appreciated by one of skill in the art, within each 24 hour period, the volume removed from the system as filtered material and the volume added to the reservoir is preferably from about 100% to about 400% (e.g. between about 100% and about 350%, between about 100% and about 300%, between about 100% and about 250%, between about 100% and about 200%, between about 100% and about 150%, between about 150% and about 400%, between about 150% and about 350%, between about 150% and about 300%, between about 150% and about 250%, between about between about 150% and about 200%, between about 200% and about 400%, between about 200% and about 350%, between about 200% and about 300%, or between about 200% and about 25% of the reservoir volume or the total cell culture volume at the beginning of the method run.
[000147] Those skilled in the art will appreciate that the liquid culture medium removed from the system as filtered material and the liquid culture medium added to the reservoir can be of the same type of medium. In other examples, the liquid culture medium removed from the system as filtered material and the liquid culture medium added to the reservoir may be substantially different. The volume of liquid culture medium can be added either manually or using an automated system, eg by a perfusion pump. Isolation of recombinant protein from filtered material
[000148] Any of the methods described herein may further include a step of isolating the secreted recombinant protein (for example, any of the recombinant proteins described herein) from the filtered material. Many methods for isolating a polypeptide (for example, a secreted polypeptide) from a fluid are known in the art. For example, methods for isolating a recombinant protein can include one or more steps of: capturing, purifying, polishing and/or filtering a fluid containing the recombinant protein. As is well known in the art, the specific methods used to isolate a recombinant protein will depend on the biophysical properties of the recombinant protein. For example, a recombinant antibody can be purified using, in part, an antibody capture step using a protein A resin.
[000149] In some examples, a recombinant protein present in the filtered material is isolated using an integrated and continuous process that includes isolation through at least one multi-column chromatography (MCCS) system (eg, any one of or plus MCCS described in this document). The integrated and continuous process can be performed using any of the exemplary biological manufacturing systems described in this document. Exemplary integrated and continuous processes for isolating a recombinant protein and biological manufacturing systems to be used in such processes are described in U.S. Patent Application no. Serial 61/775,060, filed March 8, 2013, and in U.S. Patent Application no. Serial 61/856,390, filed July 19, 2013.
[000150] The resulting isolated recombinant protein may be at least or else about 50% by weight pure, for example at least or else about 55% by weight pure, at least 60% by weight pure, at least 65% by pure weight, at least 70% by weight pure, at least 75% by weight pure, at least 80% by weight pure, at least 85% by weight pure, at least 90% by weight pure, at least 95% by weight pure , at least 96% by weight pure, at least 97% by weight pure, at least 98% by weight pure, or at least or about 99% by weight pure, or more than 99% by weight pure.
[000151] Some methods also include a step of formulating a therapeutic drug substance by mixing the isolated recombinant protein with a pharmaceutically acceptable excipient or buffer. Mixing can be performed by mixing a fluid containing the isolated recombinant protein with a buffered solution. In other examples, mixing can be performed by adding a solid buffering agent to a fluid containing the recombinant protein isolated with a buffered solution. Another form of mixing, as encompassed herein, is the dissolution of a solid composition (for example, a lyophilized powder or a cake) containing the isolated recombinant protein with a buffered solution (for example, injectable sterile saline solution). The therapeutic drug substance can be formulated for any route of administration known in the art (for example, oral administration, intravenous administration, intra-arterial administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, intrathecal administration, or inhalation). EXAMPLES
[000152] The invention is also described in the following examples, which do not limit the scope of the invention described in the claims. Example 1. Comparison of processing achieved by open circuit filtration systems provided in this document versus processing achieved by ATF™ (Refine Technology)
[000153] A set of experiments was carried out to compare the cell culture processing obtained by an open circuit filtration system provided herein with the cell culture processing obtained by ATPTM (Refine Technology) (a tangential filtration system closed circuit alternating flow). The device used to perform these experiments is generally described in FIGURE 5. Specifically, the reservoir used in the open circuit filtration system is a 15 liter Broadly-James bioreactor, where the first conduit and the second conduit consist of a Biocompatible weldable transfer tubing with an inner diameter of 0.5 inch, the TPP unit contains a single tubular cross-flow filter (composed of polyether sulfone fibers with a length of 30 cm and an inner diameter of 1 mm, and with an average pore size of 0.2 µm, a fiber density of 830 fibers/filter, and a filtration area of 0.77 m2), at least one pump is a single Watson-Marlow peristaltic pump capable of pushing a fluid in the first and second flow directions, with a pump column volume between 50 ml to 100 ml with a dual-channel GORE Sta-Pure tubing that has an inner diameter of 16 mm and a wall diameter of 4 mm. Materials and methods
[000154] A summary of the experimental parameters used for the comparison of the processing obtained by the presently provided open circuit filtration systems and ATPTM by Refine Technology is summarized in Tables 3 and 4 (below). A further detailed summary of the methods used to perform these experiments is provided below. Table 3. Experimental Parameters


[000155] The conditions used to operate the perfusion bioreactor are listed in Table 3. The bioreactors were maintained at 40 x 106 cell with 10 liters working volume and 2 reactors/day replacement volume by means of CD-CHO culture . The tested open circuit filtration system provided in this document contained the same filter and casing as the ATF4, but used a Watson-Marlow 620 Du peristaltic pump with a pump column volume between 50 ml to 100 ml as a recirculation pump of culture to reversibly flow cell culture through the system (shown in FIGURE 5) and an open loop system (rather than a closed system used in ATF4). The perfusion rate of the ATF4 bioreactor was changed from a volume of 2 reactors/day to a volume of 1 reactor/day on the 20th day of culture, while the tested open circuit filtration system provided in this document was changed from a rate of perfusion from the volume of 2 reactors/day to the volume of 1 reactor/day on the 32nd day, and 10% Efficient Feed B (Gibco, Invitrogen) were also supplemented. Results
[000156] The tested open circuit filtration system provided herein achieved a viable cell density of 40 x 106 cells on days 9 and 10, and achieved a cell density of 40 x 106 cells earlier than the ATF system corresponding (FIGURE 10). The viable cell percentage of the tested open circuit filtration system provided herein was about 90%, once the culture reached 40 x 106 cells, and continued to decrease until it stabilized at 70% for three weeks (FIGURE 11 ). The capacitance of cell culture in the tested open circuit filtration system provided herein was high compared to cell culture in the ATF system (FIGURE 12), and the mean viable cell diameter of the cell culture in the filtration system open circuit tests provided herein and the cell culture of the ATF system were similar (FIGURE 13).
[000157] The productivity profiles of cell cultures in the open loop filtration system provided herein and in the cell culture of the ATF system are shown in FIGURE 14, FIGURE 15, FIGURE 16 and FIGURE 17. IgG concentration produced by culturing cells in the open loop filtration system provided herein was increased at later time points compared to the ATF system (FIGURE 14). The volumetric productivity and the specific productivity of cell culture in the open loop filtration system provided herein were increased compared to cell culture in the ATF system (FIGURE 15 and FIGURE 16, respectively). The cell culture sieve coefficient in the tested open circuit filtration system provided herein remained at about 90% after three weeks of culture, and was higher than the cell culture sieve coefficient in the TFA system ( FIGURE 17).
[000158] The glucose and lactate production profiles of each system tested are shown in FIGURE 18, FIGURE 19, FIGURE 20 and FIGURE 21. The glucose consumption rate and the specific rate of lactate production of the culture of specific cells in the tested open circuit filtration system provided herein were greater than the glucose consumption rate and the specific lactate production rate of the specific cell culture in the ATF system (FIGURE 18 and FIGURE 19, respectively ). In addition, the specific glucose aerobic consumption rate and glucose lactate yield were higher in cell culture in the tested open loop filtration system provided herein than the glucose aerobic consumption rate and lactate yield glucose levels in cell culture in the ATF system (FIGURE 20 and FIGURE 21, respectively).
[000159] These data indicated that the presently provided open circuit filtration systems result in a cell culture with comparable improved cell culture properties such as an improved or comparable capacitance, an increased or comparable volumetric and specific productivity, a coefficient of an increased or comparable sieve, and an increased or comparable specific glucose consumption compared to another closed-loop tangential filtration system (the ATFTM system from Refine Technology). Example 2. Viable cell density observed in open circuit filtration systems
[000160] An experiment is performed to determine the highest viable cell densities obtained using an open circuit filtration system provided herein and optionally comparing the determined viable cell densities to the viable cell densities obtained by use ATFTM (Refine Technology) (a closed loop alternating flow tangential filtration system) under similar conditions. The device to be used in these experiments is shown generally in FIGURE 5. Specifically, the reservoir to be used in the open loop filtration system is a 15 liter Broadly-James bioreactor, the first conduit and the second conduit consists of a Biocompatible weldable transfer tubing with an inner diameter of 0.5 inch, where the TFF unit contains a single tubular cross-flow filter (composed of polyether sulfone fibers with a length of 30 cm and an inner diameter of 1 mm, and with an average pore size of 0.2 µm, a fiber density of 830 fibers/filter, and a filtration area of 0.77 m2), where at least one pump is a single Watson-Marlow capable peristaltic pump to propel a fluid in the first and second flow directions, with a pump column volume between 50 ml to 100 ml with a Gore Sta-Pure dual channel tubing that has an inner diameter of 16 mm and a wall diameter of 4 mm. Materials and methods
[000161] A summary of the experimental parameters for determining the highest cell densities that can be obtained using the presently provided open circuit filtration systems (and optionally ATFTM from Refine Technology) is shown in Table 5. A further detailed summary of the methods to be used in these experiments are provided below. Table 5. Experimental Parameters


[000162] The conditions to be used to operate the perfusion bioreactor are listed in Table 5. Cells are grown in the bioreactors, with a working volume of 10 liters, and a sufficient replacement with CD CHO culture medium for maintain a specific cell perfusion rate of 0.05 nL/cell-d. The open loop filtration system contains the same filter and casing as the ATF4, but uses a Watson-Marlow 620 Du peristaltic pump with a pump column volume between 50 ml to 100 ml as a culture recirculation pump to propel from Reversibly culturing cells through the system (shown in FIGURE 5) and an open loop system (rather than a closed system used in ATF4). The viable cell density of the cell culture is determined once a day for the duration of the cell culture process. OTHER MODALITIES
[000163] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the above description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

权利要求:
Claims (16)
[0001]
1. A method of processing a mammalian cell culture, the method characterized in that it includes: (a) providing an open loop filtration system (1), wherein the open loop filtration system includes a bioreactor ( 2) which includes a mammalian cell culture, a tangential flow filtration unit (TFF) having first and second inlets (4, 5), a first conduit (6) in fluid communication between the bioreactor (2) and the first inlet (4) of the TFF unit (3), and a second conduit (7) in fluid communication between the bioreactor (2) and the second inlet (5) of the TFF unit (3), and one or more pump(s ) (8) arranged within the system (1) to flow fluid through the system (1), wherein the system (1) is configured so that the mammalian cell culture can reversibly flow through the system (1) to or from the bioreactor (2) and through the first and second conduits (6, 7) and the TFF unit (3) through the one or more pump(s) (8 ), and filtered material can be collected from the TFF unit (3); (b) flowing mammalian cell culture from the bioreactor (2) through the first and second conduits (6, 7) and the TFF unit (3) in a first flow direction for a first period of time; (c) reversing the first flow direction and flowing the mammalian cell culture through the first and second conduits (6, 7) and the TFF unit (3) in a second flow direction for a second period of time; (d) reversing the second flow direction and flowing the mammalian cell culture through the first and second conduits (6, 7) and the TFF unit (3) in the first flow direction for a third period of time; (e) repeat steps (c) - (d) two or more times; and (f) collecting the filtered material.
[0002]
2. Method according to claim 1, characterized in that the TFF unit (3) includes a single cross-flow filter (12), for example a tubular cross-flow filter.
[0003]
3. Method according to claim 1, characterized in that the TFF unit (3) includes two or more cross-flow filters (12).
[0004]
4. Method according to claim 1, characterized in that the system (1) includes one or more additional TFF units (3) disposed in the first conduit (6), in the second conduit (7), or in both .
[0005]
5. Method according to claim 1, characterized in that the one or more pump(s) (8) is arranged in the first conduit (6) or in the second conduit (7), or both.
[0006]
6. Method according to claim 4, characterized in that the one or more pump(s) (8) is arranged in the system (1) between any two TFF units (3).
[0007]
7. Method according to claim 1, characterized in that the one or more pump(s) (8) is disposed in the bioreactor (2) and proximal to the first (6) or second fluid conduit (7) .
[0008]
8. Method according to any one of claims 1 or 5 to 7, characterized in that the one or more pump(s) (8) is a low turbulence pump (LTP), for example, a peristaltic pump.
[0009]
9. Method according to claim 8, characterized in that the system (1) includes a first and a second LTP, wherein the first LTP drains the mammalian cell culture in the first direction and the second LTP drains the mammalian cell culture in the second direction.
[0010]
10. Method according to claim 8, characterized in that the system (1) includes a single LTP, wherein the single LTP drains the mammalian cell culture in the first direction during the first and third time periods and flows the mammalian cell culture in the second direction during the second time period.
[0011]
11. Method according to claim 1, characterized in that: (i) the filtered material does not contain a mammalian cell; and/or (ii) the mammalian cell culture contains a secreted recombinant protein and the filtered material contains the secreted recombinant protein.
[0012]
12. Method according to claim 11, characterized in that the secreted recombinant protein is an antibody or an antigen-binding fragment thereof, a growth factor, a cytokine, or an enzyme, or a combination thereof.
[0013]
13. Method according to claim 12, characterized in that the enzyme is an alpha-galactosidase.
[0014]
14. Method according to claim 11, characterized in that it further includes isolating the secreted recombinant protein from the filtered material; and optionally, wherein isolation is performed using an integrated, continuous process that includes isolation through one or more multiple column chromatography (MCCS) systems.
[0015]
15. Method according to claim 14, characterized in that it further includes formulating a therapeutic drug substance by mixing the isolated recombinant protein with a pharmaceutically acceptable excipient or buffer.
[0016]
16. Method according to claim 1, characterized in that the mammalian cell culture or the filtered material, or both, are sterile.

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法律状态:
2019-10-08| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-11-03| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-04-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-01| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 16/09/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361878502P| true| 2013-09-16|2013-09-16|

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US61/878,502|2013-09-16|

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PCT/US2014/055897|WO2015039115A1|2013-09-16|2014-09-16|Methods and systems for processing a cell culture|

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