Alternating recycle tangential flow (ARTF) filtration for cell culture medium perfusion and bioreactor harvest

Abstract

The present invention relates to a combination of alternating tangential flow (ATF) filtration and recirculation tangential flow (RTF) filtration as alternating recirculation tangential flow (ARTF) filtration, overcoming the treatment challenges observed with either ATF or FTF filtration alone. ARTF filtration is a means for effecting medium perfusion or harvesting desired products as part of cell culture bioreactor process development.

Description

Technical Field

[0001] This invention generally relates to the filtration of cell culture media and methods and systems for cell perfusion culture. In particular, this invention relates to an improved tangential flow filtration system. Background Technology

[0002] With the continued growth in demand for larger quantities of therapeutic recombinant proteins, significant investment is being made in process optimization, particularly in methods and strategies for growing, feeding, and maintaining production cell cultures, which have a positive impact on cell viability and protein recovery. Developing manufacturing processes for recombinant proteins is a complex undertaking, requiring the balancing of many variables. This is especially true for upstream processes, where each element of the cell culture process can significantly influence later stages of production, particularly harvesting and downstream processing.

[0003] Typical cell culture involves a growth phase, an exponential growth period characterized by increasing cell density. Following the growth phase is a transition phase, where exponential cell growth slows and protein production begins to increase. This marks the beginning of the stationary phase (production phase), where cell density typically plateaus and product titers increase. In batch harvest systems, where cell cultures are maintained for a set number of days and then harvested all at once, most of the product can be generated in the final days before harvest, at which point the cell culture typically reaches its maximum output. While this can result in a single high-titer harvest, the cost is unproductive turnaround time to start the next run and a lag in achieving peak yield again. In continuous harvest systems, where product-containing permeate is continuously collected from the cell culture throughout the production phase, the cell culture duration is extended, but at the cost of lower product titers and a larger volume of waste cell culture fluid to be processed during the harvest and purification phases.

[0004] In summary, perfusion culture (a type of continuous harvest) has been recognized as offering relatively favorable economics for cell culture. In this operation, cells are retained in a bioreactor, and products, along with metabolic byproducts, are continuously removed using a hollow fiber filter. Nutrient-containing culture medium is continuously fed into the bioreactor. Perfusion culture enables high cell densities, and more importantly, cells can be maintained at a highly productive state for weeks or months. This results in significantly higher yields and a reduction in bioreactor size.

[0005] Because cell culture material and byproducts continuously flow through the filter, culture medium filtration is critical during perfusion. Despite extensive development of filtration technologies, they are often limited by their tendency to clog when used to filter suspensions of cultured mammalian cells. Cell culture filtration methods tend to use tangential flow filtration (also known as TFF). In TFF, the fluid to be filtered is recirculated by a pump, typically from the reservoir through the filter and back to the reservoir. The flow through the filter is parallel to the filter surface. See, for example, U.S. Patent No. 9,663,753. Another process, known as alternating tangential flow (ATF) filtration, provides yet another filtration mode. See, for example, U.S. Patent No. 10,081,788. It is similar to TFF in that it produces a flow pattern parallel to the filter membrane surface; however, it differs from TFF in that the flow direction is repeatedly alternating or reversed on the filter surface using a diaphragm pump and rapid alternating circulation (approximately ~10 seconds). When cell cultures are recirculated through the filter membrane, it is called recirculating tangential flow (RTF) filtration. See, for example, U.S. Patent Application Publications US20160222337 and US20140093952. However, the filtration efficiency of these systems is not optimal, and they remain sensitive to membrane clogging and fouling.

[0006] One method to avoid filter contamination in hollow fiber filters is by using a helical winding form. See, for example, WO 2011 / 8222550. Using helical wound membrane designs can have unacceptable effects on cell viability and overall performance. These filters are designed with complex flow paths where flow rates are highly variable.

[0007] Accordingly, an improved design of the infusion system is needed to allow for better control of the hollow fiber filtration. Summary of the Invention

[0008] This disclosure provides a system for cell perfusion culture, the system comprising:

[0009] a) At least one bioreactor;

[0010] b) At least one hollow fiber or flat sheet filter unit, the at least one hollow fiber or flat sheet filter unit comprising a first end and a second end at opposite ends of the at least one filter unit; and

[0011] c) At least one fluid pump, wherein the fluid pump is fluidly connected to the at least one filter unit and the bioreactor, such that fluid can be pumped from the bioreactor through the at least one fluid pump to the at least one filter and back to the bioreactor, and retentate is returned to the bioreactor, while membrane permeate can be collected to a separate receiver; and

[0012] Wherein i) the fluid pump is reversible or ii) the first and second ends of the filter unit are fluidly connected to the at least one fluid pump via a set of valves arranged to alternately allow flow in the direction from the at least one bioreactor to the at least one fluid pump to the first end of the at least one filter unit and back to the bioreactor, while blocking flow in the opposite direction, or allow flow in the direction from the at least one bioreactor to the at least one fluid pump to the second end of the at least one filter unit and back to the bioreactor, while blocking flow in the opposite direction.

[0013] In some embodiments, the fluid pump is reversible. In these embodiments, the fluid pump operates in a reversible mode.

[0014] In some aspects, the at least one fluid pump is a centrifugal pump, a peristaltic pump, a rotary vane pump, or a reciprocating pump. In one aspect, the pump is a fluid-driven diaphragm pump, and the system includes a drive fluid pressure transducer arranged to measure the drive fluid pressure in the at least one fluid pump.

[0015] In one embodiment, the first and second ends of the filter unit are fluidly connected to the at least one fluid pump via a set of valves (and associated piping connections), the set of valves being arranged to alternately 1) allow flow in the direction from the at least one bioreactor to the at least one fluid pump to the first end of the at least one filter unit and back to the bioreactor, while blocking flow in the opposite direction, or 2) allow flow in the direction from the at least one bioreactor to the at least one fluid pump to the second end of the at least one filter unit and back to the bioreactor, while blocking flow in the opposite direction. In other words, the set of valves only allows flow in one of the two membrane flow directions. In some aspects of this embodiment, the at least one fluid pump is unidirectional. In some aspects of this embodiment, the at least one fluid pump is reversible, operating in a unidirectional mode.

[0016] Each valve in the set of valves can be independently configured to be in an open or closed position. In some aspects, the valve positioning is automated. In one aspect, the set of valves is a four-valve assembly. In another embodiment, the set of valves is a five-valve assembly, wherein one or more valves are added to a basic four-valve assembly to bypass the filter unit. In some aspects, the four-valve assembly is configured such that two valves can be in an open position and two valves can be in a closed position. In some aspects, the two open valves are connected in parallel, and the two closed valves are connected in parallel.

[0017] In some embodiments, the means for implementing the alternating membrane crossflow mechanism may include a set of valves and piping connections, allowing flow to be configured in one of two membrane flow directions. In this case, instead of using an integrated valve assembly, piping / valve is added at both ends of the filter to allow for flow alternation. It has been observed that this design results in significant dead angles due to the length of the piping, leading to embodiments with valve assemblies that reduce dead angles.

[0018] In some embodiments, the at least one filter unit is a hollow fiber filter cartridge.

[0019] In some embodiments, the system further includes one or more pressure monitoring devices. In some aspects, the system includes at least three pressure monitoring devices configured at at least three locations: 1) one located at end 1; 2) one located at end 2; and 3) one located in the permeate flow path. The pressure devices at end 1 and end 2 will alternately indicate the feed pressure and / or stagnant pressure according to the flow direction. Pressure transmitters may be arranged such that the inlet pressure can be monitored and used to control switching in the event of an increase in the feed pressure measurement. In some embodiments, the valve reverses its position when a high inlet pressure is detected.

[0020] In some embodiments, the system further includes one or more flow meters. On one hand, the flow meters are configured to be positioned on the feed flow path and / or return flow path. On the other hand, the flow meters are configured to be positioned at at least two monitoring locations: 1) a flow meter positioned on the feed flow path and / or return flow path; and 2) a flow meter located on the permeate flow path. The positions of the flow meters on the feed flow path and / or return flow path can be effectively within the feed section or the retentate section, and the feed pressure can be mathematically calculated by adding and subtracting values ​​from the permeate stream, respectively. In some embodiments, the system used in the method includes a retentate flow meter positioned on a return section of the pipeline after an ARTF valve (e.g., a 4-valve assembly) for transporting retentate from the system to the bioreactor to control the amount of recirculated retentate.

[0021] In some embodiments, the at least one fluid pump is unidirectional. A reversible pump operating in unidirectional mode may also be employed. In some aspects, the at least one fluid pump is a centrifugal pump, peristaltic pump, rotary vane pump, or reciprocating pump. In one aspect, the pump is a fluid-driven diaphragm pump, and the system includes a drive fluid pressure transducer arranged to measure the drive fluid pressure in the at least one fluid pump.

[0022] In some embodiments, the system further includes a permeate pump that can be combined with automation to remove permeate at a specified flow rate and maintain the applicable pressure within a preferred range.

[0023] In some embodiments, the system further includes a control unit with a graphical display unit configured to perform control of key process parameters (such as flow rate and pressure settings) and monitoring of alarm conditions. The system monitors the flow direction so that it can adjust the input used to correctly calculate the transmembrane pressure (TMP) and monitors the recirculation inlet pressure, the monitoring point of which alternates from end 1 to end 2 according to the flow direction. In some aspects, the system may also display the flow direction and record the flow direction along with the key process parameters in the data.

[0024] In some embodiments, the system further includes a control unit configured to control the at least one fluid pump and the set of valves based on pressure data received from the pressure transducer. In some aspects, the at least one control unit is arranged to control each valve in the set of valves.

[0025] In some embodiments, the system further includes a Y-shaped element or other means to allow a second filter to be configured as a backup. This configuration allows the backup filter to be used with the instrumentation and pump when the primary filter is unavailable.

[0026] The present invention also provides a method for perfusion culture of cells, the method comprising:

[0027] a) Provide a system as described herein;

[0028] b) Add cell culture medium and cells to the at least one bioreactor;

[0029] c) Culture the cells under stirring in the at least one bioreactor;

[0030] d) During cultivation, operate the at least one fluid pump to draw fluid via piping from the at least one bioreactor to the set of valves, then to the first end of the at least one filter unit, and return the fluid from the second end of the at least one filter unit to the at least one bioreactor; and

[0031] e) Reverse the flow path via the set of valves, such that the at least one fluid pump draws fluid from the at least one bioreactor to the set of valves and then to the second end of the at least one filter unit via a pipe, and returns the fluid from the first end of the at least one filter unit to the at least one bioreactor.

[0032] In some embodiments, the method further includes controlling the set of valves to reverse fluid flow once the pressure reading from one of the pressure transmitters exceeds a preset value.

[0033] In some embodiments, the method provides a method that achieves a preset high P 进料 The value reverses the triggered flow path. An increased P value... 进料 The value indicates a decline in filter performance and triggers an automatic change in flow direction, depending on process-specific factors and equipment design.

[0034] In some embodiments, the flow reverses at preset time intervals. The specific timing depends on process-specific factors and equipment design. Typical alternation timing can be several minutes or several hours; generally, durations exceeding 4 hours will result in high P-values. 进料 Switching. Attached Figure Description

[0035] Figure 1 This is a schematic representation of an ARTF system utilizing a single four-valve assembly.

[0036] Figure 2 This is a schematic representation of an ARTF system utilizing six valves. It represents a preliminary design for changing the flow direction of the hollow fiber by establishing two flow paths parallel to the hollow fiber.

[0037] Figure 3 This is a schematic representation of an ARTF system that utilizes a four-valve assembly with a fifth valve as a bypass valve.

[0038] Figure 4 A to Figure 4 B is a schematic representation of an ARTF system having multiple filter units connected in parallel (A) or in series (B).

[0039] Figure 5 This is a schematic representation of an ARTF system incorporating one or more Y-shaped elements. The Y-shaped elements are included to allow for the installation of secondary hollow fiber units in the event of negative consequences (such as contamination) from the primary filter. This enables quick replacement and process restart.

[0040] Figures 6A to 6B (A) A small-scale ARTF (simulated ATF, 38 cm) is shown. 2 Small-scale ATF (APS-10 225cm) 2 ATF and ATF 2, 850 cm 2 Small-scale RTF (TFF, 38 cm) 2 (B) Screening performance of full-scale 2000 L ATF; and small-scale ARTF (simulated ATF, 38 cm⁻¹)2 Small-scale ATF (1508-CR6 225 cm) 2 ATF and ATF 2,850 cm 2 Small-scale RTF (TFF, 38 cm) 2 ) and full-scale 2000 L ATF yield.

[0041] Figure 7 Images showing the filter cavities at the RTF inlet and outlet compared to the filter cavities at the top and bottom of the ARTF are presented. Detailed Implementation

[0042] This invention is based in part on the finding that a combination of technologies used in ATF and RTF can overcome existing challenges and limitations observed in cell culture medium perfusion using either ATF or RTF alone. Alternating Recirculating Tangential Flow Filtration (ARTF Filtration) offers a novel approach to addressing the process challenges observed with the wider use of Alternating Tangential Flow (ATF) or Recirculating Tangential Flow (RTF) filtration as part of the development and / or execution of cell culture bioreactor processes, enabling a means of culture medium perfusion or desired product harvesting. Specifically, improved modes of cleaning the lumen and backwashing the HF (hollow fiber) inlet of the Alternating Recirculating Tangential Flow (ARTF) filtration enable higher VCD culture and higher titer output.

[0043] Improved membrane cleaning can be achieved by controlling the ARTF crossflow at higher rates than in the RTF process mode. The backwash inlet has proven effective in preventing cell debris or deposits from accumulating at the RTF inlet during unidirectional flow. In the ATF, the alternating crossflow pattern of the filter inherently prevents the accumulation of cell debris or deposits.

[0044] ARTF enables cell culture perfusion processes to avoid the co-concentration effect seen in ATF, where not all bioreactor material drawn into the ATF filter / pump space is completely returned to the bioreactor. The concentration within the ATF filter typically increases over time and becomes problematic with high-density cell cultures, which frequently contaminate or clog the filter flow path. ARTF offers advantages over the ATF method commonly used in large-scale biopharmaceutical manufacturing because it enables higher feed / residue cross-flow rates due to the extended circulation of independently controlled pumps, while eliminating co-concentration, which has been observed to cause ATF filter contamination when bioreactor cultures exceed 30% cell volume due to high process VCD.

[0045] The ARTF systems described in this paper were tested using hollow fiber membrane designs because they offer the optimal configuration for managing bioreactor recirculation. An unacceptable impact was found on cell viability and overall performance when using helical wound membrane designs. These helical wound filters have complex flow paths where flow rates are highly variable.

[0046] Tangential flow filtration (TFF) is a separation process that uses membranes to separate components in a liquid solution or suspension based on differences in size, molecular weight, or other parameters. In perfusion processes, TFF is used to remove target proteins from cell culture media while retaining the cells within the medium. In a TFF process, fluid is pumped tangentially along the membrane surface, and particles, molecules, or cells too large to pass through the membrane are trapped and returned to the process tank (i.e., the bioreactor). TFF processes can involve multiple passes of fluid through the membrane (e.g., recirculation) until the process fluid is sufficiently clarified, concentrated, or purified. The cross-flow nature of TFF minimizes membrane fouling, thus allowing for large-scale processing per batch.

[0047] ATF is designed to allow cross-flow to alternate with limited circulation time and flow rate (e.g., every 10 seconds ~ 9.8 mL / min / cavity). RTF allows for higher cross-flow in a single unidirectional flow, which should allow for improved cavity ID scavenging. Therefore, RTF achieves a certain steady state that allows separation to be maintained under process operating performance conditions. In RTF mode, the flow is unidirectional, and any deposits / debris are drawn into the inlet and trapped on the HF filter inlet, accumulating over time until cell debris blocks some (or all) of the channels, causing the feed pressure to rise to unsafe levels and requiring a shutdown.

[0048] While RTF provides robust performance for many biopharmaceuticals, HF (hollow fiber) membrane fouling can still occur, especially in biopharmaceuticals where higher VCD (which leads to higher titers) is expected and concentrated culture media are used. For example, enhanced high VCD processes have demonstrated HF membrane fouling when run in RTF mode, while the same HF membranes run in ATF mode did not fail.

[0049] Like RTFs, ARTFs deliver high crossflow to improve membrane performance and, like ATFs, allow flow alternation to address fouling. With ARTFs, the long-term accumulation of cell debris can be potentially eliminated or significantly reduced when the flow direction is reversed. When the flow is reversed, any material accumulated in one direction is pushed back into the bioreactor. ARTFs provide the benefits of RTFs while also adding the contribution of ATFs.

[0050] ARTF offers the same advantages as RTF in the following ways: improved process economy, more efficient process execution (less setup time, faster changeover), improved process performance due to higher crossflow rates, and a wider selection of hollow fiber (HF) membranes from suppliers that are not compatible with ATF.

[0051] Small peristaltic laboratory pumps can be easily reversed, but changing the overall flow path by reversing the pump introduces much greater complexity, making it impractical in commercial manufacturing processes.

[0052] Perfusion culture (sometimes called continuous culture) is a culture in which cell cultures receive fresh medium added to the bioreactor via a feed line, while used medium is removed from the bioreactor via a membrane perfusion unit. As the perfused cell medium undergoes periodic filtration to remove target proteins and / or waste, fresh medium can be replenished periodically or continuously. Perfusion can be continuous, stepwise, intermittent, or any combination of these. Perfusion can be performed by adding fresh medium to the bioreactor at the same rate as removing permeate from the TFF system, a process referred to in the art as continuous or constant-volume perfusion. The perfusion rate can range from less than one working volume to multiple working volumes per day. The term “perfusion flow rate” is the amount of medium that passes through (added and removed) from the bioreactor in a given time, typically expressed as a portion or multiple working volumes. “Working volume” refers to the volume of the bioreactor used for cell culture. Perfusion feed media can be formulated to maximize perfusion nutrient concentrations, thereby minimizing the perfusion rate.

[0053] Perfusion can be accomplished by a variety of methods, including centrifugation, sedimentation, or filtration. See, for example, Voisard et al., (2003), Biotechnology and Bioengineering 82:751-65.

[0054] Preferably, the cells are retained in the culture, and the removed used culture medium is substantially cell-free or has significantly fewer cells than the cell culture. The recombinant protein expressed by the cell culture may be retained or removed from the cell culture, depending on the retention system used. Sometimes it is preferred that the host cells and the expressed recombinant protein are retained in the retentate in the bioreactor, while the permeate is substantially free of both or has significantly fewer of both (“component-free permeate”). At other times it may be preferred to retain the cells but allow the expressed protein to enter the permeate (“harvest permeate”).

[0055] Perfusion is a type of dialysis filtration in which continuous bioprocessing occurs. For perfusion or dialysis filtration, a TFF system may include a reservoir or container for fresh culture medium or dialysis filtration solution and one or more conduits for transporting the fresh culture medium or dialysis filtration solution from the fresh culture medium or dialysis filtration solution container to the bioreactor.

[0056] definition

[0057] Although the terminology used herein is standard in the art, definitions of certain terms are provided herein to ensure clarity and definiteness of the meaning of the claims. Units, prefixes, and symbols may be expressed in their International System of Units (SI) accepted forms. The numerical ranges enumerated herein include the numbers defining the ranges and include and support every integer within the defined ranges. Unless otherwise indicated, the methods and techniques described herein may be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001); Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates (1992); and Harlow and Lane *Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1990).

[0058] As used herein, unless otherwise expressly stated, the terms “a” and “an” mean one or more. Furthermore, unless the context requires otherwise, singular terms will include plural and plural terms will include singular. Generally, the nomenclature and techniques used in conjunction with those described herein for cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are those well-known and commonly used in the art.

[0059] All documents or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, and monographs, are expressly incorporated herein by reference. The content described in the embodiments of the invention may be combined with other embodiments of the invention.

[0060] As used herein, the term "bioreactor" means any container that can be used for the growth of cell cultures. The cell cultures described herein can be grown in a bioreactor, and the bioreactor can be selected based on the application of the target protein produced by the cells grown in the bioreactor. Bioreactors can be of any size, as long as they are suitable for cell culture; typically, the size of the bioreactor is appropriate for the volume of the cell cultures grown within it. Typically, the bioreactor will be at least 1 liter and can be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or larger, or any volume between these values. The internal conditions of the bioreactor, including but not limited to pH and temperature, can be controlled during culture. Those skilled in the art will recognize and be able to select a suitable bioreactor for practicing the methods disclosed herein based on relevant considerations.

[0061] As used herein, the terms “feed,” “feed sample,” “feed stream,” and “culture medium replenishment,” and similar terms refer to (e.g., continuously or in batches) a solution delivered to the filtration module for filtration. The feed delivered to the filtration module for filtration may be, for example, feed from a feed container (e.g., a vessel) outside the system, or retentate from a previous filtration module within the same system.

[0062] As used herein, the terms “feed line” or “feed channel” refer to a conduit used to deliver feed from a feed source (e.g., a feed container) to one or more processing units in a filtration assembly.

[0063] As used in this article, the term "filtration" generally refers to the action of using a membrane to separate a feed sample into two streams (permeate and retentate).

[0064] As used herein, the terms "permeate" and "filtrate" refer to the portion of the feed that has permeated through the membrane.

[0065] As used herein, the terms "permeate line" or "permeate channel" refer to the conduits in a filtration assembly used to transport permeate.

[0066] As used in this article, the term "retention" refers to the portion of a solution that has been retained by a membrane, and retention is a stream rich in retained substances.

[0067] As used herein, the terms “residue line” or “residue channel” refer to the conduits in the filter assembly used to transport residue.

[0068] As used herein, the term "filtration membrane" refers to a selective permeation membrane used in TFF processes to separate feed into permeate and retained streams. Filtration membranes include, but are not limited to, ultrafiltration (UF) membranes, microfiltration (MF) membranes, reverse osmosis (RO) membranes, and nanofiltration (NF) membranes.

[0069] As used herein, the term "flow path" refers to a channel that supports the flow of liquids (e.g., feed, retentate, permeate) through all or part of a system. Flow paths can have any topology that supports tangential flow (e.g., straight, coiled, zigzag arrangement). Flow paths can be in parallel or in series. A flow path can also refer to a path that allows for a single passage through the system or a path for recirculating retentate through the TFF system. Furthermore, flow paths can be open, as in the example of channels formed, for example, by hollow fiber membranes. Exemplary flow paths are depicted in the accompanying drawings.

[0070] As used herein, the term "fluid connection" refers to the connection of two or more components of a TFF system via one or more conduits (e.g., feed channels, retention channels, permeate channels) that allow liquid to flow from one component to another.

[0071] As used herein, the term "processing" refers to the act of filtering a feed containing a target product and subsequently recovering the product in a concentrated form. The concentrated product can be recovered from the filtration system in either the stagnant or permeate stream, depending on the size of the product and the pore size of the filter membrane. The terms "parallel processing," "concurrent processing," "parallel operation," and "concurrent operation" refer to the simultaneous or rapid continuous distribution of liquid from a TFF system to two or more filtration units (e.g., filter modules, TFF cartridges) within an assembly for subsequent tangential flow filtration.

[0072] As used herein, the terms “tandem processing,” “serial processing,” “tandem operation,” and “serial operation” refer to distributing liquid in a TFF system one at a time to a filtration unit (e.g., a filter module, TFF cartridge), such that the stagnant stream from the previous unit is used as the feed stream for the subsequent adjacent unit.

[0073] As used herein, the term "product" refers to the target compound in the feed. Typically, the product will be the target biomolecule (e.g., a protein), such as a monoclonal antibody (mAb).

[0074] As used herein, the term "tangential flow filtration" (abbreviated as "TFF") refers to the tangential pumping of a liquid solution or suspension along the surface of a membrane. This is also known as cross-flow filtration.

[0075] As used herein, the term “cell density” refers to the number of cells in a given volume of culture medium. “Viable cell density” refers to the number of viable cells in a given volume of culture medium, as determined by standard viability assays such as trypan blue exclusion assays.

[0076] As used herein, the term "cell viability" refers to the ability of cells in a culture to survive under a given set of culture conditions or experimental variations. The term also refers to the number of living cells at a given time, relative to the total number of cells (living and dead) in the culture at that time.

[0077] As used herein, “cell volume” (PCV), also known as “cell volume percentage” (%PCV), is the ratio of the volume occupied by cells to the total volume of the cell culture, expressed as a percentage. See, for example, Stettler et al., 2006, Biotechnol Bioeng. [Biotechnology and Bioengineering] 95:1228-33. Cell volume is a function of cell density and cell diameter; an increase in cell volume can be caused by an increase in cell density or cell diameter, or both. Cell volume is a measure of the solids content in a cell culture. Solids are removed during harvesting and downstream purification. More solids mean more effort required to separate solid material from the desired product during harvesting and downstream purification steps. Furthermore, the desired product can be trapped in solids and lost during harvesting, resulting in a reduced product yield. Due to the varying sizes of host cells and the presence of dead and dying cells, as well as other cell debris, cell volume is a more accurate way to describe the solids content within a cell culture compared to cell density or live cell density. For example, a cell density of 50 × 10⁶ cells / year... 6 A 2000 L culture of cells / ml will have drastically different cell compactions depending on the cell size. Additionally, when in a state of growth arrest, some cells will increase in size, so the cell compactions before and after growth arrest may differ due to the increased biomass resulting from the increased cell size.

[0078] As used herein, “titer” refers to the total amount of a target polypeptide or protein (which may be naturally occurring or recombinant) produced by a cell culture in a given volume of culture medium. Titer can be expressed in milligrams or micrograms of polypeptide or protein per milliliter of culture medium (or other volumetric measure). “Cumulative titer” is the titer produced by the cells during culture and can be determined, for example, by measuring the daily titer and using those values ​​to calculate the cumulative titer.

[0079] As used herein, “working volume” refers to the volume of a bioreactor used for cell culture. For example, the perfusion flow rate may be one working volume per day or less. Perfusion feed media can be formulated to maximize the concentration of perfusion nutrients, thereby minimizing the perfusion rate.

[0080] This invention discloses a system for perfusion culture of cells. The system includes: a) at least one bioreactor; b) at least one hollow fiber or flat sheet filter unit, the at least one hollow fiber or flat sheet filter unit including a first end and a second end at opposite ends of the at least one filter unit; and c) at least one fluid pump, wherein the fluid pump is fluidly connected to the at least one filter unit and the bioreactor such that fluid can be pumped from the bioreactor through the at least one fluid pump to the at least one filter unit and back to the bioreactor, and wherein i) the fluid pump is reversible or ii) the first end and the second end of the filter unit are fluidly connected to the at least one fluid pump via a set of valves arranged to alternately allow flow in the direction from the at least one bioreactor to the at least one fluid pump to the first end of the at least one filter unit and back to the bioreactor while blocking flow in the opposite direction, or allow flow in the direction from the at least one bioreactor to the at least one fluid pump to the second end of the at least one filter unit and back to the bioreactor while blocking flow in the opposite direction. In this way, an apparatus for controlled circulation of flow path direction is provided, wherein the two ends of the filter unit alternate between an inlet and an outlet.

[0081] In some embodiments, the system disclosed herein allows cell culture medium to flow through one or more filter units in alternating directions while utilizing a reversible fluid pump.

[0082] In some embodiments, the systems disclosed herein allow cell culture medium to flow in alternating directions through one or more filter units, while utilizing a unidirectional fluid pump combined with a set of valves configured to allow alternating flow directions. In these embodiments, a reversible pump operating in unidirectional mode may also be employed.

[0083] Typically, it is preferred that the system operates primarily in one direction, for example, where the flow is drawn from the bioreactor, pumped through a fluid pump, passes through a filter unit, and returns to the bioreactor. Operating in this manner tends to remove any air bubbles introduced from the bioreactor. When the system operates primarily in one direction, switching to the other direction for short periods will reduce filter fouling.

[0084] Fluid pumps can suitably include any circulating pump, including but not limited to peristaltic pumps, centrifugal pumps, reciprocating pumps, or rotary vane pumps. Reciprocating pumps can include diaphragms, membranes, or pistons. A fluid pump can be, for example, a fluid-driven diaphragm pump, wherein a pump chamber and a drive chamber filled with drive fluid are separated by a flexible diaphragm that constitutes the reciprocating motion member. The drive fluid can be a gas (e.g., air) or a liquid. When fluid pressure is applied to the drive chamber via a drive fluid supply line, the diaphragm discharges liquid from the pump chamber during an inward stroke, and when the fluid pressure is released, the diaphragm flexes backward and draws liquid into the pump chamber during an outward stroke. The reciprocating motion member can move back and forth relative to the pump chamber (also called a cylinder when the motion member is a piston), thereby forcing fluid (e.g., culture medium) out of the pump chamber during the inward stroke of the motion member and drawing fluid (e.g., culture medium) into the pump chamber during the outward stroke. The stroke volume of the fluid pump corresponds to the fluid (culture medium). During each stroke, the volume moves out of or into the pump chamber.

[0085] The pump chamber can be connected, for example, directly to the sludge inlet compartment of the filter unit at a joint. Alternatively, it can be connected via a fluid connector. A short section of pipe, with a diameter large enough not to impede fluid flow and a volume significantly smaller than the stroke volume of the fluid pump (e.g., less than 20%, 10%, or 5% of the stroke volume), can optionally be connected via a sterile connector.

[0086] Examples of suitable magnetic levitation pumps include the Levitronix® Puralev® series pumps (Levitronix Technologies, Framingham, MA). Examples of suitable diaphragm pumps include the Repligen XCell™ ATF pumps (Repligen, Waltham, MA). Examples of suitable peristaltic pumps include the Watson Marlow Series 500 and Series 600 pumps (Watson Marlow, Wilmington, MA).

[0087] In embodiments involving a set of valves, the set of valves is designed to allow the system to operate in either of two directions, i.e., either end of the filter unit can be an inlet. Each valve in the set can be independently configured to be in an open or closed position. In some embodiments, these valves are manually operated. In some embodiments, these valves are automatically operated. Each valve can operate independently and allow flow in either direction while blocking flow in the opposite direction. In this respect, each valve will be in an open or closed position. In one embodiment, a four-valve switching assembly allows for changing the flow direction of the hollow fiber while reducing unnecessary valves and additional parallel flow path piping. During the transition from the initial direction to the opposite direction, all valves can be open for a short period of time.

[0088] In one embodiment, these valves are modified chromatographic valves that are simplified for use in only one of two directions. By adding disposable tubing, an automatically switchable design is achieved that keeps the pump running in one direction (as required for low-shear centrifugal pumps), and the pump can remain stationary for purposes outside the switching window, thus simplifying the control system design.

[0089] The valve is preferably a non-invasive valve residing outside the conduit carrying the return flow. In one embodiment, the valve "squeezes" the conduit to limit and control the flow rate. This type of valve is non-invasive and offers commercial advantages because the return line to the reactor passes through the valve and is positioned to regulate the pressure applied to the membrane.

[0090] The initial valve assembly design aimed to achieve hollow fiber switching capability for an initial proof-of-concept demonstration, resulting in an initial six-valve design. This design was functional but offered several opportunities for improvement. These included combining the number of valves and simplifying single-use piping.

[0091] On one hand, this valve group is a four-valve assembly. Figure 1 The image depicts an exemplary set of valves. Alternatively, this set of valves is a four-valve assembly having one or more valves for bypassing a filter unit. Figure 3 The document describes a set of exemplary valves with bypass valves.

[0092] In some respects, the four-valve assembly is configured such that two valves can be in the open position and two valves can be in the closed position. In other respects, the two open valves are connected in parallel, and the two closed valves are connected in parallel.

[0093] In some embodiments, the means for achieving an alternating membrane crossflow mechanism may include a set of valves and piping connections such that the flow is configured in one of two membrane flow directions. In this case, instead of using an integrated valve assembly, piping / valve is added at both ends of the filter to allow for flow alternation. It has been observed that this design results in significant dead angles due to the length of the piping, leading to embodiments with valve assemblies that reduce dead angles.

[0094] The filter unit can be any suitable filter unit, such as a hollow fiber filter unit or a flat sheet filter unit. In some embodiments, the filter unit is not a spiral wound filter unit.

[0095] Filter units are used in mammalian cell perfusion culture for the retention of cells and / or recombinant protein products. When cell cultures (including cell culture medium, cells (intact and lysed), soluble expressed recombinant proteins, host cell proteins, waste, etc.) are introduced into the filter, the filter unit material can retain certain cell culture components on the inner lumen side (internal) based on pore size or molecular weight cutoff (MWCO), and allow certain components to pass through the filter (permeate) based on the pore size or MWCO of the filter material. The retained material (permeate) is returned to the bioreactor. Fresh perfused cell culture medium is added to the bioreactor, and permeate is removed from the filter at predetermined time intervals or continuously to maintain the desired or constant bioreactor volume. Permeate can be discarded, stored in storage tanks, bags, or transport boxes, or directly transferred to another unit operation, such as filtration, centrifugation, and / or other downstream purification methods.

[0096] In some embodiments, the at least one filter unit is a hollow fiber filter. In some aspects of these embodiments, the at least one filter unit is a hollow fiber filter cartridge. Hollow fiber filter units for microfiltration typically have a pore size ranging from 0.1 pm to 5-10 pm, or a molecular weight cutoff of 500 kDa or greater, and can be used to allow proteins to pass through the permeate. Ultrafiltration hollow fibers typically have a pore size range of 0.01 pm to 0.1 pm, or a molecular weight cutoff of 300 kDa or less, and can be used to retain desired proteins in the retentate and return it to the bioreactor. This can be used, for example, to concentrate recombinant protein products for harvesting. These filters are commercially available, such as Xampler UFP-750-E-4MA, Xampler UFP-30-E-4MA (GE Healthcare, Pittsburgh, PA) and Midikros TC modules T02-E030-10, T02-050-10, T02-E750-05, T02-M10U-06 (Spectrum Laboratories, Inc., Dominguez, CA).

[0097] In some embodiments, the at least one filter unit is a flat sheet.

[0098] In some embodiments, the filter unit is selected from a group consisting of hollow fiber, flat sheet TFF box or plate, and frame design.

[0099] For the systems and methods of the present invention employing hollow fiber filter units, the hollow fiber filter is typically manufactured as a filter cartridge comprising multiple hollow fibers (HF) extending parallel to the length of the filter cartridge and embedded (preferably with a potting compound) at each end of the filter cartridge; the cavities at the ends of the HF remain open, thereby forming a continuous passage through each cavity from one end of the filter cartridge to the other (i.e., from the filter cartridge inlet end to the filter cartridge outlet end). The hollow fibers are enclosed by the outer wall of the filter cartridge (i.e., the filter cartridge wall) and the potting compound at their ends. Thus, a chamber exists defined by the filter cartridge wall and the outer wall of the HF. This chamber can be used as a filtrate chamber. The space within the HF cavities (internal space) is collectively considered to constitute part of the retention chamber in each system of the present system.

[0100] In the system described herein, the flow between the ends is reversible. In other words, depending on the flow direction, one end of the hollow fiber filter unit can be considered either the inlet or outlet end. One end can be directly connected to a fluid pump at a connector.

[0101] In one embodiment, the filter system includes at least one hollow fiber filter element having a pore size or molecular weight cutoff (MWCO) that retains the recombinant protein in the bioreactor and at least one hollow fiber filter element having a pore size or MWCO that does not retain the recombinant protein in the bioreactor. In another embodiment, the at least one hollow fiber filter element retaining the recombinant protein in the bioreactor has a MWCO of 300 kDa or less. In another embodiment, the at least one hollow fiber filter element not retaining the recombinant protein in the bioreactor has a MWCO of at least 500 kDa. In another embodiment, the at least one hollow fiber filter element retaining the recombinant protein in the bioreactor is an ultrafilter, and the at least one hollow fiber filter element not retaining the recombinant protein in the bioreactor is a microfilter. In another embodiment, the filter system is contained within a housing. In another embodiment, the filter system further includes a spacer between at least two of the hollow fiber filter elements.

[0102] In some embodiments, the bioreactor is a production bioreactor. In one embodiment, the bioreactor has a capacity of at least 500 L. In one embodiment, the bioreactor has a capacity of at least 500 L to 2000 L. In one embodiment, the bioreactor has a capacity of at least 1000 L to 2000 L. In some embodiments, the bioreactor is made of stainless steel.

[0103] In some embodiments, the bioreactor includes an inflatable flexible bag resting on a movable support, and the piping section is a flexible conduit. The system described herein is particularly suitable for bioreactors agitated by moving the entire bioreactor container (e.g., the inflatable flexible bag) because only one conduit section is required to connect to the bioreactor, which keeps the number of moving connections to the container small and reduces tangling of these connections.

[0104] In some embodiments, the system further includes an osmosis pump. This is to allow the removal of used culture medium.

[0105] In some embodiments, the system further includes one or more pressure monitoring devices. For example, the system may also include a drive fluid pressure transducer arranged to measure the drive fluid pressure in the at least one fluid pump.

[0106] In some embodiments, the system includes an inlet pressure transducer arranged to measure the pressure at the end primarily for which filtrate enters. This transducer may be located at the end of the filter unit primarily used as a filtrate inlet, or at any point between that end and an inlet control valve. In embodiments without an inlet control valve, the transducer may be located at any point between the filtrate inlet end and a branch point. The system may also (or alternatively) include an outlet pressure transducer arranged to measure the pressure at the end primarily used as a filtrate outlet. This transducer may be located at the end of the filter unit primarily used as a filtrate outlet, or at any point between that end and an outlet control valve. In embodiments without an outlet control valve, the transducer may be located at any point between the end primarily used as a filtrate outlet and a branch point.

[0107] In some aspects, the system includes at least three pressure monitoring devices configured at at least three locations: 1) one located at end 1; 2) one located at end 2; and 3) one located in the permeate flow path. The pressure devices at end 1 and end 2 will alternately indicate the feed pressure and / or stagnant pressure according to the flow direction. Pressure transmitters can be arranged such that the inlet pressure can be monitored and used to control switching in the event of an increase in the feed pressure measurement. In some embodiments, the valve reverses its position when a high inlet pressure is detected.

[0108] Pressure transducers can be manually readable pressure gauges or transducers arranged to transmit pressure data to a control unit, for example, by electrical, electromagnetic, or optical means. Pressure transducers, primarily used at the outlet end, can be used, for example, to accurately determine the transmembrane pressure on the filter unit and the membrane, respectively.

[0109] In some embodiments, the fluid pump is a fluid-driven diaphragm pump, and the system includes a drive fluid pressure transducer arranged to measure the pressure of the drive fluid in the fluid pump and mounted, for example, on a drive fluid supply line. The drive fluid pressure transducer may be a manually readable pressure gauge or a transducer arranged to transmit pressure data to a control unit, for example, by electrical, electromagnetic, or optical means. The drive fluid may be a gas (e.g., air) or a liquid.

[0110] In some embodiments, the fluid pump is a gas-driven diaphragm pump, and the system includes a gas pressure transducer arranged to measure the gas pressure in the pump, mounted, for example, on a gas supply line. The gas may in particular be air, in which case the gas (driving fluid) supply source is a compressed air source.

[0111] In some embodiments, the fluid pump is designed as a diaphragm pump with a highly flexible diaphragm (e.g., a soft silicone rubber diaphragm), thereby reducing mechanical energy loss between the fluid pump on one side of the fluid supply line and one side of the filter unit and its retentate inlet end. The pressure at the end of the filter unit, which primarily serves as the inlet side, will be equal to the pressure measured at the pressure transducer on one side of the fluid supply line. Therefore, the fluid pressure of the bioreactor fluid at the end primarily serving as the retentate inlet can be effectively measured and controlled by employing a drive fluid pressure transducer that does not contact the bioreactor fluid, thereby measuring and controlling the filtration process and transmembrane pressure. This design is advantageous when designing systems with disposable components that come into contact with the bioreactor fluid. It reduces cost and complexity because the drive fluid pressure transducer can be reusable and does not require sterilization. Therefore, non-sterilizable high-performance sensors can also be used, and if sterilizable high-performance sensors are used, recalibration after sterilization is not required.

[0112] In some embodiments, the permeate outlet port includes a permeate pressure transmitter or transducer. The permeate pressure transducer can be capable of measuring the pressure at the end of the filter unit that primarily serves as the permeate side, and this pressure, combined with data from a pressure transducer measuring the inlet and / or outlet pressures, can be used to calculate the transmembrane pressure on the filter unit. The permeate pressure transducer can also be used in a feedback loop to control the transmembrane pressure via the operation of the permeate pump or via some other permeate pressure control means, such as hydrostatic pressure control. The transducer can also be used to control the backwash cycle during the outward stroke of the fluid pump.

[0113] In some embodiments, the system further includes a pressure transmitter located at each end of the filter unit. The pressure transducers can be arranged such that the inlet pressure can be monitored and used to control switching in the event of an increase in the feed pressure measurement. In some embodiments, the valve reverses its position when a high inlet pressure is detected.

[0114] In some embodiments, the system further includes one or more flow meters. On one hand, the flow meters are configured to be positioned on the feed flow path and / or return flow path. On the other hand, the flow meters are configured to be positioned at at least two monitoring locations: 1) a flow meter positioned on the feed flow path and / or return flow path; and 2) a flow meter located on the permeate flow path. The positions of the flow meters on the feed flow path and / or return flow path can be effectively within the feed section or the retentate section, and the feed pressure can be mathematically calculated by adding and subtracting values ​​from the permeate stream, respectively. In some embodiments, the system used in this method includes a retentate flow meter positioned at a return section of the piping after the ARTF valve (4-valve assembly) for transporting retentate from the system to the bioreactor to control the amount of recirculated retentate.

[0115] In some embodiments, the system includes at least one control unit arranged to control at least one of a fluid pump and an inlet / outlet control valve based on pressure data received from at least one of the inlet / outlet pressure transducers. The control unit may be suitably electrically, electromagnetically (e.g., via wireless communication), optically (e.g., via fiber optic cable), or pneumatically connected to at least one of the fluid pump and the inlet / outlet control valve, as well as at least one of the inlet / outlet pressure transducers. The control unit may be, for example, a computer, a programmable logic controller, or any similar device capable of a) receiving input signals from one or more pressure transducers, b) calculating one or more output parameters from the input signals according to a predetermined method, and c) transmitting the output parameters as one or more signals to one or more control valves and / or pumps. The control unit may be an integrated control unit arranged to control both the fluid pump and the inlet / outlet control valve, or the system may include a main control unit (or valve control unit) and a pump control unit (not shown). The pump control unit may, for example, be arranged to control the flow and pressure profiles generated by the fluid pump via one or more of the stroke frequency, stroke length, and speed of the moving member during inward and outward strokes. In the case of a fluid-driven diaphragm pump, the stroke frequency, stroke length, and diaphragm speed can be controlled, for example, via one or more valves on the drive fluid supply line. As mentioned above, the features of the pump control unit can also be integrated into the main control unit, or vice versa—the control valve can also be controlled by the integrated pump control unit. Alternatively, the control valve can be preset or manually adjusted so that only the pump control unit is needed. The control unit can also be connected to the permeate pump and / or permeate pressure transducer to control the transmembrane pressure.

[0116] In some embodiments, the system has a graphical display to indicate the flow direction. In one embodiment, the graphical display shows an arrow or other indicator of the flow direction. In one embodiment, the graphical display is utilized via a distributed control system (DCS) (such as DeltaV™ (Emerson, St. Louis, Missouri)).

[0117] In some embodiments, the system further includes a control unit with a graphical display unit, configured to perform control of critical process parameters such as flow rate and pressure settings, and monitoring of alarm conditions. The system monitors the flow direction so that it can adjust the input used to correctly calculate the transmembrane pressure (TMP), and monitors the recirculation inlet pressure, the monitoring point of which alternates from end 1 to end 2 according to the flow direction. In some aspects, the system may also display the flow direction and record the flow direction along with the critical process parameters in the data.

[0118] In some embodiments, the system further includes a control unit configured to control the at least one fluid pump and / or the group of valves based on pressure data received from the pressure transducer. In some aspects, the control unit is arranged to control each valve within the group of valves.

[0119] In some embodiments, the system further includes a Y-shaped element or other means to allow a second filter to be configured as a backup. This configuration allows the backup filter to be used with the instrumentation and pump when the primary filter is unavailable.

[0120] The connections between the various components of the system can be achieved, for example, through piping (such as flexible or stainless steel tubing), with valves aligned with the piping or located near branch points or filter unit ends. Branch points can be, for example, tee connectors or manifolds. The connection between the branch point and the bioreactor can be a single line of tubing (such as flexible tubing). This tubing section can be connected to the internal volume of the bioreactor vessel via ports on the vessel wall. Valves can be any suitable check valve type, such as valves, ball valves, slit disc valves, etc.

[0121] During the outward stroke of the fluid pump, the culture medium is drawn from the bioreactor via the pipe section and branch point to the end of the filter unit that serves as the retentate inlet. Simultaneously, the outward stroke creates negative pressure at the end of the filter unit that serves as the retentate side, causing a certain amount of permeate to backwash the filter membrane and thus remove clogging cells and other material. During the inward stroke of the fluid pump, the culture medium is propelled through the end of the filter unit that serves as the retentate side, producing extractable permeate, which is then returned to the bioreactor via the end that serves as the retentate outlet, branch point, and pipe section. In some embodiments, the systems described herein have the advantage of not requiring a reversible pump.

[0122] The construction materials used in this system can be appropriately compatible with commonly used sterilization methods, such as gamma radiation and / or autoclaving. For reusable components, stainless steel (e.g., with corrosion resistance at least equivalent to 316 L) or engineering plastics (e.g., polysulfone, PEEK, etc.) can be used, while for disposable components, plastics such as polysulfone, polypropylene, polyethylene, or ethylene copolymers can be used.

[0123] To prevent cell culture contamination, the bioreactor and all components in contact with the culture medium can be sterilized prior to culturing. The system or its components can be assembled and sterilized by autoclaving or radiation, or one or more components can be pre-sterilized and assembled in a sterile system. For ease of assembly, sterilized system parts or components can be equipped with sterile connectors, such as those from the Ready Mate type (GE Healthcare). Alternatively, sterilized system parts / components can be contained in sterile packaging and assembled in a sterile cleanroom.

[0124] An ARTF system that can be used to perform the methods described herein may further include one or more additional components that can be used to perform the ARTF process, including, but not limited to, examples of which are known in the art: one or more sampling ports, T-lines (e.g., for online buffer addition), pressure sensors, diaphragms for pressure sensors, or valve sensors for indicating whether any valve in the system is open or closed. In a particular embodiment, the ARTF system includes sampling ports (e.g., sanitary sampling ports) at one or more locations in the system. For example, sampling ports may be included at the end of a retentate line, a permeate line, or both. Typically, the sampling port will be located on a manifold section in a filtration module.

[0125] Existing TFF devices used in perfusion systems include the XCell™ ATF system (Repligen, Waltham, MA) for hollow fiber devices and the KrosFlo® perfusion system (Spectrum Laboratories, Inc., Dominguez, CA). These devices contain open feed channels to limit physical damage to cells in the feed stream, and both require high crossflow rates to minimize contamination (i.e., particle buildup along the membrane wall).

[0126] In some embodiments, the system comprises, in part or in part, disposable components that may be pre-sterilized, for example, by gamma irradiation or autoclaving, and then connected together using sterile connectors such as ReadyMate (GE Healthcare). Filter units, check valves, control valves, and inlet / outlet pressure transducers (not shown) may also be included in the housing. Fluid pumps may also be included in the same housing or in a separate housing. The fluid pump may be disposable or reusable, and it may even be assembled from a disposable portion including a pump chamber and a diaphragm, and a reusable portion including a drive chamber and a drive fluid supply line with a drive fluid pressure transducer. In this case, the disposable portion of the fluid pump may also be integrated with the filter unit.

[0127] In an exemplary embodiment, the bioreactor material stream is transferred from the bioreactor vessel to a filtration system, and the bioreactor fluid return stream is transferred from the filtration system back to the bioreactor vessel. Permeate streams (e.g., metabolic waste material streams) are separated from the bioreactor material stream by a hollow fiber permeation filtration system and carried away through a waste material conduit. Metabolic waste and associated proteins are drawn from the hollow fiber permeation tangential flow system into a waste container via a permeate pump.

[0128] Figure 1 A depicts a representative system with a single hollow fiber filter, as described in this disclosure. Solid black lines indicate unidirectional flow. Specifically, a pump circulates bioreactor fluid through the pump and back to this set of valves. Bioreactor fluid is also pumped from this set of valves and returned to the bioreactor. Solid lines indicate bidirectional flow, meaning flow can occur in either direction. Figure 1In reactor A, there are four valves operating in pairs. When the valve depicted in gray is in the open position (and the valve depicted in black is closed), the flow proceeds through the first gray valve to the hollow fiber tangential flow filter in upward flow mode, through the second gray valve, and back to the bioreactor vessel. The flow changes the direction of the fluid in the flow path. When the valve depicted in black is in the open position (and the valve depicted in gray is closed), the flow proceeds through the first black valve to the hollow fiber tangential flow filter in downward flow mode, through the second black valve, and back to the bioreactor vessel. Permeate is continuously removed from the bioreactor fluid flowing through the tangential flow hollow fiber filter.

[0129] Figure 1 B depicts a representative system of this disclosure with two pumps and two hollow fiber filters connected in parallel. Solid black lines indicate unidirectional flow. Specifically, the pumps circulate the bioreactor fluid through each of the two pumps and to each of the two sets of valves. The bioreactor fluid is also pumped from both sets of valves and returned to the bioreactor. Solid lines indicate bidirectional flow, meaning flow can occur in either direction. Figure 1 In section B, each group contains six valves, two depicted in gray and four in black. When the gray-depicted valves are open (and the black-depicted valves are closed), the flow proceeds through the first gray valve to the hollow fiber tangential flow filter in upward flow mode, through the second gray valve, and back to the bioreactor vessel. When the four black-depicted valves are open (and the gray-depicted valves are closed), the flow proceeds through the first pair of black valves to the hollow fiber tangential flow filter in downward flow mode, through the second pair of black valves, and back to the bioreactor vessel. Permeate is continuously removed from the bioreactor fluid flowing through the tangential flow hollow fiber filters.

[0130] This disclosure also provides a method for cell perfusion culture, the method comprising:

[0131] a) Provide a system as described herein;

[0132] b) Add cell culture medium and cells to the at least one bioreactor;

[0133] c) Culture the cells under stirring in the at least one bioreactor;

[0134] d) During cultivation, operate the at least one fluid pump to draw fluid via piping from the at least one bioreactor to the set of valves, then to the first end of the at least one hollow fiber filter unit, and return the fluid from the second end of the at least one hollow fiber filter unit to the at least one bioreactor; and

[0135] e) Reverse the valve set such that the fluid pump draws fluid from the at least one bioreactor to the valve set and then to the second end of the at least one hollow fiber via a pipe, and returns fluid from the first end of the at least one hollow filter unit to the at least one bioreactor.

[0136] In some embodiments, the method includes adding at least one fluid, such as cell culture medium, to the bioreactor during cultivation. This has the advantages of replenishing the culture medium removed via a filter unit and supplying fresh nutrients and other reagents to the culture. The liquid feed can be any liquid (e.g., a biological fluid) containing particles to be filtered (e.g., viral particles, host cell proteins). For example, the liquid feed may contain a target molecule (e.g., a target protein, such as a recombinant protein) and one or more impurities (e.g., non-target proteins). Typically, the liquid feed is obtained from a source of the target molecule (e.g., a hybridoma expressing a monoclonal antibody (mAb) or other host cells). In a particular embodiment, the target molecule in the liquid feed is a mAb, and the non-target molecule is a host cell protein (HCP) (e.g., a protein derived from a host hybridoma cell). Non-target proteins are typically a heterogeneous mixture of proteins of different sizes, hydrophobicities, and charge densities. In another embodiment, the liquid feed contains one or more viruses (e.g., for virus filtration processes). In yet another embodiment, the liquid feed includes plasma products.

[0137] The bioreactor can be inoculated with at least 0.5 × 10⁻⁶ cells using serum-free medium. 6 Up to 3.0 × 10 6 live cells / mL, for example 1.0 × 10⁶ 6 live cells / mL.

[0138] In the method disclosed herein, the flow can be reversed based on manual input or automatically based on one or more predetermined parameters or time points.

[0139] Predetermined parameters can be achieved by realizing some desired characteristics, properties, or performance milestones of cell cultures (such as viable cell density, cell hematocrit, or titer) or some characteristics of the ARTF system (such as pressure), or the predetermined parameters can be time points.

[0140] In one embodiment, the predetermined parameters can be set at a live cell density greater than or equal to 1 × 10⁻⁶. 6 This can be achieved at a live cell / ml density. In one embodiment, the predetermined parameters can be achieved at a live cell density of at least 20 × 10⁶ cells / ml. 6 live cells / ml up to 30 × 10⁶ 6The predetermined parameter can be achieved when the cell volume is less than or equal to 35%. In another embodiment, the predetermined parameter can be achieved when the cell volume is less than or equal to 30%.

[0141] In one embodiment, the predetermined parameter is pressure. Specifically, P can be monitored. 进料 To see if it rises above a certain level. For example, ARTF pressure can be monitored, and once P... 进料 Once 10 psig is reached, the flow is reversed. The ARTF flow can be circulated based on predetermined times. Predetermined times can be 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, or 4 hours or longer. The ARTF flow can also be circulated based on the parameters of the ARTF system. For example, the switching time for the ARTF flow can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the time it takes for the hollow fiber filter to become fouled. If P 进料 If the rise is faster than the predetermined time or the calculated time, the ARTF stream can also be reversed.

[0142] The predetermined parameter can be based on a specific point in time. This point in time can be measured in hours, days, weeks, or months after a triggering event or action. The triggering event or action can be hours or days after culture, or hours or days after an event such as reaching a certain viable cell density, cell hematocrit, titer, inoculation of a bioreactor, or collection of harvested permeate. In one embodiment, the predetermined parameter can be reached within 12 hours to 25 days after the triggering event or action. In one embodiment, the predetermined parameter can be reached within 24 to 72 hours after the triggering event or action. In one embodiment, the predetermined parameter can be reached within 4 days after the triggering event or action. In one embodiment, the predetermined parameter can be reached 5 days or more after the triggering event or action. In one embodiment, the predetermined parameter can be reached at least 25 days after the triggering event or action. In one embodiment, a first predetermined parameter can be reached within 5 to 25 days after inoculation of a bioreactor. In one embodiment, a first predetermined parameter can be reached within 10 to 12 days after inoculation of a bioreactor. In one embodiment, a second predetermined parameter can be reached within 12 to 72 hours after collection of harvested permeate. In one embodiment, a second predetermined parameter can be reached within 24 to 72 hours after collection of harvested permeate. In one embodiment, the second predetermined parameter can be reached within 24 to 48 hours after the harvested permeate is collected.

[0143] Once the predetermined parameters have been reached, the flow is reversed for a set time to allow the filter to be backwashed. The flow reversal time can be from 10 minutes, 20 minutes, 30 minutes, or up to 1 hour, 2 hours, 3 hours, 4 hours or longer.

[0144] In some embodiments, the method further includes controlling the set of valves to reverse fluid flow once the pressure reading from one of these pressure transmitters exceeds a preset value. In some embodiments, the method includes calculating transmembrane pressure based on data provided by the inlet / outlet pressure transducer or based on data provided by at least one of the inlet / outlet pressure transducer and the permeate pressure transducer, and controlling at least one of the fluid pump, the permeate pump, and the inlet / outlet control valve to maintain the transmembrane pressure between a preset upper and lower limit. This has the advantage of maximizing mass transfer across the filter membrane and minimizing fouling and concentration polarization. Pressure can also be controlled using more sophisticated algorithms, such as generating a desired transmembrane pressure versus time profile during both the outward and inward strokes.

[0145] In some embodiments, the method provides a method for achieving a preset high P 进料 The value reverses the triggered flow path. An increased P value... 进料 The value indicates a decline in filter performance and triggers an automatic change in flow direction, depending on process-specific factors and equipment design.

[0146] In some embodiments, the flow reverses at preset time intervals. The specific timing depends on process-specific factors and equipment design. Typical alternation durations can be minutes or hours; generally, durations exceeding 4 hours will result in high P-values. 进料 Switching.

[0147] In some embodiments, the cell culture medium is a serum-free cell culture medium with a defined chemical composition. In one embodiment, the cell culture medium is a perfusion cell culture medium. In one embodiment, the mammalian cells are Chinese hamster ovary (CHO) cells. In one embodiment, the recombinant protein is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, recombinant fusion proteins, or cytokines.

[0148] Samples from cell cultures can be monitored and evaluated using any analytical technique known in the art. A variety of parameters, including recombinant proteins and culture medium quality and characteristics, can be monitored throughout the culture period. Samples can be acquired and monitored intermittently at desired frequencies, including continuous monitoring, real-time or near real-time.

[0149] In some embodiments, the recombinant protein is purified from the harvested permeate by one or more of flocculation, precipitation, centrifugation, deep filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed-mode anion exchange chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography. In one embodiment, the method described herein further includes sampling during purification and evaluating the samples to quantitatively and / or qualitatively monitor the characteristics of the recombinant protein and / or the purification process. In one embodiment, process analysis techniques are used to quantitatively and / or qualitatively monitor the samples. In one embodiment, the recombinant protein is formulated into a pharmaceutically acceptable preparation.

[0150] The purified protein can then be "formulated" (meaning buffer exchange, sterilization, batch packaging, and / or packaging for the end user). Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 18th edition, 1995, Mack Publishing Company, Easton, Pennsylvania.

[0151] As used herein, “cell culture” or “culture” means the growth and reproduction of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See, for example, *Animal cellculture: A Practical Approach*, edited by D. Rickwood, Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid substrate.

[0152] As used herein, the term “cell culturing medium” (also known as “culture medium,” “cell culture media,” or “tissue culture medium”) refers to any nutrient solution used to grow cells (such as animal or mammalian cells) and typically provides at least one or more of the following components: energy (usually in the form of carbohydrates, such as glucose); one or more of all essential amino acids, typically twenty basic amino acids plus cysteine; vitamins and / or other organic compounds typically required in low concentrations; lipids or free fatty acids; and trace elements, such as inorganic compounds or naturally occurring elements, typically required in very low concentrations (usually in the micromolar range).

[0153] Nutrient solutions may optionally be supplemented with additional components to optimize cell growth, such as hormones and other growth factors, such as insulin, transferrin, epidermal growth factor, serum, etc.; salts (such as calcium salts, magnesium salts, and phosphates) and buffers (such as HEPES); nucleosides and bases, such as adenosine, thymidine, hypoxanthine; and proteins and tissue hydrolysates, such as hydrolyzed plant or animal proteins (peptones or peptone mixtures, which may be obtained from animal by-products, purified gelatin, or plant material); antibiotics, such as gentamicin; polyamines (such as putrescine, spermidine, and spermine, see WIPO Publication No. WO 2008 / 154014) and pyruvate (see US Patent No. 8,053,238), and anti-apoptotic compounds (such as MDL). 28170, cypermethrin, cyclosporine A, BBMP, bongkrekic acid, S-15176 difumarate, cyclopyrfenidone-a, pirfenidone mu, BI-6C9, NSCI, NS3694 or necrotizing agent-1 (see WIPO Publication No. WO 2014 / 022102), depending on the requirements of the cells to be cultured and / or the desired cell culture parameters.

[0154] "Perfusion" cell culture media are typically used to maintain cell cultures via perfusion or continuous culture methods and are sufficiently complete to support cell culture during the process. Perfusion cell culture medium formulations can be richer or more concentrated than basal cell culture medium formulations to suit the methods used for removing used medium. Perfusion cell culture media can be used during both the growth and production phases.

[0155] “Production” cell culture medium refers to a cell culture medium that is typically used for cell culture during the transition period from the end of exponential growth to the beginning of protein production, the “transition” and / or “product” phase, and is sufficiently complete to maintain the desired cell density, viability and / or product titer during that phase.

[0156] Concentrated cell culture media may contain some or all of the nutrients necessary to maintain cell culture; in particular, concentrated media may contain nutrients identified or known to be consumed during the production phase of cell culture. Concentrated media can be based on virtually any cell culture medium formulation. Such concentrated feed media may contain some or all of the components of cell culture media, for example, in normal amounts of about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even about 1000X.

[0157] The components used to prepare cell culture media can be completely ground into powdered culture medium formulations; partially ground together with liquid supplements to be added to the cell culture media as needed; or added to the cell culture media in completely liquid form.

[0158] Cell cultures can also be supplemented with separate concentrated feeds containing specific nutrients that may be difficult to formulate in cell cultures or are rapidly depleted in them. Such nutrients can be amino acids such as tyrosine, cysteine, and / or cystine (see, for example, International Patent Application Publication No. 2012 / 145682). For example, a concentrated tyrosine solution can be separately fed into cell cultures grown in a tyrosine-containing cell culture medium such that the tyrosine concentration in the cell culture does not exceed 8 mM. In another example, concentrated tyrosine and cysteine ​​solutions are separately fed into cell cultures grown in a cell culture medium lacking tyrosine, cysteine, or cysteine. Separate feeding can be initiated before or during the production phase. Separate feeding can be accomplished by feeding the cell culture medium in batches on the same day or different days as the concentrated feed medium. Separate feeding can also be performed on the same day or different days as the perfusion medium.

[0159] "Serium-free" refers to cell media that do not contain animal serum, such as fetal bovine serum. Various tissue media, including defined media, are commercially available. For example, any one or a combination of the following cell media can be used: RPMI-1640, RPMI-1641, Dürbeco Modified Eagle Medium (DMEM), Eagle Minimum Essential Medium, F-12K, Ham F12, Iskov Modified Dürbeco Medium, McCoy 5A, Leibovitz L-15, and serum-free media such as EX-CELL. TM Examples of such media include the 300 series (JRH Biosciences, Lenexa, Kansas) and MCDB 302 (Sigma Aldrich Corp., St. Louis, MO). Serum-free forms of these media are also available. Depending on the needs of the cells being cultured and / or the required cell culture parameters, the cell culture medium can be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, and trace elements. Custom-made cell culture media can also be used.

[0160] As used herein, the term “host cell” should be understood to include cells that have been genetically engineered to express a target polypeptide. Genetic engineering of cell lines involves transfecting, transforming, or transducing cells with a nucleic acid encoding a recombinant polynucleotide molecule (“target gene”), and / or otherwise altering (e.g., through homologous recombination and gene activation or fusion of recombinant and non-recombinant cells) to induce the host cell to express the desired recombinant polypeptide. Methods and vectors for genetically engineering cells and / or cell lines to express target polypeptides are well known to those skilled in the art; for example, various techniques are described in Current Protocols in Molecular Biology, edited by Ausubel et al. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); and Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15–69. This term includes the offspring of the parent cells, regardless of whether the offspring are morphologically or genetically identical to the original parent cells, as long as the target gene is present. Cell cultures may contain one or more host cells.

[0161] Animal or mammalian cells are cultured in a medium suitable for the specific cells being cultured, and this medium can be determined by those skilled in the art without excessive experimentation. Commercially available media can be used, including but not limited to: Iskov modified Dürbeco medium; RPMI 1640; α-minimum essential medium (MEM-α); Dürbeco modified Eagle medium (DMEM); DME / F12; α MEM; basal Eagle medium containing El BSS; glutamine-containing DMEM high-glucose; glutamine-free DMEM high-glucose; glutamine-free DMEM low-glucose; glutamine-containing DMEM:F12 1:1; GMEM (Glasgow MEM); GMEM containing glutamine; Grace Complete Insect Medium; Grace Insect Medium without FBS; Flame F-10 containing glutamine; Flame F-12 containing glutamine; IMDM containing HEPES and glutamine; IMDM containing HEPES but without glutamine; IP41 Insect Medium; 15 (Leibovitz) (2x) without glutamine or phenol red; 15 (Leibovitz) without glutamine; McCoy 5A Modified Medium; Medium 199; MEM Eagle without glutamine or phenol red (2x); MEM Eagle-Earl BSS containing glutamine; MEM Eagle-Earl BSS without glutamine; MEM Eagle-Franks BSS without glutamine; NCTC-109 containing glutamine; Richter CM Medium containing glutamine; RPMI containing HEPES, glutamine, and / or penicillin-streptomycin. RPMI 1640; RPMI 1640 with glutamine; RPMI 1640 without glutamine; Schneider insect medium; or any other medium known to those skilled in the art and formulated for a particular cell type. Supplementary components or ingredients, including optional components, may be added to the foregoing exemplary mediums in appropriate concentrations or amounts as required or desired, and as known and practiced by those skilled in the art using conventional techniques.

[0162] The various mammalian cell lines suitable for growth in the systems and methods disclosed herein are available from the American Type Culture Collection (Manassas, Virginia) and commercial suppliers. Examples of cell lines commonly used in the industry include monkey kidney CVl lines transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 cells or subclones for growth in suspension culture) (Graham et al., J. Gen Virol. [Journal of General Virology] 36: 59, 1977); juvenile hamster kidney cells (BHK, ATCC CCL10); mouse Setolly cells (TM4, Mather, Biol. Reprod. [Reproductive Biology] 23: 243-251, 1980); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); and Buffalo rat hepatocytes (BRL 3A, ATCCCRL). 1442); human lung cells (W138, ATCC CCL 75); human liver cancer cells (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells, as well as many other cell lines and Chinese hamster ovary (CHO) cells.

[0163] Large-scale production of proteins for commercial applications typically takes place in suspension culture. Therefore, the mammalian host cells used to generate the recombinant mammalian cells described herein can, but need not, be adapted for growth in suspension culture. Several host cells are known to be adapted for growth in suspension culture, including mouse myeloma NS0 cells and CHO cells from the CHO-S, DG44, and DXB11 cell lines. Other suitable cell lines include mouse myeloma SP2 / 0 cells, juvenile hamster BF1K-21 cells, and human PER.C6 cells. ® Cells, human embryonic kidney F1EK-293 cells, and cell lines derived from or engineered from any cell line disclosed herein.

[0164] CHO cells are widely used for the production of complex recombinant proteins, including CHOK1 cells (ATCC CCL61). Dihydrofolate reductase (DHFR) deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences] 77: 4216-4220) DXB11 and DG-44 are ideal CHO host cell lines because efficient DHFR-selective and amplifiable gene expression systems allow for high-level expression of recombinant proteins in these cells (Kaufman RJ (1990), Meth Enzymol [Enzyme Methodology] 185:537-566). Also included are glutamine synthetase (GS) knockout CHOK1SV cell lines selected using methionine sulfoxide imine (MSX) based on glutamine synthetase (GS). Other suitable CHO host cells may include, but are not limited to, the following (ECACC accession numbers are in parentheses): CHO (85050302), CHO (protein-free) (00102307), CHO-K1 (85051005), CHO-K1 / SF (93061607), CHO / dhFr- (94060607), CHO / dhFr-AC-free (05011002), and RR-CHOKI (92052129).

[0165] This disclosure provides systems and methods for culturing cells expressing a “target protein”; a “target protein” includes naturally occurring proteins, recombinant proteins, and engineered proteins (e.g., proteins that do not exist in nature and have been designed and / or produced by humans). A target protein may be, but does not have to be, a protein known or suspected of having therapeutic relevance. Specific examples of target proteins include antigen-binding proteins (as described and defined herein), peptide bodies (i.e., molecules comprising a peptide fused directly or indirectly to the Fc domain of other molecules such as antibodies, wherein the peptide portion binds specifically to a desired target; the peptide may be fused to an Fc region or inserted into an Fc ring or a modified Fc molecule, such as as described in U.S. Patent Application Publication No. 2006 / 0140934, which is incorporated herein by reference in its entirety), fusion proteins (e.g., Fc fusion proteins, wherein an Fc fragment is fused to a protein or peptide, including a peptide body), cytokines, growth factors, hormones, and other naturally occurring secreted proteins, as well as mutant forms of naturally occurring proteins.

[0166] As used herein, the terms “peptide” and “protein” (e.g., as used in the context of a target protein or target peptide) are used interchangeably to refer to polymers containing amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are analogs or mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. These terms may also cover amino acid polymers that have been modified, for example, by adding carbohydrate residues to form glycoproteins or by phosphorylation. Peptides and proteins may be produced by naturally occurring and non-recombinant cells, or by genetically engineered or recombinant cells. Peptides and proteins may comprise molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acids with a natural sequence that have been omitted, added, and / or substituted.

[0167] The terms “peptide” and “protein” encompass molecules containing only naturally occurring amino acids, as well as molecules containing non-naturally occurring amino acids. Examples of non-naturally occurring amino acids (which may, as needed, replace any naturally occurring amino acid found in any sequence disclosed herein) include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the peptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.

[0168] As used herein, the term "antigen-binding protein" is used in its broadest sense and refers to a protein that includes a portion that binds to an antigen or target, optionally including a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes antigen-binding protein binding to an antigen. Examples of antigen-binding proteins include human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; biantibodies; triantibodies; tetraantibodies; Fab fragments; F(ab')2 fragments; IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies, and fragments thereof. Antigen-binding proteins may include, for example, alternative protein scaffolds or artificial scaffolds with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein; and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics. 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. [Advances in Biotechnology] 20 :639-654 (2004). Alternatively, peptide antibody mimics (“PAMs”) and scaffolds based on antibody mimics utilizing fibronectin components as scaffolds can be used.

[0169] Antigen-binding proteins can have structures such as those of naturally occurring immunoglobulins. An immunoglobulin is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light chain" (approximately 25 kDa) and a "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region, primarily responsible for effector functions. Human light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively.

[0170] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FRs) linked by three hypervariable regions (also called complementarity-determining regions or CDRs). From the N-terminus to the C-terminus, both the light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Each domain can be assigned amino acids according to the definition in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, (1991). CDRs can also be assigned according to alternative nomenclature schemes, such as Chothia's (see Chothia and Lesk, 1987, J. Mol. Biol.). 196 :901-917; Chothia et al., 1989, Nature 342 :878-883 or Honegger and Pluckthun, 2001, J. Mol. Biol. [Journal of Molecular Biology] 309 :657-670)) Redefining.

[0171] Unless otherwise stated, the term "antibody" includes any isotype or subclass of glycosylated and non-glycosylated immunoglobulin, or its antigen-binding region that competes with an intact antibody for specific binding. Additionally, unless otherwise stated, the term "antibody" refers to an intact immunoglobulin or its antigen-binding portion that competes with an intact antibody for specific binding. Antigen-binding portions can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies and can form elements of the target protein. Antigen-binding portions particularly include Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), fragments including complementarity-determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, biantibodies, triantibodies, tetraantibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific binding of an antigen to the polypeptide.

[0172] Fab fragments are those with V L V H C L and C H 1. A monovalent segment of a structural domain; F(ab')2 segment is a divalent segment having two Fab segments connected by a disulfide bridge in the hinge region; Fd segment has V H and C H 1. Structural domain; the Fv fragment has a V-shaped arm for the antibody. L and V H Structural domain; and the dAb fragment has V H Structural domain, V L Domain, or V H or V L Antigen-binding fragments of the domain (US Patent Nos. 6,846,634, 6,696,245, US Application Publication Nos. 2005 / 0202512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958, Ward et al., 1989, Nature 341:544-546).

[0173] Single-chain antibodies (scFv) are antibodies in which V L and V HRegions are linked via linkers (e.g., synthetic sequences of amino acid residues) to form continuous protein chains, wherein the linkers are long enough to allow the protein chains to fold back and form monovalent antigen-binding sites (see, for example, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). Biantibodies are divalent antibodies comprising two polypeptide chains, each containing V-type antigens linked by a linker. H and V L The linker is too short to allow pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:6444-48; and Poljak et al., 1994, Structure [Structure] 2:1121-23). ​​If the two polypeptide chains of a biantibody are identical, the biantibody produced by pairing them will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to prepare biantibodies with two different antigen-binding sites. Similarly, triantibodies and tetraantibodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which may be the same or different.

[0174] One or more CDRs can be covalently or non-covalently incorporated into a molecule to make it an antigen-binding protein. Antigen-binding proteins can be incorporated into CDRs as part of a larger polypeptide chain, covalently linked to another polypeptide chain, or non-covalently incorporated into CDRs. CDRs allow antigen-binding proteins to bind specifically to a particular target antigen.

[0175] Antigen-binding proteins may have one or more binding sites. If more than one binding site is present, these binding sites may be the same or different from each other. For example, naturally occurring human immunoglobulins typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two different binding sites.

[0176] For clarity, and as described herein, note that antigen-binding proteins may, but do not have to, be of human origin (e.g., human antibodies), and in some cases will contain non-human proteins, such as rat or mouse proteins, and in other cases antigen-binding proteins may contain hybrids of human and non-human proteins (e.g., humanized antibodies).

[0177] The target protein may include a human antibody. The term "human antibody" includes all antibodies having one or more variable and constant regions derived from a human immunoglobulin sequence. In one embodiment, all variable and constant domains are derived from a human immunoglobulin sequence (a fully human antibody). Such antibodies can be prepared in a variety of ways, including by immunizing mice genetically modified to express antibodies derived from human heavy and / or light chain encoding genes, such as those derived from Xenomouse, with the target antigen. ® UltiMab™ or Velocimmune ® The system can be used in mice. A phage-based approach can also be used.

[0178] Alternatively, the target protein may include a humanized antibody. The sequence of a “humanized antibody” differs from that of an antibody derived from a non-human species in that one or more amino acid substitutions, deletions, and / or additions are made such that, when administered to a human subject, the humanized antibody is less likely to induce an immune response and / or induce a less severe immune response compared to a non-human species antibody. In one embodiment, certain amino acid mutations are made in the framework and constant domains of the heavy and / or light chains of a non-human species antibody to produce a humanized antibody. In another embodiment, one or more constant domains from a human antibody are fused to one or more variable domains from a non-human species. Examples of how humanized antibodies can be prepared can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293.

[0179] The “Fc” region, as used in this text, contains the C-cell containing the antibody. H 2 and C H The three-domain structure consists of two heavy-chain segments. These two heavy-chain segments are composed of two or more disulfide bonds and C... H The hydrophobic interactions of the three domains maintain their cohesion. Target proteins containing the Fc region (including antigen-binding proteins and Fc fusion proteins) form another aspect of this disclosure.

[0180] A "half-antibody" is an immunofunctional immunoglobulin construct comprising a complete heavy chain, a complete light chain, and a second heavy chain Fc region paired with the Fc region of the complete heavy chain. A linker may, but is not necessary, connect the heavy chain Fc region and the second heavy chain Fc region. In a particular embodiment, the half-antibody is a monovalent form of the antigen-binding protein disclosed herein. In other embodiments, one Fc region may be associated with a second Fc region using paired charged residues. In the context of this disclosure, the half-antibody may be the target protein.

[0181] The target peptides and proteins may have scientific or commercial significance, including protein-based therapeutics. Target proteins particularly include secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Target peptides and proteins can be produced using cell culture methods through recombinant animal cell lines and may be referred to as “recombinant proteins.” One or more expressed proteins can be produced intracellularly or secreted into a culture medium from which they can be recovered and / or collected. The terms “isolated protein” or “isolated recombinant protein” refer to a target peptide or protein purified from proteins or peptides or other contaminants that would interfere with its therapeutic, diagnostic, preventative, research, or other uses. Target proteins include proteins that exert therapeutic effects by binding to targets, particularly those listed below, including targets derived from them, associated targets, and modifications thereof.

[0182] The target protein includes "antigen-binding proteins." An "antigen-binding protein" is a protein or polypeptide that includes an antigen-binding region or portion that has an affinity for another molecule (antigen) to which it binds. Antigen-binding proteins encompass antibodies, peptides, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including single-chain variable fragments (scFv), double-chain (bivalent) scFv, and IgG scFv) (see, for example, Orcutt et al., 2010, Protein EngDes Sel [Protein Engineering, Design & Selection] 23:221-228), heterologous IgG (see, for example, Liu et al., 2015, JBiol Chem [Journal of Biochemistry] 290:7535-7562), mutant proteins, and XmAbs. ® (Xencor, Inc., Monrovia, California). Also includes bispecific T-cell connectors (BiTE). ® Bispecific T-cell connectives with extended durations (such as extended half-life) (e.g., HLE BiTE, HeteroIg BITE, and others), chimeric antigen receptors (CAR, CAR T), and T-cell receptors (TCR).

[0183] scFv is a single-chain antibody fragment containing variable regions of linked heavy and light antibody chains. See U.S. Patents 7,741,465 and 6,319,494 and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136. scFv retains the ability of the parent antibody to specifically interact with the target antigen.

[0184] The term "antibody" includes any isotype or subclass of glycosylated and non-glycosylated immunoglobulin, or its antigen-binding region that competes with intact antibodies for specific binding. Unless otherwise stated, antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclonal, heterologous IgG, bispecific antibodies, and their oligomers or antigen-binding fragments. Antibodies include IgG1, IgG2, IgG3, or IgG4. It also includes proteins having antigen-binding fragments or antigen-binding regions, such as Fab, Fab', F(ab')2, Fv, biantibodies, Fd, dAb, macrobody, single-chain antibody molecules, and single-domain V. H H, complementarity-determining region (CDR) fragments, scFv, biantibodies, triantibodies, tetraantibodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to bind a specific antigen to a target polypeptide.

[0185] It also includes human, humanized, and other antigen-binding proteins, such as human antibodies and humanized antibodies, which do not produce a significantly harmful immune response when administered to humans.

[0186] It also includes modified proteins, such as those chemically modified by non-covalent, covalent, or both covalent and non-covalent bonds. It further includes proteins containing one or more post-translational modifications, which can be prepared by modification through cellular modification systems or by in vitro introduction or other means by enzymatic and / or chemical methods.

[0187] The target protein may also include recombinant fusion proteins, which include, for example, polymerized domains such as leucine zippers, coiled helices, and the Fc portion of immunoglobulins. It also includes proteins containing all or part of the amino acid sequence of the differentiating antigen (called CD proteins) or their ligands, or proteins substantially similar to any of these.

[0188] In some embodiments, the target protein may include a colony-stimulating factor, such as granulocyte colony-stimulating factor (G-CSF). Such G-CSF reagents include, but are not limited to, Neupogen. ® (Fegrostine) and Neulasta ® (Pegfilgrastim). Also includes erythropoiesis stimulants (ESAs), such as Epogen. ® (Ibertin α), Aranesp ® (Dabenostatin α), Dynepo ® (Ibertinδ), Mircera ® (Methoxylated polyethylene glycol-Ibertin β), Hematide ® ,MRK-2578,INS-22,Retacrit ®(Iberdin ζ), Neorecormon ® (Iberine β), Silapo ® (Ibertin ζ), Binocrit ® (Iberine α), Iberine α Hexal, Abseamed ® (Ibertin α), Ratioepo ® (Eporatio) ® (Ibertinθ), Biopoin ® (Ebertinθ), epolinα, epolinβ, epolinζ, epolinθ and epolinδ, epolinω, epolinι, tissue plasminogen activator, GLP-1 receptor agonist, and molecules of any of the foregoing substances or their variants or analogs and biosimilars.

[0189] In some embodiments, the target protein may include proteins that specifically bind to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGF), insulin-like growth factors, bone-inducing factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factors (CSF), other blood and serum proteins, and blood group antigens; receptors, receptor-related proteins, growth hormone, growth hormone receptors, and T-cell receptors; neurotrophic factors, neurotrophic proteins, relaxin, interferon, interleukin, viral antigens, lipoproteins, integrins, rheumatoid factor, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0190] In some embodiments, the target protein may be combined alone or in any combination with one or more of the following proteins: CD proteins (including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174), HER receptor family proteins (including, for example, HER2, HER3, HER4, and EGF receptors), EGFRvIII, cell adhesion molecules (e.g., LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, and αv / β3 integrin), growth factors (including but not limited to, for example, vascular endothelial growth factor (“VEGF”); VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, and Müllerian-inhibiting substances. substance), human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factors (such as NGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor (including, for example, aFGF and bFGF), epidermal growth factor (EGF), Cripto, transforming growth factor (TGF) (especially including TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5)), insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone-inducing factor, insulin and insulin-related proteins (including but not limited to insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding protein); (coagulation proteins and coagulation-related proteins, especially, such as factor VIII, tissue factor, van Wilbond) Willebrand factor, protein C, α-1-antitrypsin, plasminogen activators (such as urokinase and tissue plasminogen activator (“t-PA”)), bombazine, thrombin, thrombopoietin and thrombopoietin receptor, colony-stimulating factor (CSF) (especially including M-CSF, GM-CSF and G-CSF), other blood and serum proteins (including but not limited to albumin, IgE and blood group antigens), receptors and receptor-associated proteins (including, for example, flk2 / flt3 receptors, obesity (OB) receptors, growth hormone receptors and T-cell receptors); neurotrophic factors, including but not limited to bone-derived neurotrophic factor (BDNF) and neurotrophin-3, neurotrophin-4, neurotrophin-5 or neurotrophin-6 (NT-3, NT-4, NT-5 or NT-6).Relaxin A chain, relaxin B chain and pro-relaxin, interferons (including, for example, interferon α, interferon β and interferon γ), interleukins (ILs) (e.g. IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL-1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2 or IL-17 receptor, IL-1RAP); viral antigens, including but not limited to AIDS envelope virus antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic peptide, pulmonary surfactant. Agents, tumor necrosis factor-α and tumor necrosis factor-β, enkephalin, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (activated and regulated normal T cell expression and secretion factors), mouse gonadotropin-related peptide, DNase, FR-α, inhibin and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, decay accelerator factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressin, regulatory protein, immunoadhesin, antigen-binding protein, growth hormone, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv [PE38] conjugate, Legionella pneumophila (lly), IFN γ, interferon-gamma inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein IIb / IIIb (PAC-1), transforming growth factor β (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin, and any bioactive fragments or variants of the foregoing substances.

[0191] In another embodiment, the target protein includes abciximab, adalimumab, adelimumab, aflibercept, alenmab, alicurumab, anakinase, asceticumab, bailiximab, belimumab, bevacizumab, biotinylate, bonatumab, bentuximab, brodatumab, mocantozumab, konatumab, cetuximab, tertuximab, konatumab, and dalizumab. Antibiotics, denosumab, eculimab, eculoxetine, efalizumab, epazolizumab, etanercept, evokulumab, galiliximab, genitalumab, gemutuzumab, golimumab, teimomab, infliximab, ipilimumab, levofloxacin, levofloxacin, levofloxacin (lxdkizumab), mapamumumab, motesanib phosphate diphosphate), morotumab-CD3, natecillatab, nimotuzumab, nivolumab, olizumab, olrezumab, olfamumab, olmalizumab, interleukin, palizumab, panitumab, pembrolizumab, pertuzumab, pectizumab, ranituzumab, rituximab, rituximab, romistastatin, lomoxoluzumab, saxaglastin, tocilizumab, tosimomab, trastuzumab, uterotumab, vedozazumab, vexizumab, voloximab, zalumab, zalumab, and any biosimilars of the foregoing substances.

[0192] The target protein according to the present invention encompasses all the foregoing and further includes antibodies comprising 1, 2, 3, 4, 5, or 6 complementarity-determining regions (CDRs) of any of the aforementioned antibodies. Variations are also included that comprise regions of amino acid sequences having 70% or higher, particularly 80% or higher, more particularly 90% or higher, even more particularly 95% or higher, especially 97% or higher, even more particularly 98% or higher, even more particularly 99% or higher identity with the reference amino acid sequence of the target protein. This identity can be determined using a variety of well-known and readily available amino acid sequence analysis software. Preferred software includes those implementing the Smith-Waterman algorithm, which is considered a satisfactory solution to the problem of searching and aligning sequences. Other algorithms may also be employed, particularly where speed is an important consideration. Commonly used programs for DNA, RNA and peptide alignment and homology matching that can be used in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE and MPSRCH, the latter being an implementation of the Smith-Waltman algorithm for execution on massively parallel processors manufactured by MasPar.

[0193] Target proteins can also include genetically engineered receptors, such as chimeric antigen receptors (CARs or CAR-Ts) and T-cell receptors (TCRs), as well as other proteins containing antigen-binding molecules that interact with the target antigen. CARs can be engineered to bind antigens (such as cell surface antigens) by incorporating antigen-binding molecules that interact with the target antigen. CARs typically tandem an antigen-binding domain (e.g., scFv) with one or more co-stimulatory (“signaling”) domains and one or more activation domains.

[0194] Preferably, the antigen-binding molecule is its antibody fragment, and more preferably one or more single-chain antibody fragments (“scFv”). scFvs are preferred for use in chimeric antigen receptors because they can be engineered to be expressed as part of a single chain along with other CAR components. See Krause et al., 1998, J. Exp. Med., 188(4): 619–626; Finney et al., 1998, J Immunol 161: 2791-2797.

[0195] Chimeric antigen receptors contain one or more co-stimulatory (signal transduction) domains to enhance their potency. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Krause et al. and Finney et al. (ibid.), Song et al., 2012, Blood 119:696-706; Kalos et al., 2011, Sci Transl. Med. 3:95; Porter et al., 2011, N. Engl. J. Med. 365:725-33, and Gross et al., 2016, Annu. Rev. Pharmacol. Toxicol. 56:59-83.Suitable co-stimulatory domains can be derived from (among other sources) CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ξ), CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig α (CD79a), DAP-10, Fc γ receptor, MHC Class I molecules, TNF, TNFr, integrins, signal transduction lymphocyte activating molecules, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7R α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl-ld, ITGAE, CD103, ITGAL, CDl-la, LFA-1, ITGAM, CDl-lb, ITGAX, CDl-l c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, 41-BB, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligands, or fragments or combinations thereof. The co-stimulatory domain may include one or more extracellular, transmembrane, and intracellular portions.

[0196] CARs also include one or more activation domains. CD3ζ is a component of the T cell receptor on natural T cells and has been shown to be an important intracellular activation element within CARs.

[0197] CARs are transmembrane proteins containing an extracellular domain, typically including an antigen-binding protein that recognizes and binds to the target antigen, and also including a "hinge" region. Other domains include a transmembrane domain and an intracellular (cytoplasmic) domain.

[0198] Extracellular domains facilitate signaling and effective responses of lymphocytes to antigens derived from any of the proteins or combinations thereof described herein. Extracellular domains can be derived from synthetic or natural sources, such as the proteins described herein. Extracellular domains typically contain a hinge portion. This is a part of the extracellular domain and is sometimes referred to as a “spacer” region. The hinge can be derived from these proteins as described herein, particularly the aforementioned co-stimulatory proteins, as well as immunoglobulin (Ig) sequences or other suitable molecules to achieve the specific distance required from the target cell.

[0199] The transmembrane domain can fuse with the extracellular domain of the CAR. It can similarly fuse with the intracellular domain of the CAR. The transmembrane domain can be derived from synthetic or natural sources, such as proteins described herein, particularly the aforementioned co-stimulatory proteins.

[0200] Intracellular (cytoplasmic) domains can fuse with transmembrane domains and can provide activation of at least one of the normal effector functions of immune cells. Effector functions of T cells can include, for example, cytolytic or helper activities involving cytokine secretion. Intracellular domains can be derived from proteins described herein, particularly CD3.

[0201] The polynucleotides, peptides, vectors, host cells, immune cells, compositions, etc., according to the present invention can be prepared using a variety of known techniques.

[0202] This invention is not limited in scope to the specific embodiments described herein, which are intended as individual illustrations of various aspects of the invention, and functionally equivalent methods and components are also within the scope of the invention. In fact, various modifications to the invention will become apparent to those skilled in the art from the foregoing description and drawings, in addition to those shown and described herein. Such modifications are intended to fall within the scope of the appended claims. Example

[0203] Example 1

[0204] Full-scale filtration in a 2000L bioreactor was simulated using three different small-scale reduction models by matching the UF and MF filter loading levels, flux rates, and / or crossflow (mL / min / fiber). A 225 cm⁻¹ filter was used.2 A 4.9L working volume ATF model was established using a hollow fiber filter and a Magma APS-10 diaphragm pump (PendoTECH). An 850 cm³ / h diaphragm pump was used. 2 A 1.5L working volume ATF model was established using a hollow fiber filter and an XCell™ ATF 2 disposable unit, including a diaphragm pump (Repligen Corporation). A 38 cm³ ATF was used. 2 Hollow fiber filters and Masterflex ® L / S ® A peristaltic pump with a working volume RTF model of 0.9 was established.

[0205] Small-scale perfusion cell culture experiments were conducted using different small-scale models and several test antibodies. Proprietary cell lines expressing the test antibodies were cultured in proprietary basal medium and supplemented with proprietary feed medium via perfusion. The culture period was 15 days in total, after which the cells were harvested.

[0206] Viability and cell density were measured using the Vi-CELL™ BLU Cell Viability Analyzer (Beckman Coulter, Indianapolis, Indiana).

[0207] Yield was defined as the percentage ratio of the cumulative product mass in the permeate to the sum of the cumulative product mass in the permeate, the cumulative product mass in the exudate, and the daily product mass in the bioreactor.

[0208] Screening performance can be determined as a percentage using the product titer in the permeate relative to the product titer in the bioreactor.

[0209] For all test conditions, comparable viable cell density (VCD) was observed. The small-scale model showed slightly higher viability and lower cell volume per unit volume (PCV). The titer of the small-scale model was also slightly lower.

[0210] The sieving attenuation and yield of the small-scale ATF model were comparable to those of the full-scale 2000L bioreactor. The sieving and yield of the small-scale RTF model were slightly lower.

[0211] To test the Alternating Reverse Tangential Flow (ARTF) strategy, a 0.9 working volume RTF model was used, with the feed flow direction reversed every 60 minutes.

[0212] like Figure 6A As shown, the ARTF strategy resulted in improved screening performance. This led to increased yield, such as... Figure 6B As shown.

[0213] Example 2

[0214] The feasibility of ARTF compared to RTF and ATF at a 200L scale was tested. Three perfusion systems were connected to the bioreactor and perfused simultaneously for the duration of the process. The same hollow fiber membrane (0.48 m², 30 cm HFM, with 520 lumens) was used in each system. The crossflow rate was the same for all systems (4.3 LPM), and the maximum permeate flux used for all systems was 3.8 LMH. For the ARTF system, automatic flow path control and pneumatic valves were used to reverse the flow every two hours to redirect the flow.

[0215] To apply maximum stress to the system, allowing foam buildup to attempt to clog the perfusion system, cell cultures are concentrated, and the cell culture duration is extended from 15 days to 20 days. The increased foaming inside the bioreactor can facilitate the formation of cell debris, which, once incorporated into the cell culture, can enter the perfusion system and clog the HFM lumen.

[0216] Although no perfusion system malfunctioned, cell concentration increased the feed pressure on the ARTF and RTF systems.

[0217] Table 1: Feed Pressure

[0218]

[0219] Both the ARTF and RTF systems showed that the feed pressure increased by 2.1 times and 3.6 times, respectively.

[0220] The permeate trend curves of the ATF, ARTF, and RTF systems show similar pressure curves. At high percentage PCV, the ATF does show a decrease in permeate pressure as the cell culture becomes more concentrated within the ATF apparatus, indicating some filter fouling. The recirculation flow in the ARTF and RTF systems prevents cell culture concentration within the hollow fiber membrane.

[0221] The transmembrane pressure changes and differential pressure changes between ARTF and RTF are comparable.

[0222] Since the pressure curves indicate that RTF has minimal impact on filter contamination, visual inspection of the filters in both the ARTF and RTF systems is performed.

[0223] Figure 7 An image of the filter's internal lumen is shown. The percentage of clogged lumens is determined by visual inspection, and the clogged lumens are counted against the unclogged lumens; the percentage of clogged lumens is then calculated. In some cases, each lumen may be difficult to see due to the presence of cell debris. In such cases, the number of unclogged lumens is compared to the total number of lumens provided by the hollow fiber membrane manufacturer.

[0224] Table 2: Percentage of Blockage in the Lumen

[0225]

[0226] Nearly three-quarters of the lumen of the RTF inlet is blocked by cell culture debris. For the ARTF system, the inlet was originally on the bottom side, but this system is reversed as described above. The ARTF system exhibits significantly less blockage at the inlet.

Claims

1. A system for perfusion culture of cells, the system comprising: a) At least one bioreactor; b) At least one hollow fiber or flat sheet filter unit, the at least one hollow fiber or flat sheet filter unit comprising a first end and a second end at opposite ends of the at least one filter unit; and c) At least one fluid pump, wherein the fluid pump is fluidly connected to the at least one filter unit and the bioreactor, such that fluid can be pumped from the bioreactor through the at least one fluid pump to the at least one filter, back to the bioreactor, and retentate can be returned from the bioreactor, while membrane permeate can be collected to a separate receiver; and, Wherein i) the fluid pump is reversible or ii) the first and second ends of the filter unit are fluidly connected to the at least one fluid pump via a set of valves arranged to alternately allow flow in the direction from the at least one bioreactor to the at least one fluid pump to the first end of the at least one filter unit and back to the bioreactor, while blocking flow in the opposite direction, or allow flow in the direction from the at least one bioreactor to the at least one fluid pump to the second end of the at least one filter unit and back to the bioreactor, while blocking flow in the opposite direction.

2. The system of claim 1, wherein, The fluid pump is reversible.

3. The system of claim 1, wherein, The at least one fluid pump is unidirectional, wherein the first and second ends of the filter unit are fluidly connected to the at least one fluid pump via a set of valves (and associated piping connections), the set of valves being arranged to alternately allow flow in the direction from the at least one bioreactor to the at least one fluid pump to the first end of the at least one filter unit and back to the bioreactor, while blocking flow in the opposite direction, or allow flow in the direction from the at least one bioreactor to the at least one fluid pump to the second end of the at least one filter unit and back to the bioreactor, while blocking flow in the opposite direction.

4. The system of claim 1, wherein, The at least one fluid pump is a centrifugal pump, a peristaltic pump, a rotary vane pump, or a reciprocating pump.

5. The system of claim 3, wherein, Each valve in the group can be independently configured to be in an open or closed position.

6. The system of any one of claims 3 to 5, wherein, The valve position is automated.

7. The system of any one of claims 3 to 6, wherein, The set of valves is a four-valve assembly.

8. The system of claim 7, wherein, The four-valve assembly is configured such that two valves can be in the open position and two valves can be in the closed position.

9. The system of claim 8, wherein, The two valves that are open are connected in parallel, and the two valves that are closed are connected in parallel.

10. The system of any one of claims 3 to 9, wherein, The set of valves further includes a bypass valve.

11. The system of any one of claims 1 to 10, wherein, The at least one filter unit is a hollow fiber filter cartridge.

12. The system of any one of claims 1 to 11, wherein the system further comprises one or more pressure transmitters.

13. The system of claim 12, wherein, The valves in the set of valves reverse their positions when a high inlet pressure is detected.

14. The system of any one of claims 1 to 13, wherein the system further comprises one or more flow meters.

15. The system of any one of claims 1 to 13, further comprising a control unit configured to control the at least one fluid pump and the set of valves.

16. The system of claim 15, wherein, The control unit is configured to control the at least one fluid pump and the set of valves according to pressure data received from the pressure transducers.

17. The system of any one of claims 1 to 16, further comprising a graphical display unit configured to display flow direction.

18. The system of any one of claims 1 to 17, further comprising a Y-piece.

19. A method for perfusion culture of cells, the method comprising: a) providing a system according to claim 1 ; b) adding cell culture medium and cells to the at least one bioreactor; c) culturing cells in the at least one bioreactor under agitation; d) operating the at least one fluid pump during culturing to draw fluid from the at least one bioreactor via tubing to the set of valves, to the first end of the at least one filter unit, and to return fluid from the second end of the at least one filter unit to the at least one bioreactor; and e) reversing the flow path via the set of valves such that the at least fluid pump draws fluid from the at least one bioreactor via tubing to the set of valves, to the second end of the at least one filter unit, and to return fluid from the first end of the at least one filter unit to the at least one bioreactor.

20. The method of claim 19, further comprising controlling the set of valves to reverse fluid flow once a pressure reading from one of the pressure transducers exceeds a pre-set value.

21. The method of claim 19, further comprising reversing the triggered flow path when a preset high P 进料 value is reached.

22. The method of claim 21, wherein the pre-set P 进料 value is 10 psig.

23. The method of any one of claims 19 to 22, wherein flow is reversed every 1 to 4 hours at set intervals or at random intervals.

24. The method of claim 19, wherein flow is reversed for about 1 hour.

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