AU2024294028A1 - Alternating recirculating tangential flow (artf) filtration for cell culture media perfusion and bioreactor harvesting - Google Patents

Abstract

The present invention relates to a combination of Alternating Tangential Flow (ATF) filtration and Recirculating Tangential Flow (RTF) filtration as an Alternating Recirculating Tangential Flow (ARTF) filtration that overcomes processing challenges observed with either ATF or FTF filtration alone. ARTF filtration is sued to achieve a means of media perfusion or harvesting of desired product as part of a cell culture bioreactor process development.

Description

ALTERNATING RECIRCULATING TANGENTIAL FLOW (ARTF) FILTRATION

FOR CELL CULTURE MEDIA PERFUSION AND BIOREACTOR HARVESTING

FIELD OF THE INVENTION

[0001] The present invention relates generally to filtration of cell culture media and methods and systems for perfusion culture of cells. In particular, the present invention relates to an improved tangential flow filtration system.

BACKGROUND OF THE INVENTION

[0002] As the demand for greater quantities of therapeutic recombinant proteins continues to grow, much effort is being placed on process optimization, particularly 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 production of recombinant proteins is a complex endeavor where many variables must be balanced. This is particularly true for upstream processes, where every element of the cell culture process can have a large impact on the later stages of production, particularly harvest and downstream processing.

[0003] A typical cell culture undergoes a growth phase, i.e.. a period of exponential growth where cell density increases. The growth phase is followed by a transition phase where exponential cell growth is slows and protein production starts to increase. This marks the start of the stationary phase, a production phase, where cell density typically levels off and product titer increases. In batch harvest systems, where the cell culture is maintained for a set number of days followed by harvesting the entire culture all at once, the majority of the product may be produced in the last few days prior to harvest when the cell culture typically has reached its greatest output. While this may result in a single high titer harvest, it is at the expense of a non-productive turnaround time to initiate the next run and the lag time to once again achieve peak production. In a continuous harvest systems, where product containing permeate is collected from the cell culture on a continuous basis throughout the production phase, the cell culture duration is extended, but at the expense of lower product titers and higher volumes of waste cell culture fluid to be dealt with during the harvest and purification stages.

[0004] On balance, it has been recognized that perfusion culture, a kind of continuous harvest, offers relatively good economics for cell cultures. In this operation, cells are retained in the bioreactor, and the product is continuously removed along with metabolic byproducts through the use of hollow fiber filters. Media feed, containing nutrients, is added continually to the bioreactor. Perfusion culture operation is capable of achieving high cell densities and more importantly, the cells can be maintained in a highly productive state for weeks or months. This achieves much higher yields and reduces the size of the bioreactor. [0005] Media filtration is more critical in perfusion processes because of the continuous flow of cell culture material and byproducts through a filter. Despite tire extensive developments in filter technology, they are generally limited by their tendency to clog when used to filter a suspension of cultured mammalian cells. Filtration approaches with cell culture tend to use Tangential Flow Filtration (also known as TFF). In TFF, fluid to be filtered is recirculated with a pump, typically, from a reservoir through a filter and back to the reservoir. The flow through the filter is parallel to the surface of the filter. See, e.g., U.S. Pat. No. 9.663.753. Another process, known as alternating tangential flow (ATF) filtration, offers yet another mode of filtration. See. e.g., U.S. Patent No. 10,081.788. It is similar to TFF, in that it generates a flow pattern parallel to the filtration membrane surface: however, it differs from TFF in that the direction of flow is repeatedly alternating or reversing across the filter surface through the use of a diaphragm pump and rapid alternating cycling (on the order of ~10 seconds). When the cell culture is recirculated through the filtration membrane it is known as recirculating tangential flow (RTF) filtration. See, e.g., U.S. Patent Application Publication Nos. US20160222337 and US20140093952. However, the filtration efficiency of these systems is not optimal, and they are still sensitive to clogging and fouling of the membranes. [0006] One approach to avoid filter fouling in hollow fiber filters is through the use of a spiral wound format. See. e.g., WO2011/8222550. Use of spiral wound membrane designs can have unacceptable impacts on cell viability and overall performance. The designs of these filters have complicated flow paths where flow rate is highly variable.

[0007] Accordingly, there is a need for improved designs of perfusion systems allowing better control of the hollow fiber filtration.

SUMMARY OF THE INVENTION

[0008] The present disclosure provides a system for perfusion culture of cells comprising: a) at least one bioreactor; b) at least one hollow fiber or flat sheet filter unit comprising a first end and a second end on opposite ends of the at least one filter emit; and c) at least one fluidic pump, wherein said fluidic pump is fluidically connected to said at least one filter unit and said bioreactor such that fluid can be pumped from the bioreactor through the at least fluidic pump to the at least one filter back to the bioreactor and retentate returned to the bioreactor, with the ability to collect membrane permeate to a separate receiver; and wherein i) said fluidic pump is reversible or ii) said first end and second end of the filter unit are fluidically connected to said at least one fluidic pump via a set of valves arranged to alternatively allow flow in the direction from the at least one bioreactor to the at least one fluidic pump to the first end of the at least one filter unit and back to the bioreactor while blocking flow in the reverse direction or allow flow in the direction from the at least one bioreactor to the at least one fluidic pump to the second end of the at least one filter unit and back to the bioreactor while blocking flow in the opposite direction.

[0009] In certain embodiments, the fluidic pump is reversible. In these embodiments, the fluidic pump is operated in reversible mode.

[0010] In certain aspects, the at least one fluidic pump is a centrifugal, peristaltic, rotary lobe, or reciprocating pump. In one aspect, the pump is a fluid-driven diaphragm pump and the system comprises a drive fluid pressure transducer arranged to measure a drive fluid pressure in the at least one fluidic pump.

[0011] In one embodiment, said first end and second end of the filter unit are fluidically connected to said at least one fluidic pump via a set of valves (and associated tubing connections) arranged to alternatively 1) allow flow in the direction from the at least one bioreactor to the at least one fluidic pump to the first end of the at least one filter unit and back to the bioreactor while blocking flow in the reverse direction or 2) allow flow in the direction from the at least one bioreactor to the at least one fluidic 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 permits only flow in one of the two membrane flow directions. In certain aspects of this embodiment, the at least one fluidic pump is unidirectional. In certain aspects of this embodiment, the at least one fluidic pump is reversible, operating in a unidirectional mode.

[0012] Each valve in the set of valves can be independently configurable in an open position or a closed position. In certain aspects, the position of the valves is automatable. In one aspect, the set of valves is a four-valve assembly. In another embodiment, the set of valves is a five-valve assembly with one or more valves added to the basic four valve assembly for bypassing the filter unit. In certain aspects the four-valve assembly is configured such that tw o valves can be in the open position while two valves can be in the closed position. In certain aspects, the two valves which are open are parallel, and the two valves which are closed are parallel.

[0013] In certain embodiments, the means for achieving an alternating membrane cross flow mechanism can comprise a set of valves and tubing connections such that configuration of flow can be in one of the two membrane flow directions. In this instance, a consolidated valve assembly is not used but instead tubing/valving has been added to the two ends of the filter to allow alternating of flow. It was observed that this design resulted in significant deadlegs due to lengths of tubing leading to the embodiment of the valve assembly that reduced the deadlegs.

[0014] In certain embodiments, the at least one filter unit is a hollow fiber cartridge. [0015] In certain embodiments, the system further comprises one or more pressure monitoring devices. In certain aspects, the system comprises at least three pressure monitoring devices configured in at least three positions: 1) one positioned at end 1; 2) one positioned at end 2; and 3) one positioned in the permeate flow path. The pressure devices at ends 1 and end 2 will alternately indicate feed and/or retentate pressure depending on the flow direction. The pressure transmitter can be arranged so that inlet pressure can be monitored and used to control switching in the event of elevated feed pressure measurement. In certain embodiments, the valves reverse position upon detection of high inlet pressure.

[0016] In certain embodiments, the system further comprises one or more flow meters. In one aspect, a flow meter is configured to be positioned in the feed and/or return flow path. In another aspect, flow meters are configured to be positioned in at least two monitoring positions: 1) a flow meter that is positioned in the feed and/or return flow path; and 2) a flow meter that is in the permeate flow path. The position of the flow meter in the feed and/or return flow path can effectively be either in the feed or retentate section with the ability to calculate mathematically feed pressure using the value from permeate flow to add or subtract respectively. In certain embodiments, the method utilizes a system which includes a retentate flow meter that is positioned on the return portion of the tubing after the ARTF valve (e.g.. 4- valve assembly) carrying retentate from the system to the bioreactor to control the amount of retentate that is recirculated.

[0017] In certain embodiments, the at least one fluidic pump is unidirectional. Reversible pumps operating in a unidirectional mode can also be employed. In certain aspects, the at least one fluidic pump is a centrifugal, peristaltic, rotary lobe, or reciprocating pump. In one aspect, the pump is a fluid-driven diaphragm pump and the system comprises a drive fluid pressure transducer arranged to measure a drive fluid pressure in the at least one fluidic pump. [0018] In certain embodiments, the system further comprises a permeate pump that can be combined with automation to remove permeate at a specified flow rate as well as maintain applicable pressures within preferred ranges.

[0019] In certain embodiments, the system further comprises a control unit with a graphical display unit configured to perform control of critical process parameters like flow rates, pressure settings and monitoring for alarm conditions. This system monitors the direction of flow so that it can adjust the inputs being used to properly calculate transmembrane pressure (aka TMP) and monitors the recirculating inlet pressure whose monitoring point will alternate from End 1 to End 2 depending on flow direction. In certain aspects, this system can also display the direction of flow and records flow direction in data along with critical process parameters.

[0020] In certain embodiments, the system further comprises a control unit configured to control the at least one fluidic pump and the set of valves depending on pressure data received from said pressure transducer. In certain aspects, the at least one control unit is arranged to control each valve of the set of valves.

[0021] In certain embodiments, the system further comprises a wye(s) or other means to allow a second fdter to be set up as a backup. This configuration allows the backup filter to be used with the instrumentation and pump if the primary filter becomes unusable.

[0022] The present invention also provides a method for perfusion culture of cells, comprising: a) providing a system as described herein; b) adding cell culture medium and cells to said at least one bioreactor; c) cultivating cells in said at least one bioreactor under agitation; d) operating, during the cultivating, said at least one fluidic pump to withdraw fluid from said at least one bioreactor via tubing to said set of valves to said first end of said at least one filter unit and to return fluid from said second end of said at least one filter unit to said at least one bioreactor and; e) reversing the flow path via the set of valves so that the at least fluidic pump withdraws fluid from said at least one bioreactor via tubing to said set of valves to said second end of said at least one filter unit and to return fluid from said first end of said at least one filter unit to said at least one bioreactor.

[0023] In certain embodiments, the method further comprises controlling the set of valves to reverse the flow of fluid once a pressure reading from one of the pressure transmitters exceeds a preset value.

[0024] In certain embodiments, the method provides for tire reversing the flow path which is triggered upon reaching a preset High Pfeed value. Elevated Pfeed values are indicative of filter performance degradation and triggers an automated flow direction change, dependent upon process specific factors and equipment design.

[0025] In certain embodiments, the flow is reversed at a preset time interval. Specific timing is dependent upon process specific factors and equipment design. Typical alternating timing can be minutes or hours; generally more than 4 hours or more will result in switching based on High Pfeed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic representation of an ARTF system utilizing a single four- valve assembly.

[0027] Figure 2 is a schematic representation of an ARTF system utilizing six valves. This was a preliminary design that enabled hollow fiber flow direction change by establishing two flow paths parallel to the hollow fiber. [0028] Figure 3 is a schematic representation of an ARTF system utilizing a four-valve assembly with a fifth valve as a bypass valve.

[0029] Figures 4A-B are schematic representations of ARTF systems with multiple filter units in (A) parallel, or (B) in series.

[0030] Figure 5 is a schematic representation of an ARTF system incorporating one or more wyes. Wyes were incorporated to allow secondary hollow fiber units to be installed should there be a negative outcome to the primary filter like fouling. This enabled rapid change and process restart.

[0031] Figures 6A-B show (A) sieving performance of small-scale ARTF (simulated ATF, 38cm²), small-scale ATF (APS-10 225cm² ATF and ATF 2, 850 cm²), small-scale RTF (TFF, 38cm²) and a full-scale 2000 L ATF; and (B) yield for small-scale ARTF (simulated ATF. 38cm²). small-scale ATF (1508-CR6 225cm² ATF and ATF 2. 850 cm²), small-scale RTF (TFF. 38cm²) and a full-scale 2000 L ATF.

[0032] Figure 7 shows images of the filter lumens for a RTF inlet and outlet vs. an ARTF top and bottom.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is based in part on the discovery that a combination of the techniques used in ATF and RTF can overcome existing challenges and limitations observed in cell culture media perfusion using ATF or RTF alone. Alternating Recirculating Tangential Flow Filtration (ARTF Filtration) presents a novel way to address processing challenges observed with more widespread use of Alternating Tangential Flow (ATF) or Recirculating Tangential Flow (RTF) filtration to achieve a means of media perfusion or harvesting of desired product as part of a cell culture bioreactor process development and/or execution. Specifically, with Alternating Recirculating Tangential Flow (ARTF) filtration's improved mode of sweeping the lumen and backflushing a HF (hollow fiber) inlet, higher VCD cultures and higher titer output can be achieved.

[0034] The ability to control ARTF crossflow at higher rates than a RTF process pattern can enable improved membrane sweeping. Backflushing the inlet has been demonstrated to be effective at preventing cell debris or precipitate that would otherwise build up at an RTF inlet during unidirectional flow. In ATF. the alternating mode of filter cross flow inherently prevents cell debris or precipitate build up.

[0035] ARTF enables cell culture perfusion processes to avoid the co-concentration effect that is seen with ATF where not all of the bioreactor material that is drawn into the ATF filter/pump space is fully returned back to the bioreactor. Concentration within the ATF filter typically increases over time and becomes problematic with high density cell cultures that often foul or plug filter flow paths. ARTF provides an advantage over the ATF approach commonly used in large-scale biologies manufacturing because it enables higher feed/retentate cross flow rates due to the extended circulation with an independently controllable pump while eliminating the co-concentration phenomena that has been observed to cause ATF filter fouling as the bioreactor culture exceeds 30% packed cell volume due to high process VCD.

[0036] The ARTF systems described herein were tested using hollow fiber membrane designs since they provide an optimal configuration for managing bioreactor recirculation. Use of spiral wound membrane designs was found to have unacceptable impacts on cell viability and overall performance. The design of these spiral wound filters have complicated flow paths where flow rate is highly variable.

[0037] Tangential flow filtration (TFF) is a separation process that uses membranes to separate components in a liquid solution or suspension on the basis of size, molecular weight or other differences. TFF is used in perfusion processes to remove target proteins from cell culture media, while retaining cells within the media. In TFF processes, fluid is pumped tangentially along the membrane surface and particles, molecules, or cells that are too large to pass through the membrane are rejected and returned to a process tank (i.e., bioreactor). TFF processes can involve additional passes of the fluid across 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 permitting high volume processing per batch.

[0038] ATF alternates crossflow at constrained cycle times and flow rates based on design (e.g. ~9.8 mL/min/lumen every 10 seconds). RTF allows for higher cross flows in a single, unidirectional flow that should allow improved sweeping of lumen ID. Hence. RTF achieves a sort of steady state that allows separation to be sustained at an operational process performance condition. In RTF mode, flow is unidirectional and any precipitate/debris would be sucked into inlet and get caught up on the HF filter inlet building over time until cell debris is blocking some (or all) of the channels so the feed pressure increases to an unsafe level and needs to be shut down.

[0039] While RTF provides robust performance with many biologies, HF (hollow fiber) membrane fouling can occur particularly in biologies where a higher VCD (results in higher titer) is desired and where concentrated media is used. For example, an intensified high VCD process has demonstrated fouling of the HF membrane when run in RTF mode while the same HF membrane ran in ATF mode did not fail.

[0040] ARTF delivers high crossflows like RTF for improved membrane performance with alternating flow like ATF to address fouling. With ARTF, long term build- up of cell debris can potentially be eliminated or significantly reduced when flow direction is alternated. Whatever has built up in one direction will be pushed back into the bioreactor when flow is reversed. With ARTF, the benefits of RTF are obtained with the added contribution that ATF provides.

[0041] ARTF provides the same advantages that RTF presents in terms of improved process economics, more efficient process execution (less prep time, faster change over), improved process performance due to higher cross flow rates and a broader selection of Hollow Fiber (HF) membranes from suppliers that don't work with ATF.

[0042] Small peristaltic lab pumps can easily reverse but changing overall flow path by reversing pumps would bring far too much complexity for it to be practical in a commercial manufacturing process.

[0043] Perfusion culture, sometimes known as continuous culture, is one in which the cell culture receives the addition of fresh medium to the bioreactor via a feed line and spent medium is removed from the bioreactor via a membrane perfusion unit. As the perfusion cell culture media undergoes periodic filtration to remove target proteins and/or waste products, fresh media can be periodically or continuously resupplied. Perfusion can be continuous, step- wise, intermittent, or a combination of any or all of any of these. Perfusion can be performed by adding the fresh media to the bioreactor at the same rate that permeate is removed from the TFF system, a process which is known in the art as continuous, or constant-volume, perfusion. Perfusion rates can be less than a working volume to many working volumes per day. The term ‘‘perfusion flow rate” is the amount of media that is passed through (added and removed) from a bioreactor, typically expressed as some portion of or a multiple of the working volume, in a given time. “Working volume” refers to the amount of bioreactor volume used for cell culture. Perfusion feed medium can be formulated to maximize perfusion nutrient concentration to minimize perfusion rate.

[0044] Perfusion can be accomplished by a number of means including centrifugation, sedimentation, or filtration. See c.g. Voisard ct al., (2003), Biotechnology and Bioengineering 82:751-65.

[0045] Preferably the cells are retained in the culture and the spent medium that is removed is substantially free of cells or has significantly fewer cells than the cell culture. Recombinant proteins expressed by tire cell culture may be retained or removed from the cell culture, depending on the retention system used. Sometimes it is preferable for the host cells and the expressed recombinant proteins to remain in the retentate in the bioreactor and for the permeate be substantially free of or have significantly less of either (“null permeate”). Other times it may be preferable to retain cells but allow the expressed proteins to pass into the permeate (“harvest permeate”).

[0046] Perfusion is a type of diafiltration in which continuous bioprocessing occurs. To perform perfusion or diafiltration, the TFF system can include a reservoir or container for fresh media or diafiltration solution and one or more conduits for carrying fresh media or diafdtration solution from the fresh media or diafiltration solution container to the bioreactor.

Definitions

[0047] While the terminology used in this application is standard within the art, definitions of certain terms are provided herein to assure clarity and definiteness in the meaning of the claims. Units, prefixes, and symbols may be denoted in their SI (International System of Units) accepted form. Numeric ranges recited herein are inclusive of the numbers defining the range and include and are supportive of each integer within the defined range. The methods and techniques described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See. e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and 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, N.Y. (1990).

[0048] As used herein, the terms “a” and “an” mean one or more unless specifically indicated otherw ise. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of. cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0049] All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, arc hereby expressly incorporated by reference. What is described in an embodiment of the invention can be combined with other embodiments of the invention.

[0050] As used herein, the term “bioreactor” means any vessel useful for the growth of a cell culture. The cell cultures described herein can be grown in a bioreactor, which can be selected based on the application of a protein of interest that is produced by cells growing in the bioreactor. A bioreactor can be of any size so long as it is useful for the culturing of cells; typically, a bioreactor is sized appropriate to the volume of cell culture being grown inside of it. Typically, a bioreactor will be at least 1 liter and may be 2, 5, 10, 50. 100, 200, 250, 500, 1.000. 1500. 2000. 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. The internal conditions of the bioreactor, including, but not limited to pH and temperature, can be controlled during the culturing period. Those of ordinary skill in the art will be aware of, and will be able to select, suitable bioreactors for use in practicing the methods disclosed herein based on the relevant considerations.

[0051] As used herein, the terms “feed,” “feed sample” “feed stream” and “media feed” and like terms refer to the solution that is delivered (e.g., continuously, or as a batch) to a filtration module to be filtered. The feed that is delivered to a filtration module for filtration can be, for example, feed from a feed container (e.g., vessel, tank) external to the system, or retentate from a preceding filtration module in the same system.

[0052] As used herein, the terms “feed line” or “feed channel” refers to a conduit for conveying a feed from a feed source (e.g.. a feed container) to one or more processing units in a filtration assembly.

[0053] As used herein, the term “filtration” generally refers to the act of separating the feed sample into tw o streams, a permeate and a retentate. using membranes.

[0054] As used herein, the terms “permeate” and "filtrate” refer to that portion of the feed that has permeated through the membrane.

[0055] As used herein, the terms “permeate line” or “permeate channel” refers to a conduit in a filtration assembly for carrying permeate.

[0056] As used herein, the term “retentate” refers to the portion of the solution that has been retained by the membrane, and the retentate is the stream enriched in a retained species.

[0057] As used herein, the terms “retentate line” or “retentate channel” refers to a conduit in a filtration assembly for carrying retentate.

[0058] As used herein, the term “filtration membrane” refers to a selectively permeable membrane for separating a feed into a permeate stream and a retentate stream using a TFF process. Filtration membranes include, but are not limited to, ultrafiltration (UF) membranes, microfiltration (MF) membranes, reverse osmosis (RO) membranes and nanofiltration (NF) membranes.

[0059] As used herein, the term “flow path” refers to a channel supporting the flow of a liquid (e.g., feed, retentate, permeate) through all or part of a system. The flow path can have any topolog}' which supports tangential flow (e.g., straight, coiled, arranged in zigzag fashion). The flow path can be parallel or serial. A flow path can also refer to a path resulting in a single pass through a system or a path for recirculating retentate through a TFF system. Furthermore, a flow path can be open, as in an example of channels fonned by, for example, hollow fiber membranes. Exemplary flow paths are depicted in the Figures.

[0060] As used herein, the term “fluidly connected” refers to two or more components of a TFF system that are connected by one or more conduits (e.g., a feed channel, a retentate channel, a permeate channel) such that a liquid can flow from one component to the other. [0061] As used herein, the term “processing” refers to the act of filtering a feed containing a product of interest and subsequently recovering the product in a concentrated form. The concentrated product can be recovered from the filtration system in either the retentate stream or permeate stream depending on the product's size and the pore size of the filtration membrane. The terms “parallel processing”, “processing in parallel”, “parallel operation” and “operation in parallel” refer to distributing a liquid in a TFF system to two or more filtration units (e.g., filtration modules, TFF cassettes) in the assembly concurrently, or in rapid succession, for subsequent tangential flow filtration.

[0062] As used herein, the terms “serial processing”, “processing in series”, “serial operation” and “operation in series” refer to distributing a liquid in a TFF system to one filtration unit (e.g.. filtration module, TFF cassette) at a time, such that the retentate flow of a preceding unit sen es as the feed flow for a subsequent, adjacent unit.

[0063] As used herein, the term “product” refers to a target compound in a feed. Typically, a product will be a biomolecule (e.g.. protein) of interest, such as a monoclonal antibody (mAb).

[0064] As used herein, the term “Tangential Flow Filtration”, abbreviated as “TFF”, refers to the pumping of a liquid solution or suspension tangentially along the surface of the membrane. This is also referred to as cross-flow filtration.

[0065] 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 live cells in a given volume of culture medium, as determined by standard viability assays (such as in pan blue dye exclusion method).

[0066] As used herein, the term “cell viability” means the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term also refers to that portion of cells which are alive at a particular time in relation to the total number of cells, living and dead, in the culture at that time.

[0067] As used herein, “packed cell volume” (PCV), also referred to as “percent packed cell volume” (%PCV), is the ratio of the volume occupied by the cells, to the total volume of cell culture, expressed as a percentage. See, e.g., Stettler et al., 2006, Biotechnol Bioeng. 95:1228- 33. Packed cell volume is a function of cell density and cell diameter: increases in packed cell volume could arise from increases in either cell density or cell diameter or both. Packed cell volume is a measure of the solid content in the cell culture. Solids are removed during harvest and downstream purification. More solids mean more effort to separate the solid material from the desired product during harvest and downstream purification steps. Also, the desired product can become trapped in the solids and lost during the harvest process, resulting in a decreased product yield. Since host cells vary in size and cell cultures also contain dead and dying cells and other cellular debris, packed cell volume is a more accurate way to describe the solid content within a cell culture than cell density or viable cell density. For example, a 2000L culture having a cell density of 50 x IO⁶ cells/ml would have vastly different packed cell volumes depending on the size of the cells. In addition, some cells, when in a growth- arrested state, will increase in size, so the packed cell volume prior to growth-arrest and post growth-arrest will likely be different, due to increase in biomass as a result to cell size increase.

[0068] As used herein, “titer” means the total amount of a polypeptide or protein of interest (which may be a naturally occurring or recombinant protein of interest) produced by a cell culture in a given amount of medium volume. Titer can be expressed in units of milligrams or micrograms of polypeptide or protein per milliliter (or other measure of volume) of medium. “Cumulative titer” is the titer produced by the cells during the course of the culture, and can be determined, for example, by measuring daily titers and using those values to calculate the cumulative titer.

[0069] As used herein, “working volume” refers to the amount of bioreactor volume used for cell culture. For example, a perfusion flow rate can be one working volume or less per day. Perfusion feed medium can be formulated to maximize perfusion nutrient concentration to minimize perfusion rate.

[0070] The present invention discloses a system for perfusion culture of cells. The system comprises a) at least one bioreactor; b) at least one hollow fiber or flat sheet filter unit comprising a first end and a second end on opposite ends of the at least one filter unit; and c) at least one fluidic pump, wherein said fluidic pump is fluidically connected to said at least one filter unit and said bioreactor such that fluid can be pumped from the bioreactor through the at least fluidic pump to the at least one filter back to the bioreactor, and wherein i) said fluidic pump is reversible or ii) said first end and second end of the filter unit are fluidically connected to said at least one fluidic pump via a set of valves arranged to alternatively allow flow in the direction from the at least one biorcactor to the at least one fluidic pump to the first end of the at least one filter unit and back to the bioreactor while blocking flow in the reverse direction or allow flow in the direction from the at least one bioreactor to the at least one fluidic pump to tire second end of the at least one filter unit and back to the bioreactor while blocking flow in the opposite direction. In this way, a means for controlled cycling of flow path direction is provided where the two ends of the filter unit alternate between being an inlet and an outlet.

[0071] In certain embodiments, the systems disclosed herein allow for the flow of cell culture fluid in alternate directions through the one or more filter units while utilizing a reversible fluidic pump.

[0072] In certain embodiments, the systems disclosed herein allow for the flow of cell culture fluid in alternate directions through the one or more filter units while utilizing a unidirectional fluidic pump in combination with a set of valves configured to allow the direction of the flow to be alternated. In these embodiments, reversible pumps operating in a unidirectional mode can also be employed.

[0073] In general, it is preferable for the system to primarily operate in one direction, e.g., where the flow is drawn from the bioreactor through the fluidic pump, through the filter unit and returned to the bioreactor. Operation in this manner will tend to clear any air bubbles pulled in from the bioreactor. When the system is primarily operated in one direction, switching to the other direction for a short period of time will reduce fdter fouling.

[0074] The fluidic pump can suitably comprise any circulation pump, including, but not limited to. peristalic. centrifugal, reciprocating, or rotary lobe. A reciprocating moving pump can include a diaphragm, a membrane or a piston. The fluidic pump may e.g. be a fluid driven diaphragm pump, with a pump chamber and a drive fluid-filled drive chamber separated by a flexible diaphragm, which constitutes the reciprocating moving 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 expels liquid from the pump chamber in an inward stroke and when the fluid pressure is released, the diaphragm flexes back and draws liquid into the pump chamber in an outward stroke. The reciprocating moving member can move back and forth in relation to a pump chamber (also called a cylinder, when the moving member is a piston), forcing fluid (e.g. culture liquid) out from the pump chamber during an inward stroke of the moving member and sucking fluid (e.g. culture liquid) into the pump chamber during an outward stroke. The stroke volume of the fluidic pump corresponds to the fluid (culture liquid). Volume is displaced out from or into the pump chamber during each stroke.

[0075] The pump chamber may e.g. be directly connected to the retentate inlet compartment of the filter unit at a joint. Alternatively, it may be connected via a fluid connector. Such as a short piece of tubing with a diameter large enough not to impede the liquid flow and a volume significantly smaller than the stroke volume of the fluidic pump (e.g. less than 20% of the stroke volume, such as less than 10% or less than 5% of the stroke volume), optionally via an aseptic connector.

[0076] Examples of suitable magnetic levitation pumps include Levitronix® Puralev® Series pump (Levitronix Technologies, Framingham. MA). Examples of suitable diaphragm pumps include Repligen XCell™ ATF pump (Repligen, Waltham, MA). Examples of suitable peristaltic pumps include Watson Marlow Series 500 and Series 600 pumps (Watson Marlow, Wilmington, MA).

[0077] In embodiments involving a set of valves, the set of valves is designed to allow the system of operate in either one of two directions, i.e., where either end of the fdter unit can be the inlet. Each valve in the set of valves can be independently configurable in an open position or a closed position. In certain embodiments, the valves are operated manually. In certain embodiments, the valves are operated automatically. Each valve can be independently operated and allow flow in either direction, while blocking flow in the opposite direction. In this regard, each valve will be in an open position or a closed position, hi one embodiment, a four-valve switching assembly allows for changing hollow fiber flow direction while reducing unneeded valves and reducing the extra parallel flow path lines. During the transition from an initial direction to the opposite direction, all the valves may be open for a brief period of time. [0078] In one embodiment, the valves are modified chromatography valves simplified for use in only one of two directions. By adding single use tubing, an automatable switching design that keeps the pump running in one direction (required for the centrifugal pumps used for low shear) can be obtained and the pump can remain fixed for purposes outside the switching window so the control system design is simple.

[0079] The valves are preferably non-invasive valves which reside outside of the tubing carrying the return flow. In one embodiment, the valve “squeezes” the tubing to restrict and control the flow. Such a valve is non-invasive and provides a commercial advantage since the return line to the reactor is situated through the valve to regulate the applied pressure on the membrane.

[0080] Initial valving designs were intended to enable the hollow fiber switching capability for initial proof of concept demonstration which resulted in the initial six -valve design. This design was functional but presented several opportunities for improvement. These included consolidating the number of valves as well as simplification of the single use tubing.

[0081] In one aspect, the set of valves is a four-valve assembly. An exemplary set of valves is depicted in Fig. 1. In another aspect, the set of valves is a four-valve assembly with one or more valves for bypassing the filter unit. An exemplary set of valves with a bypass valve is depicted in Fig. 3.

[0082] In certain aspects, the four-valve assembly is configured such that two valves can be in the open position while two valves can be in the closed position. In certain aspects, the two valves which are open are parallel, and the two valves which are closed are parallel.

[0083] In certain embodiments, the means for achieving alternating membrane cross flow mechanism can comprise a set of valves and tubing coimections so that configuration of flow is in one of the two membrane flow directions. In this instance, a consolidated valve assembly is not used but instead tubing/valving has been added to die two ends of the filter to allow alternating of flow. It was observed that this design resulted in significant deadlegs due to lengths of tubing leading to the embodiment of the valve assembly that reduced the deadlegs.

[0084] Filter units can be any suitable filter unit, such as a hollow fiber filter unit or flat sheet filter unit. In certain embodiments, the filter unit is not a spiral wound filter unit.

[0085] Filter units are used in mammalian cell perfusion culture for cell and/or recombinant protein product retention. When the cell culture, including cell culture media, cells (whole and lysed), soluble expressed recombinant proteins, host cell proteins, waste products and the like, are introduced to the fdter, depending on the pore size or molecular weight cutoff (MWCO) the filter unit material may retain certain cell culture components on the lumen side (inside) and allow certain components to pass through the filter (permeate) based on the pore size or molecular weight cutoff of tire filter material. The material that is retained (retentate) is returned to the bioreactor. Fresh perfusion cell culture media is added to the bioreactor and permeate is withdrawn from the filter at predetermined intervals or continuously to maintain a desired or constant bioreactor volume. The permeate can be discarded, stored in holding tanks, bags or totes or transferred directly to another unit operation, such as filtration, centrifugation and/or other downstream purification methods or the like.

[0086] In certain embodiments, the at least one filter unit is a hollow fiber filter. In certain aspects of these embodiments, the at least one filter unit is a hollow fiber 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 cut off of 500 kDa or more and can be used to allow the protein to pass through into the permeate. Ultrafiltration hollow fibers typically have a pore size range of 0.01 pm to 0.1 pm or a molecular weight cut off of 300 kDa or less, and can be used to retain the desired protein in the retentate and return it back to the bioreactor. This can be used, for example, to concentrate the recombinant protein product for harvest. Such filters are available commercially, 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, Calif.).

[0087] In certain embodiments, the at least one filter unit is a flat sheet.

[0088] In certain embodiments, the filter unit is selected from the group consisting of a hollow fiber, flat sheet TFF cassette or plate and frame design

[0089] For the systems and methods of the invention employing hollow fiber filter units, the hollow fiber filter is generally made as a cartridge tiiat comprises multiple hollow fibers (HF) tiiat run, in parallel, the length of the cartridge and are embedded at each end of the cartridge (preferably with a potting agent); the lumens at the end of the HFs are retained open, thus forming a continuous passage through each of the lumens from one end of tiie cartridge to the other, i.e., from a cartridge entrance end to a cartridge exit end. The hollow fibers are enclosed by the outer wall of the cartridge (i.e., the cartridge wall) and a potting layer at their ends. As a result, there is a chamber bounded by the cartridge wall and the outer walls of the HFs. That chamber can be used as the filtrate chamber. The intraluminar (internal) spaces of the HFs are considered collectively to constitute part of the retentate chamber in each of the present systems.

[0090] In the systems described herein, the flow between the ends is reversible. In other words, one end of the hollow fiber filter unit can be considered the inlet end or outlet end depending on the direction of the flow. One end may be directly connected to the fluidic pump at a joint.

[0091] In one embodiment, the filter system comprises at least one hollow fiber filter component having a pore size or molecular weight cut off (MWCO) that retains the recombinant protein in the bioreactor and at least one hollow fiber filter component having a pore size or molecular weight cut off that does not retain the recombinant protein in the bioreactor. In another embodiment, the molecular weight cutoff of at least one hollow fiber filter component that retains the recombinant protein in the bioreactor is 300 kDa or less. In another embodiment the molecular weight cutoff of at least one hollow fiber filter component that does not retain the recombinant protein in the bioreactor is at least 500 kDa. In another embodiment at least one hollow fiber filter component that retains the recombinant protein in the bioreactor is an ultrafilter and at least one hollow fiber filter component that does not retain 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 comprises a spacer between at least two of the hollow fiber filter components.

[0092] In certain 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 certain embodiments, the bioreactor is made of stainless steel. [0093] In certain embodiments, the bioreactor comprises an inflatable flexible bag, resting on a movable support, and the length of tubing is a flexible length of tubing. The system described herein is particularly suitable for bioreactors which are agitated by moving the entire bioreactor vessel (e.g., an inflatable flexible bag), since only one length of tubing is needed to connect to the bioreactor, which keeps the number of moving connections to the vessel low and reduces entangling of these.

[0094] In certain embodiments, the system further comprises a permeate pump. This is to allow removal of spent media.

[0095] In certain embodiments, the system further comprises one or more pressure monitoring devices. For example, the system may also comprise a drive fluid pressure transducer arranged to measure a drive fluid pressure in the at least one fluidic pump. [0096] In certain embodiments, the system comprises an inlet pressure transducer arranged to measure the pressure at the end that is primarily for retentate inlet. This pressure transducer can be located at the end of the filter unit primarily serving as the retentate inlet or at any point between that end and the inlet control valve. In embodiments without an inlet control valve, it can be located at any point between the retentate inlet end and the branch point. The system can also (or alternatively) comprise an outlet pressure transducer arranged to measure the pressure at the end primarily serving as the retentate outlet. This pressure transducer can be located at the end of the filter unit primarily serving as the retentate outlet or at any point between that end and the outlet control valve. In embodiments without an outlet control valve, it can be located at any point betw een the end serving primarily as the retentate outlet and the branch point.

[0097] In certain aspects, the s stem comprises at least three pressure monitoring devices configured in at least three positions: 1) one positioned at end 1; 2) one positioned at end 2; and 3) one positioned in the permeate flow path. The pressure devices at ends 1 and end 2 will alternately indicate feed and/or retentate pressure depending on the flow direction. The pressure transmitter can be arranged so that inlet pressure can be monitored and used to control switching in the event of an elevated feed pressure measurement. In certain embodiments, the valves reverse position upon detection of high inlet pressure.

[0098] The pressure transducers can be either manually readable manometers or transducers arranged to transmit pressure data to a control unit, e.g. by electrical, electromagnetic or optical means. The pressure transducer at the end serving primarily as an outlet can e.g. be employed for an exact determination of the transmembrane pressure over the filter unit and membrane, respectively.

[0099] In some embodiments, the fluidic pump is a fluid-driven diaphragm pump and the system comprises a drive fluid pressure transducer arranged to measure the driv e fluid pressure in the fluidic pump, mounted e.g. in the drive fluid supply line. The drive fluid pressure transducer can be either a manually readable manometer or a transducer arranged to transmit pressure data to a control unit, e.g. by electrical, electromagnetic or optical means. The drive fluid can be a gas (e.g. air) or a liquid.

[0100] In certain embodiments, the fluidic pump is a gas-driven diaphragm pump and the system comprises a gas pressure transducer arranged to measure the gas pressure in the pump, mounted e.g. in the gas supply line. The gas can in particular be air, in which case the gas (drive fluid) supply is a source of compressed air.

[0101] In some embodiments, the fluidic 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 tire fluidic pump at the side of the fluid supply line towards the side of the filter unit and its retentate inlet end. The pressure at the filter unit end serving primarily as the inlet side will be equal to the pressure measured at the pressure transducer at the side of the fluid supply line. Thus, the fluid pressure of the bioreactor fluid at the side of the end serving primarily as the retentate inlet, and thereby the filtration process and the transmembrane pressure, can effectively be measured and controlled by employing the drive fluid pressure transducer, which is not in contact with the bioreactor fluid. This design gives advantages when designing a system with single-use components in contact with the bioreactor fluid. It reduces cost and complexity as the drive fluid pressure transducer can be re-usable and does not need to be sterilized. Hence also non-sterilizable high-performance sensors can be used and if a sterilizable high-performance sensor is used it does not need to be recalibrated after sterilization.

[0102] In some embodiments, the permeate outlet port comprises a permeate pressure transmitter or transducer. The permeate pressure transducer can be capable of measuring the pressure on the end of the filter unit primarily serving as the permeate side, which, in combination with data from the pressure transducers measuring inlet and/or outlet pressures, can be used to calculate the transmembrane pressure over the filter unit. The permeate pressure transducer can also be used in a feedback loop for control of the transmembrane pressure by the operation of the penneate pump or by some other permeate pressure control means (e.g. hydrostatic pressure control). The transducer is also useful for control of the backflush cycle during the outward stroke of the fluidic pump.

[0103] In certain embodiments, the system further comprises a pressure transmitter located at each end of the filter unit. The pressure transducers can be arranged so that inlet pressure can be monitored and used to control switching in the event of elevated feed pressure measurement. In certain embodiments, the valves reverse position upon detection of high inlet pressure.

[0104] In certain embodiments, the system further comprises one or more flow meters. In one aspect, a flow meter is configured to be positioned in the feed and/or return flow path. In another aspect, flow meters are configured to be positioned in at least two monitoring positions: 1) a flow meter that is positioned in the feed and/or return flow path; and 2) a flow meter that is in the penneate flow path. The position of the flow meter in the feed and/or return flow path can effectively be either in feed or retentate section with the ability’ to calculate mathematically the feed pressure using the value from permeate flow to add or subtract respectively. In certain embodiments, the method utilizes a system which includes a retentate flow meter that is positioned on the return portion of tubing site after the ARTF valve (4 valve assembly) carrying retentate from the system to the bioreactor to control the amount of retentate that is recirculated.

[0105] In certain embodiments, the system comprises at least one control unit, arranged to control at least one of the fluidic pump and the inlet/outlet control valves depending on pressure data received from at least one of the inlet/outlet pressure transducers. The control unit(s) can suitably be electrically, electromagnetically (e.g. by wireless communication), optically (e.g. via optical fibers) or pneumatically connected to at least one of the fluidic pump and the inlet/outlet control valves and to at least one of the inlet/outlet pressure transducers. The control unit(s) can be e.g. 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 parameter! s) as a signal/signals to one or more control valves and/or a pump. The control unit can be one integrated control unit, arranged to control both the fluidic pump and the inlet/outlet control valve or the system may comprise a main control unit (or a valve control unit) and a pump control unit (not shown). The pump control unit may e.g. be arranged to control the flow and pressure profile generated by the fluidic pump via one or more of the stroke frequency, the stroke length and the velocity of the moving member during the inward and outward strokes. In the case of a fluid driven diaphragm pump, the stroke frequency, the stroke length and the diaphragm velocity can be controlled e.g. via one or more valves on the drive fluid supply line. As noted above, the features of the pump control unit can also be integrated into a main control unit, or vice versa — the control valve(s) may also be controlled by an integrated pump control unit. Alternatively, the control valve(s) can be preset or manually adjusted such that only a pump control unit is needed. The control unit may also be connected to the permeate pump and/or the permeate pressure transducer for control of the transmembrane pressure.

[0106] In certain embodiments, the system has a graphical display to indicate the direction of the flow. In one embodiment, the graphical display shows an arrow or other indicator of the direction of the flow. In one embodiment, a graphical display is utilized via a Distributed Control System (DCS), for example Delta V™ (Emerson. St. Louis, MO).

[0107] In certain embodiments, the system further comprises a control unit with a graphical display unit and is configured to perform control of critical process parameters like flow rates, pressure settings and monitoring for alarm conditions. This system monitors the direction of flow so that it can adjust the inputs being used to properly calculate transmembrane pressure (aka TMP) and monitors the recirculating inlet pressure whose monitoring point will alternate from End 1 to End 2 depending on flow direction. In certain aspects, this sy stem can also display the direction of flow and records flow direction in data along with critical process parameters.

[0108] In certain embodiments, the system further comprises a control unit configured to control the at least one fluidic pump and/or the set of valves depending on pressure data received from a pressure transducer. In certain aspects, the control unit is arranged to control each valve within the set of valves.

[0109] In certain embodiments, the system further comprises a wye(s) or other means to allow a second filter to be set up as a backup. This configuration allows the backup filter to be used with the instrumentation and pump if the primary filter becomes unusable.

[0110] The connections between the various components of the system can be achieved e.g. by tubing, such as flexible tubing or stainless steel tubing, with the valves in-line with the tubing or adjacent to the branch point or the filter unit ends. The branch point may be e.g. a three-way tubing connector or a manifold. The connection between the branch point and the bioreactor may be a single line in the form of a length of tubing, such as flexible tubing. The tubing length may be connected to the inner volume of the bioreactor vessel through a port in the vessel wall. The valves may be of any suitable check valve type, e.g. flap, ball, slit disk valves, etc.

[0111] During the outward stroke of the fluidic pump, culture liquid will be drawn from the bioreactor via the length of tubing and the branch point to the end of the filter unit primarily serving as retentate inlet. At the same time, the outward stroke generates a negative pressure on the end of the filter unit serving as the retentate side, which causes a certain amount of permeate to backflush the filter membrane and thus clean the membrane from clogged cells and other material. During the inward stroke of the fluidic pump, culture liquid is pushed through the end of the filter unit primarily serving as the retentate side, producing permeate that can be withdrawn, and further via the end serving primarily as the retentate outlet, the branch point and the length of tubing back to the bioreactor. In certain embodiments, the systems described herein are advantageous in that they do not require a reversible pump.

[0112] The construction materials used in the system can suitably be compatible with commonly used sterilization methods, such as e.g. gamma irradiation and/or autoclaving. For reusable components, stainless steel (e.g. with corrosion resistance at least equivalent to 316 L) or engineering plastics such as polysulfone. PEEK, etc. may be used, while for single-use components, plastics, such as e.g. polysulfone, polypropylene, polyethylene or ethylene copolymers, may be used.

[0113] To avoid infection of the cell culture, the bioreactor and all components in contact with the culture fluid may be sterilized before cultivation. The system or parts of the system may be assembled and sterilized by autoclaving or radiation, or one or more components may be presterilized and assembled in a sterile system. To facilitate assembly, the sterilized system parts or components may be equipped with aseptic connectors, e.g., of the Ready Mate ty pe (GE Healthcare). Alternatively, the sterilized system parts/components may be contained in aseptic packages and assembled in a sterile clean room.

[0114] ARTF systems useful for performing the methods described herein can further contain one or more additional components useful for performing ARTF processes including, but not limited to, the following, examples of which are known in the art: one or more sampling ports, a T-line (e.g., for in-line buffer addition), a pressure sensor, a diaphragm for a pressure sensor, or a valve sensor to indicate whether any valves in the system are open or closed. In a particular embodiment, the ARTF system includes a sampling port (e.g.. sanitary sampling port) at one or more locations in the system. For example, sampling ports can be included at the end of the retentate line, the permeate line, or both. Typically, the sampling port will be located on the manifold segment in a filtration module. [0115] Existing TFF devices used in perfusion systems include XCell™ ATF System (Repligen, Waltham, MA) and KrosFlo® Perfusion System (Spectrum Laboratories, Rancho Dominguez, CA), which are hollow fiber devices. These devices contain open feed channels, so as to limit physical damage to cells in the feed stream, and both devices require high crossflow rates to minimize fouling (i.e., the accumulation of particles along the wall of membrane).

[0116] In some embodiments, the system is partially or entirely composed of single-use components, which can suitable be presterilized e.g. by gamma irradiation or autoclaving and then connected together using aseptic connectors, such as e.g. Ready Mate (GE Healthcare). It is also possible to contain the filter unit, check valves and control valves, as well as inlet/outlet pressure transducers (not shown), in an outer housing. It is also possible to contain the fluidic pump in the same housing or in a separate housing. The fluidic pump may be single-use or reusable and it may even be assembled from a single use part comprising the pump chamber and the diaphragm and a reusable part comprising the drive chamber and the drive fluid supply line with the drive fluid pressure transducer. The single use part of the fluidic pump may in this case also be integrated with the filter unit.

[0117] In an exemplary embodiment, the flow of bioreactor material passes from the bioreactor vessel to the filtering system and the return flow- of the bioreactor fluid passes from the filtering system back to the bioreactor vessel. A permeate flow (for example, a metabolic w aste material flow) is stripped from the How of bioreactor material by the hollow fiber perfusion filtering system and carried away by waste material tubing. The metabolic waste, as well as associated proteins, are drawing from the hollow⁷ fiber perfusion tangential flow⁷ system by a permeate pump into a w aste container.

[0118] FIG. 1 A depicts a representative system of the disclosure having a single hollow fiber filter. The solid black lines represent unidirectional flow. Specifically, the pump circulates the bioreactor fluid through the pump and to the set of valves. The bioreactor fluid is also pumped from the set of valves and returned to die bioreactor. The solid lines represent bidirectional flow⁷, i.e., the flow can be in either direction. In FIG. 1 A, there are four valves which operate in pairs. When the valves depicted in grey are in the open position (and the valves depicted in black are closed), the flow proceeds through the first grey valve to the hollow fiber tangential flow filter in upflow mode, passes through the second grey valve and back to the bioreactor vessel. The flow changes direction of the fluid in the flowpath. When the valves depicted in black are in the open position (and the valves depicted in grey are closed), the flow proceeds through a first black valve to the hollow fiber tangential flow filter in downflow mode, passes through the second black valve and back to the bioreactor vessel. The permeate flow⁷ is continually removed from the bioreactor fluid which flows through the tangential flow⁷ hollow fiber filter. [0119] FIG. IB depicts a representative system of the disclosure having two pumps and two hollow fiber filters in parallel. The solid black lines represent unidirectional flow. Specifically, the pump circulates the bioreactor fluid through each of the two pumps and to each of two set of valves. The bioreactor fluid is also pumped from the sets of valves and returned to the bioreactor. The solid lines represent bidirectional flow, i.e., the flow can be in either direction. In FIG. IB, there are six valves in each set, two valves depicted in grey and four valves depicted in black. When the valves depicted in grey are in the open position (and the valves depicted in black are closed), the flow proceeds through the first grey valve to the hollow fiber tangential flow fdter in upflow mode, passes through the second grey valve and back to the bioreactor vessel. When the four valves depicted in black are in the open position (and the valves depicted in grey are closed), the flow proceeds through a first pair of black valve to the hollow fiber tangential flow filter in downflow mode, passes through the second pair of black valves and back to the bioreactor vessel. The permeate flow is continually removed from the bioreactor fluid which flows through the tangential flow hollow fiber filter. [0120] The present disclosure also provides a method for perfusion culture of cells, comprising: a) providing a system as described herein; b) adding cell culture medium and cells to said at least one bioreactor; c) cultivating cells in said at least one bioreactor under agitation; d) operating, during cultivation, said at least one fluidic pump to withdraw fluid from said at least one bioreactor via tubing to said set of valves to said first end of said at least one hollow fiber filter unit and to return fluid from said second end of said at least one hollow filter unit to said at least one bioreactor and; c) reversing the set of valves so that the at least fluidic pump w ithdraws fluid from said at least one bioreactor via tubing to said set of valves to said second end of said at least one hollow- fiber and to return fluid from said first end of said at least one hollow filter unit to said at least one bioreactor.

[0121] In some embodiments, the method comprises adding at least one fluid, such as a cell culture medium, to the bioreactor during cultivation. This has the advantage that culture liquid removed via the filter unit can be replenished and that fresh nutrients and other reagents can be supplied to the culture. The liquid feed can be any liquid (e.g., a biological liquid) that contains particles (c.g., viral particles, host cell proteins) to be filtered. For example, the liquid feed can contain a target molecule of interest (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 or other host cell expressing a monoclonal antibody (mAb)). In a particular embodiment, the target molecule in the liquid feed is a mAb and the non-target molecules are host cell proteins (HCPs) (e.g., proteins from host hybridoma cells). Non-target proteins are generally a heterogeneous mixture of proteins of varying 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.

[0122] The bioreactor can be inoculated with at least 0.5 x 10⁶ up to and beyond 3.0 x 10⁶ viable cells/mL in a serum-free culture medium, for example 1.0 x 10⁶ viable cells/mL.

[0123] In the methods disclosed herein, the flow may be reversed based on manual input or automatically based on one or more predetermined parameters or time points.

[0124] The predetermined parameters may be reached by achieving some desired characteristic, attribute or performance milestone of the cell culture, such as viable cell density, packed cell volume or titer, or some characteristic of the ARTF system, such as pressure, or the predetermined parameter may be a time point.

[0125] In one embodiment, the predetermined parameter may be reached when the viable cell density is greater than or equal to 1 x 10⁶ viable cells/ml. In one embodiment, predetermined parameter may be reached when the viable cell density is at least 20 x 10⁶ viable cells/ml to 30 x 10⁶ viable cells/ml. In one embodiment, the predetermined parameter may be reached when the packed cell volume is less than or equal to 35%. In one embodiment, the predetermined parameter may be reached when the packed cell volume is less than or equal to 30%.

[0126] In one embodiment, the predetermined parameter is pressure. In particular, the Pfeed can be monitored to see if it rises above a certain level. For example, the ARTF pressure can be monitored and the flow reversed once the Pfeed reaches 10 psig. The ARTF flow can be cycled based on a predetermined time. The predetermined time can be for 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 35 mins, 40 mins, 45 mins, 50 mins, 55 mins, 1 hour, 2 hours, 3 hours, or 4 hours or more. The ARTF flow can also be cycled based on a parameter of the ARTF system. For example, the ARTF flow can be switched for a time that is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the time it took for the hollow fiber filter to foul. The ARTF flow can also be reversed if the Pfeed goes up sooner than the predetermined time or calculated time.

[0127] The predetermined parameter may be based on a time point. The time point may be measured in hours, days, weeks, or months following a triggering event or action. A triggering event or action may be hours or days in culture, hours or days following an event such as reaching a viable cell density, packed cell volume, titer, inoculating the bioreactor or collecting a harvest permeate. In one embodiment, the predetermined parameter may be reached within 12 hours to 25 days following a triggering event or action. In one embodiment. the predetermined parameter may be reached within 24 to 72 hours following a triggering event or action. In one embodiment, the predetermined parameter may be reached within 4 days of the triggering event or action. In one embodiment, predetermined parameter may be reached 5 days or more following the triggering event or action. In one embodiment, the predetermined parameter may be reached at least 25 days following the triggering event or action. In one embodiment, a first predetermined parameter may be reached within 5 to 25 days following inoculation of the bioreactor. In one embodiment, a first predetermined parameter may be reached within 10 to 12 days following inoculation of the bioreactor. In one embodiment, a second predetermined parameter may be reached within 12 to 72 horns following the collection of a harvest permeate. In one embodiment, a second predetermined parameter may be reached within 24 to 72 hours following the collection of a harvest permeate. In one embodiment, a second predetermined parameter may be reached within 24 to 48 hours following the collection of a harvest permeate.

[0128] Once the predetermined parameter has been reached, the flow is reversed for a set time to allow the filter to be back flushed. The time for reversal of the flow can be from 10 mins, 20 mins, 30 mins, or up to 1 hour, 2 hours. 3 hours. 4 hours, or more.

[0129] In certain embodiments, the method further comprises controlling the set of valves to reverse the flow of fluid once a pressure reading from one of the pressure transmitters exceeds a preset value. In some embodiments, the method comprises calculating a transmembrane pressure from data provided by the inlet/outlet pressure transducers or from at least one of the inlet/outlet pressure transducers and the permeate pressure transducer and controlling at least one of the fluidic pump, the permeate pump and the inlet/outlet control valves to keep the transmembrane pressure betw een preset upper and lower limits. This has the advantage that mass transport across the filter membrane can be maximized and fouling and concentration polarization can be minimized. It is also possible to control the pressure according to more complex algorithms, e.g. producing desirable transmembrane pressure versus time profiles during both the outward and inward strokes.

[0130] In certain embodiments, the method provides for the reversing the flow path which is triggered upon reaching a preset High Pfeed value. Elevated Pfeed values are indicative of filter performance degradation and triggers an automated flow direction change, dependent upon process specific factors and equipment design.

[0131] In certain embodiments, the flow is reversed at a preset time interval. Specific timing is dependent upon process specific factors and equipment design. Typical alternating durations can be minutes or hours; generally more than 4 hours or more will result in switching based on High Pfeed.

[0132] In certain embodiments, the cell culture medium is a serum -free chemically defined cell culture medium. 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 a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein, or a cytokine.

[0133] Samples from the cell culture can be monitored and evaluated using any of the analytical techniques known in the art. A variety of parameters including recombinant protein and medium quality and characteristics can be monitored for the duration of the culture. Samples can be taken and monitored intermittently at a desirable frequency, including continuous monitoring, real time or near real time.

[0134] In certain embodiments, the recombinant protein is purified from the harvest permeate by one or more of flocculation, precipitation, centrifugation, depth filtration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode anion exchange chromatography, hydrophobic interaction chromatography or hydroxyapatite chromatography. In one embodiment, the methods described herein further comprise taking samples during the purification process, evaluating the samples to quantitatively and/or qualitatively monitor characteristics of the recombinant protein and the purification process. In one embodiment, the samples are quantitatively and/or qualitatively monitored using process analytical techniques. In one embodiment, the recombinant protein is formulated into a pharmaceutically acceptable formulation.

[0135] The purified proteins can then be "‘formulated”, meaning buffer exchanged, sterilized, bulk -packaged, and/or packaged for a final user. Suitable formulations for pharmaceutical compositions include those described in Remington 's Pharmaceutical Sciences, 18th ed.

1995, Mack Publishing Company, Easton. Pa.

[0136] As used herein, “cell culture” or “culture” is meant the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells arc known in the art. See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells may be cultured in suspension or while attached to a solid substrate.

[0137] As used herein, the terms “cell culturing medium" (also called “culture medium. “ “cell culture media, “ “tissue culture media, “) refers to any nutrient solution used for growing cells, e.g., animal or mammalian cells, and which generally provides at least one or more components from the following: an energy' source (usually in the form of a carbohydrate such as glucose); one or more of all essential amino acids, and generally the twenty basic amino acids, plus cysteine; vitamins and/or other organic compounds typically required at low concentrations; lipids or free fatty acids; and trace elements, e.g.. inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.

[0138] The nutrient solution may optionally be supplemented with additional components to optimize growth of cells, such as hormones and other growth factors, such as insulin, transferrin, epidermal growth factor, serum, and the like; salts, such as calcium, magnesium and phosphate, and buffers, e.g., HEPES; nucleosides and bases, such as adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysates, such as hydrolyzed plant or animal protein (peptone or peptone mixtures, which can be obtained from animal byproducts, purified gelatin or plant material); antibiotics, such as gentamycin; poly amines, such as putrescine, spermidine and spermine (see WIPO Publication No. WO 2008/ 154014) and pyruvate (see U.S. Pat. No. 8,053,238), antiapoptotic compounds, e.g., MDL 28170. cypennethrin, cyclosporine A, BBMP, Bongkrekic acid. S- 15176 difumarate, cyclic pifithrin- a. pifithrin mu, BI-6C9, NSCI, NS3694 or Necrostatin-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.

[0139] A “perfusion” cell culture medium refers to a cell culture medium that is typically used in cell cultures that are maintained by perfusion or continuous culture methods and is sufficiently complete to support the cell culture during this process. Perfusion cell culture medium formulations may be richer or more concentrated than base cell culture medium formulations to accommodate the method used to remove the spent medium. Perfusion cell culture medium can be used during both the growth and production phases.

[0140] A “production” cell culture medium refers to a cell culture medium that is typically used in cell cultures during the transition when exponential growth is ending and protein production takes over, “transition” and/or “product” phases, and is sufficiently complete to maintain a desired cell density, viability and/or product titer during this phase.

[0141] Concentrated cell culture medium can contain some or all of the nutrients necessary to maintain the cell culture; in particular, concentrated medium can contain nutrients identified as or known to be consumed during the course of the production phase of the cell culture. Concentrated medium may be based on just about any cell culture media formulation. Such a concentrated feed medium can contain some or all the components of the cell culture medium at, for example, about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X. 30X, 50X. lOOx. 200X, 400X, 600X, 800X, or even about 1000X of their nonnal amount. [0142] The components used to prepare cell culture medium may be completely milled into a powder medium formulation: partially milled with liquid supplements added to the cell culture medium as needed; or added in a completely liquid form to the cell culture.

[0143] Cell cultures can also be supplemented with independent concentrated feeds of particular nutrients which may be difficult to formulate or are quickly depleted in cell cultures. Such nutrients may be amino acids such as tyrosine, cysteine and/or cy stine (see e.g., International Patent Application Publication No. 2012/145682). For example, a concentrated solution of tyrosine can independently fed to a cell culture grown in a cell culture medium containing tyrosine, such that the concentration of tyrosine in the cell culture does not exceed 8 mM. In another example, a concentrated solution of tyrosine and cystine is independently fed to the cell culture being grown in a cell culture medium lacking tyrosine, cystine or cysteine. The independent feeds can begin prior to or at the start of the production phase. The independent feeds can be accomplished by fed batch to the cell culture medium on the same or different days as the concentrated feed medium. The independent feeds can also be perfused on the same or different days as the perfused medium.

[0144] "Serum-free" applies to a cell culture medium that does not contain animal sera, such as fetal bovine serum. Various tissue culture media, including defined culture media, are commercially available, for example, any one or a combination of the following cell culture media can be used: RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM). Minimum Essential Medium Eagle. F-12K Medium. Ham's F12 Medium. Iscove's Modified Dulbecco's Medium, McCoy's A Medium, Leibovitz's L-15 Medium, and serum-free media such as EX-CELL™ 300 Series (JRH Biosciences, Lenexa, Kansas). MCDB 302 (Sigma Aldrich Corp., St. Louis, MO), among others. Serum-free versions of such culture media are also available. Cell culture media may be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements and the like, depending on the requirements of the cells to be cultured and/or the desired cell culture parameters. Customized cell culture media can also be used.

[0145] As used herein, the term “host cell” is understood to include a cell that has been genetically engineered to express a polypeptide of interest. Genetically engineering a cell involves transfecting, transforming or transducing the cell with a nucleic acid encoding a recombinant polynucleotide molecule (a “gene of interest”), and/or otherwise altering (e g., by homologous recombination and gene activation or fusion of a recombinant cell with a nonrecombinant cell) so as to cause the host cell to express a desired recombinant polypeptide. Methods and vectors for genetically engineering cells and/or cell lines to express a polypeptide of interest are well known to those of skill in the art; for example, various techniques are illustrated in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York. 1988. and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press. 1989); Kaufman, R.J., Large Scale Mammalian Cell Culture. 1990. pp. 15-69. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic makeup to the original parent cell, so long as the gene of interest is present. A cell culture can comprise one or more host cells.

[0146] Animal or mammalian cells are cultured in a medium suitable for the particular cells being cultured and which can be determined by the person of skill in the art without undue experimentation. Commercially available media can be utilized and include, but is not limited to, Iscove’s Modified Dulbecco’s Medium, RPMI 1640, Minimal Essential Medium-alpha. (MEM-alpha). Dulbecco’s Modification of Eagle’s Medium (DMEM), DME/F12. alpha MEM, Basal Medium Eagle with Earle’s BSS, DMEM high Glucose, with Glutamine. DMEM high glucose, without Glutamine. DMEM low Glucose, without Glutamine. DMEM:F12 1:1. with Glutamine. GMEM (Glasgow’s MEM), GMEM with glutamine, Grace’s Complete Insect Medium, Grace’s Insect Medium, without FBS, Flam’s F-10, with Glutamine, Flam’s F-12. with Glutamine. IMDM with HEPES and Glutamine, 1MDM with HEPES and without Glutamine, IP41 Insect Medium, 15 (Leibovitz) (2x), without Glutamine or Phenol Red, 15 (Leibovitz), without Glutamine, McCoy’s 5 A Modified Medium, Medium 199. MEM Eagle, without Glutamine or Phenol Red (2x). MEM Eagle-Earle’s BSS, with glutamine. MEM Eagle-Earle’s BSS, without Glutamine. MEM Eagle-Flanks BSS, without Glutamine, NCTC-109. with Glutamine. Richter’s CM Medium, with Glutamine, RPMI 1640 with HEPES, Glutamine and/or Penicillin- Streptomycin, RPMI 1640, with Glutamine, RPMI 1640, without Glutamine. Schneider’s Insect Medium or any other media known to one skilled in the art, which are formulated for particular cell types. To the foregoing exemplary media can be added supplementary components or ingredients, including optional components, in appropriate concentrations or amounts, as necessary or desired, and as would be known and practiced by those having in the art using routine skill.

[0147] A wide variety of mammalian cell lines suitable for growth in the systems and methods disclosed herein arc available from the American Type Culture Collection (Manassas, Va.) and commercial vendors. Examples of cell lines commonly used in the industry include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al, J. Gen Virol. 36: 59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23 : 243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75): human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562. ATCC CCL51); TRI cells (Mather et al., Annals N.Y Acad. Sci. 383:44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells, and a number of other cell lines and Chinese hamster ovary (CHO) cells. [0148] Large-scale production of proteins for commercial applications is typically carried out in suspension culture. Therefore, mammalian host cells used to generate the recombinant mammalian cells described herein can, but need not be, adapted to growth in suspension culture. A variety' of host cells adapted to growth in suspension culture are known, including mouse myeloma NSO cells and CHO cells from CHO-S, DG44, and DXB11 cell lines. Other suitable cell lines include mouse myeloma SP2/0 cells, baby hamster kidney BF1K-21 cells, human PER.C6* cells, human embryonic kidney F1EK-293 cells, and cell lines derived or engineered from any of the cell lines disclosed herein.

[0149] CHO cells are widely used to produce complex recombinant proteins, including CHOK1 cells (ATCC CCL61). The dihydrofolate reductase (DHFR) -deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77: 4216-4220), DXB11 and DG-44, are desirable CHO host cell lines because the efficient DHFR selectable and amplifiable gene expression system allows high level recombinant protein expression in these cells (Kaufman R. J. (1990), Meth Enzymol 185:537-566). Also included are the glutamine synthase (GS)- knockout CHOK1SV cell lines, making use of glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection. Other suitable CHO host cells could include, but are not limited to the following (ECACC accession numbers in brackets): CHO (85050302), CHO (PROTEIN FREE) (00102307). CHO-K1 (85051005), CHO-K1/SF (93061607). CHO/dhFr- (94060607), CHO/dhFr-AC-free (05011002), RR-CHOKI (92052129).

[0150] The present disclosure provides systems and methods which can be used for culturing cells expressing a “protein of interest”; “protein of interest” includes naturally occurring proteins, recombinant proteins, and engineered proteins (e.g., proteins that do not occur in nature and which have been designed and/or created by humans). A protein of interest can, but need not be, a protein that is known or suspected to be therapeutically relevant. Particular examples of a protein of interest include antigen binding proteins (as described and defined herein), peptibodies (/.e., a molecule comprising peptide(s) fused either directly or indirectly to other molecules such as an Fc domain of an antibody, where the peptide moiety specifically binds to a desired target; the peptide(s) may be fused to either an Fc region or inserted into an Fc-Loop. or a modified Fc molecule, for example as described in U.S. Patent Application Publication No. US2006/0140934 incorporated herein by reference in its entirety), fusion proteins (e.g. , Fc fusion proteins, wherein a Fc fragment is fused to a protein or peptide, including a peptibody), cytokines, growth factors, hormones and other naturally occurring secreted proteins, as well as mutant forms of naturally occurring proteins.

[0151] As used herein, the terms “polypeptide” and “protein” (e.g., as used in the context of a protein of interest or a polypeptide of interest) are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms can also encompass amino acid polymers that have been modified, e.g., by the addition of carbohydrate residues to form glycoproteins, or phosphorylated. Polypeptides and proteins can be produced by a naturally -occurring and non-recombinant cell, or polypeptides and proteins can be produced by a genetically -engineered or recombinant cell. Polypeptides and proteins can comprise molecules having the amino acid sequence of a native protein, or molecules having deletions from, additions to. and/or substitutions of one or more amino acids of the native sequence.

[0152] The terms “polypeptide” and “protein” encompass molecules comprising only naturally occurring amino acids, as well as molecules that comprise non-naturally occurring amino acids. Examples of non-naturally occurring amino acids (which can be substituted for any naturally-occurring amino acid found in any sequence disclosed herein, as desired) include: 4-hydroxyproline. y-carboxyglutamate. e-N,N,N-trimethyllysine. e-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, o-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide 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.

[0153] As used herein, the term “antigen binding protein” is used in its broadest sense and means a protein comprising a portion that binds to an antigen or target and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include a human antibody, a humanized antibody; a chimeric antibody; a recombinant antibody; a single chain antibody ; a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab’)₂ fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgGl antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4 antibody, and fragments thereof. The antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody -derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, e.g., Komdorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptide antibody mimetics (“PAMs”) can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components as a scaffold.

[0154] An antigen binding protein can have, for example, the structure of a naturally occurring immunoglobulin. An “immunoglobulin” is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light’’ (about 25 kDa) and one “heavy ” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu. delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively.

[0155] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1. CDR1, FR2, CDR2. FR3, CDR3 and FR4. The assignment of amino acids to each domain can be done in accordance with the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5^th Ed., US Dept, of Health and Human Services, PHS. NIH, NIH Publication no. 91-3242, (1991). As desired, the CDRs can also be redefined according an alternative nomenclature scheme, such as that of Chothia (see Chothia & Lesk. 1987. J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883 or Honegger & Pluckthun, 2001, J . Mol. Biol. 309:657-670).

[0156] The term “antibody” includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or to an antigen-binding region thereof that competes with the intact antibody for specific binding, unless otherwise specified.

Additionally, the term “antibody” refers to an intact immunoglobulin or to an antigen binding portion thereof that competes with the intact antibody for specific binding, unless otherw ise specified. Antigen binding portions can be produced by recombinant DNA techniques or by enzy matic or chemical cleavage of intact antibodies and can form an element of a protein of interest. Antigen binding portions include, inter alia, Fab, Fab’, F(ab')2, Fv, domain antibodies (dAbs), fragments including complementarity determining regions (CDRs), singlechain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and poly peptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.

[0157] A Fab fragment is a monovalent fragment having the VL. VH. CL and CHI domains; a F(ab’)2 fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the VH and CHI domains; an Fv fragment has the VL and VH domains of a single arm of an antibody; and a dAb fragment has a V_H domain, a VL domain, or an antigen-binding fragment of a V_H or V_L domain (U.S. Pat. Nos. 6,846,634, 6.696.245, U.S. App. Pub. Nos. 2005/0202512, 2004/0202995. 2004/0038291, 2004/0009507, 2003/0039958. Ward et al., 1989, Nature 341:544-546). [0158] A single-chain antibody (scFv) is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48; and Poljak et al., 1994, Structure 2: 1121-23). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.

[0159] One or more CDRs can be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein. An antigen binding protein can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) noncovalently. The CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest.

[0160] An antigen binding protein can have one or more binding sites. If there is more than one binding site, the binding sites can be identical to one another or can be different. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, while a “bispecific” or “bifunctional” antibody has tw o different binding sites.

[0161] For purposes of clarity, and as described herein, it is noted that an antigen binding protein can, but need not, be of human origin (e.g., a human antibody), and in some cases will comprise a non-human protein, for example a rat or murine protein, and in other cases an antigen binding protein can comprise a hybrid of human and non-human proteins (e.g., a humanized antibody).

[0162] A protein of interest can comprise a human antibody. The tenn “human antibody” includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immuno globulin sequences (a fully human antibody). Such antibodies can be prepared in a variety of ways, including through the immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes, such as a mouse derived from a Xenomouse®, UltiMab™, or Velocimmune® system. Phage-based approaches can also be employed.

[0163] Alternatively, a protein of interest can comprise a humanized antibody. A “humanized antibody” has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject. In one embodiment, certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody. In another embodiment, the constant domain(s) from a human antibody are fused to the variable domain(s) of a non- human species. Examples of how to make humanized antibodies can be found in U.S. Pat. Nos. 6,054,297, 5.886.152 and 5.877.293.

[0164] An “Fc” region, as the term is used herein, comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. Proteins of interest comprising an Fc region, including antigen binding proteins and Fc fusion proteins, form another aspect of the instant disclosure.

[0165] A “hemibody” is an immunologically Junctional 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 can, but need not. be employed to join the heavy chain Fc region and the second heavy chain Fc region. In particular embodiments, a hemibody is a monovalent form of an antigen binding protein disclosed herein. In other embodiments, pairs of charged residues can be employed to associate one Fc region w ith the second Fc region. A hemibody can be a protein of interest in the context of the instant disclosure.

[0166] Polypeptides and proteins of interest can be of scientific or commercial interest, including protein-based therapeutics. Proteins of interest include, among other things, secreted proteins, non-secreted proteins, intracellular proteins or membrane -bound proteins. Polypeptides and proteins of interest can be produced by recombinant animal cell lines using cell culture methods and may be referred to as “recombinant proteins”. The expressed protein(s) may be produced intracellularly or secreted into the culture medium from which it can be recovered and/or collected. The term “isolated protein” or “isolated recombinant protein” refers to a polypeptide or protein of interest, that is purified away from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. Proteins of interest include proteins that exert a therapeutic effect by binding a target, particularly a target among those listed below, including targets derived therefrom, targets related thereto, and modifications thereof. [0167] Proteins of interest include “antigen-binding proteins”. Antigen-binding protein refers to proteins or polypeptides that comprise an antigen-binding region or antigen-binding portion that has affinity for another molecule to which it binds (antigen). Antigen-binding proteins encompass antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including single-chain variable fragments (scFvs), double-chain (divalent) scFvs. and IgGscFv (see, e.g., Orcutt et al., 2010, Protein Eng Des Sei 23:221-228), hetero-IgG (see, e.g., Liu et al.. 2015. J Biol Chem 290:7535-7562), muteins, and XmAb* (Xencor, Inc., Monrovia. CA). Also included are bispecific T cell engagers (BiTE®), bispecific T cell engagers having extensions, such as half life extensions, for example HLE BiTEs, Heterolg BITE and others, chimeric antigen receptors (CARs. CAR Ts). and T cell receptors (TCRs).

[0168] An scFv is a single chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked together. See U.S. Patent Nos. 7,741,465, and 6,319,494 as well as Eshhar et al.. Cancer Immunol Immunotherapy (1997) 45: 131-136. An scFv retains the parent antibody's ability to specifically interact with target antigen.

[0169] The term “antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or to an antigen-binding region thereof that competes with the intact antibody for specific binding. Unless otherwise specified, antibodies include human, humanized, chimeric, multi-specific, monoclonal, polyclonal, heteroIgG. bispecific, and oligomers or antigen binding fragments thereof. Antibodies include the IgGl-, lgG2- lgG3- or lgG4-type. Also included are proteins having an antigen binding fragment or region such as Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, maxibodies, single chain antibody molecules, single domain VHH, complementarity determining region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to a target polypeptide. [0170] Also included are human, humanized, and other antigen-binding proteins, such as human and humanized antibodies, that do not engender significantly deleterious immune responses when administered to a human.

[0171] Also included are modified proteins, such as are proteins modified chemically by a non-covalent bond, covalent bond, or both a covalent and non-covalent bond. Also included are proteins further comprising one or more post-translational modifications which may be made by cellular modification systems or modifications introduced ex vivo by enzymatic and/or chemical methods or introduced in other ways.

[0172] Proteins of interest may also include recombinant fusion proteins comprising, for example, a multimerization domain, such as a leucine zipper, a coiled coil, an Fc portion of an immunoglobulin, and the like. Also included are proteins comprising all or part of the amino acid sequences of differentiation antigens (referred to as CD proteins) or their ligands or proteins substantially similar to either of these.

[0173] In some embodiments, proteins of interest may include colony stimulating factors, such as granulocyte colony-stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to, Neupogen® (filgrastim) and Neulasta® (pegfilgrastim). Also included are erythropoiesis stimulating agents (ESA), such as Epogen® (epoetin alfa), Aranesp* (darbepoetin alfa), Dynepo® (epoetin delta). Mircera® (methyoxy polyethylene glycol-epoetin beta), Hematide®, MRK-2578, INS-22, Retacrit® (epoetin zeta), Neorecormon® (epoetin beta), Silapo® (epoetin zeta), Binocrit* (epoetin alfa), epoetin alfa Hexal, Abseamed® (epoetin alfa), Ratioepo® (epoetin theta), Eporatio® (epoetin theta), Biopoin® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta, and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator. GLP-1 receptor agonists, as well as the molecules or variants or analogs thereof and biosimilars of any of the foregoing.

[0174] In some embodiments, proteins of interest may include proteins that bind specifically 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, osteoinductive factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony stimulating factors (CSFs), other blood and serum proteins blood group antigens; receptors, receptor-associated proteins, growth hormones, growth honn one receptors. T-cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0175] In some embodiments proteins of interest bind to one of more of the following, alone or in any combination: CD proteins including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD 138, CD 171, and CD 174, HER receptor family proteins, including, for instance, HER2, HERB, HER4, and the EGF receptor, EGFRvIII. cell adhesion molecules, for example, LFA- 1. Mol, pl50,95. VLA-4. ICAM-1, VCAM, and alpha v/beta 3 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, mullerian- inhibiting substance, human macrophage inflammatory protein (MIP-1 -alpha), erythropoietin (EPO), nerve growth factor, such as NGF-beta, platelet-derived growth factor (PDGF), fibroblast growth factors, including, for instance, aFGF and bFGF, epidermal growth factor (EGF), Cripto. transforming growth factors (TGF), including, among others, TGF-a and TGF- P, including TGF- i, TGF- 2, TGF- 3, TGF- 4, or TGF-P5, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(l-3)-IGF-I (brain IGF-I), and osteoinductive factors, insulins and insulin-related proteins, including but not limited to insulin, insulin A-chain, insulin B-chain, proinsulin, and insulin-like growth factor binding proteins; (coagulation and coagulation- related proteins, such as, among others, factor VIII, tissue factor, von Willebrand factor, protein C, alpha- 1 -antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator (“t-PA”), bombazine, thrombin, thrombopoietin, and thrombopoietin receptor, colony stimulating factors (CSFs), including the following, among others, 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, Hk2/flt3 receptor, obesity (OB) receptor, growth hormone receptors, and T-cell receptors; neurotrophic factors, including but not limited to, bone-derived neurotrophic factor (BDNF) and neuro trophin-3. -4, -5. or -6 (NT-3. NT-4, NT-5, or NT-6); relaxin A-chain, relaxin B-chain, and prorelaxin, interferons, including for example, interferon-alpha, -beta, and -gamma, interleukins (ILs), e.g.. IL-1 to IL-10. IL-12, IL-15, IL-17. IL-23. IL- 12/IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and/or IL-13 to the receptor, IL-13RA2, or IL- 17 receptor, IL-1RAP; viral antigens, including but not limited to. an AIDS envelope viral antigen, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase. BCM A, IgKappa, ROR-1, ERBB2. mesothelin, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-associated peptide. DNase, FR-alpha, inhibin, and activin, integrin, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, MIC (MIC -a, MIC-B), ULBP 1-6, EPCAM, addressins, regulatory’ proteins, immunoadhesins, antigen-binding proteins, somatropin, 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 rcccptor/hCGp. hepatitis-C virus, mesothelin dsFv[PE38] conjugate. Legionella pneumophila (lly), IFN gamma, interferon gamma induced protein 10 (IP 10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP). proprotein convertase subtilisin/Kexin Type 9 (PCSK9), stem cell factors, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, a4p7, platelet specific (platelet glycoprotein Ilb/IIIb (PAC-1), transforming growth factor beta (TFGP). Zona pellucida sperm-binding protein 3 (ZP-3). TWEAK, platelet derived growth factor receptor alpha (PDGFRa), sclerostin, and biologically active fragments or variants of any of the foregoing. [0176] In another embodiment, proteins of interest include abciximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosozumab, blinatumomab, brentuximab vedotin, brodalumab, cantuzumab mertansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lerdelimumab, lumiliximab, Ixdkizumab, mapatumumab, motesanib diphosphate, muromonab-CD3. natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelvekin. palivizumab, panitumumab, pembrolizumab. pertuzumab. pexelizumab, ranibizumab, rilotumumab. rituximab, romiplostim, romosozumab, sargamostim, tocilizumab, tositumomab. trastuzumab, ustekinumab, vedolizumab, visilizumab, volociximab. zanolimumab. zalutumumab. and biosimilars of any of the foregoing.

[0177] Proteins of interest according to the invention encompass all of the foregoing and further include antibodies comprising 1, 2. 3. 4, 5, or 6 of the complementarity determining regions (CDRs) of any of the aforementioned antibodies. Also included are variants that comprise a region that is 70% or more, especially 80% or more, more especially 90% or more, yet more especially 95% or more, particularly 97% or more, more particularly 98% or more, yet more particularly 99% or more identical in amino acid sequence to a reference amino acid sequence of a protein of interest. Identity in this regard can be determined using a variety of well-known and readily available amino acid sequence analysis software. Preferred software includes those that implement the Smith-Waterman algorithms, considered a satisfactory⁷ solution to the problem of searching and aligning sequences. Other algorithms also may be employed, particularly where speed is an important consideration. Commonly employed programs for alignment and homology' matching of DNAs, RNAs, and polypeptides that can be used in this regard include FAST A, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being an implementation of the Smith- Waterman algorithm for execution on massively parallel processors made by MasPar.

[0178] Proteins of interest 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 comprising an antigen binding molecule that interacts with that targeted antigen. CARs can be engineered to bind to an antigen (such as a cell-surface antigen) by incorporating an antigen binding molecule that interacts with that targeted antigen. CARs typically incorporate an antigen binding domain (such as scFv) in tandem with one or more costimulatory (“signaling”) domains and one or more activating domains. [0179] Preferably, the antigen binding molecule is an antibody fragment thereof, and more preferably one or more single chain antibody fragment (“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 the other CAR components. See Krause et al.. 1998, J. Exp. Med., 188(4): 619-626; Finney et al.. 1998. J Immunol 161: 2791-2797.

[0180] Chimeric antigen receptors incorporate one or more costimulatory (signaling) domains to increase their potency. See U.S. Patent Nos. 7,741,465, and 6,319.494. as well as Krause et al. and Finney et al. (supra), 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 costimulatory domains can be derived from, among other sources. CD28, CD28T, 0X40. 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4. CD5. CD7. CD8, CD9, CD16. CD22, CD27, CD30. CD 33, CD37. CD40, CD 45. CD64, CD80. CD86, CD134, CD137, CD154, PD-1, ICOS. lymphocyte function-associated antigen-1 (LFA-1 (CD1 la/CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin. signaling lymphocytic activation molecule, BTLA. Toll ligand receptor. ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT. HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4. CD8alpha, CDSbeta, IL-2R beta. IL-2R gamma, IL-7R alpha. ITGA4. VLA1, CD49a, ITGA4, IA4. CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl-ld, ITGAE, CD 103. ITGAL, CDl-la, LFA-1, ITGAM, CDLlb. ITGAX, CDLlc, ITGB1, CD29, ITGB2, CD 18, 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 ligand, or fragments or combinations thereof. The costimulatory domain can comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion.

[0181] CARs also include one or more activating domains. CD3 zeta is an element of the T cell receptor on native T cells and has been shown to be an important intracellular activating element in CARs.

[0182] CARs are transmembrane proteins, comprising an extracellular domain, typically containing an antigen binding protein that it is capable of recognizing and binding to the antigen of interest, and also including a “hinge” region. In addition is a transmembrane domain and an intracellular(cytoplasmic) domain.

[0183] The extracellular domain is beneficial for signaling and for an efficient response of lymphocytes to an antigen from any protein described herein or any combination thereof. The extracellular domain may be derived either from a synthetic or from a natural source, such as the proteins described herein. The extracellular domains often comprise a hinge portion. This is a portion of the extracellular domain, sometimes referred to as a “spacer’’ region. Hinges may be derived from the proteins as described herein, particularly the costimulatoiy proteins described above, as well as immunoglobulin (Ig) sequences or other suitable molecules to achieve the desired special distance from the target cell.

[0184] A transmembrane domain may be fused to the extracellular domain of the CAR. It can similarly be fused to the intracellular domain of the CAR. The transmembrane domain may be derived either from a synthetic or from a natural source, such as the proteins described herein, particularly the costimulatory proteins described above.

[0185] An intracellular (cytoplasmic) domain may be fused to the transmembrane domain and can provide activation of at least one of the normal effector functions of the immune cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Intracellular domains can be derived from the proteins described herein, particularly from CD3.

[0186] A variety of known techniques can be utilized in making the polynucleotides, polypeptides, vectors, host cells, immune cells, compositions, and the like according to the invention.

[0187] The present invention is not to be limited in scope by the specific embodiments described herein that are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications arc intended to fall within the scope of the appended claims.

EXAMPLES

Example 1

[0188] Three different small scale down models were used to simulate full scale filtration in 2000L bioreactors by matching UF and MF filter loading levels, flux rates and/or cross flow (mL/min/fiber). A 4.9L working volume ATF model was established using a 225cm² hollow fiber filter and a Magma APS-10 diaphragm pump (PendoTECH). A 1 ,5L working volume ATF model was established using a 850cm² hollow fiber filter and a XCell™ ATF 2 single use device including a diaphragm pump (Repligen Corporation). A 0.9 working volume RTF model was established using a 3 cm² hollow fiber filter and a Masterflex® L/S® peristaltic pump.

[0189] Small-scale perfusion cell culture perfusion cell culture experiments were conducted with several test antibodies using the different small-scale models. A proprietary cell line expressing a test antibody was cultured in a proprietary basal media and supplemented with a proprietary feed media through perfusion. The culture was performed for a total of 15 days after which the cells were harvested.

[0190] Viability and cell density were measured with Vi-CELL™ BLU cell viability analyzer (Beckman Coulter, Indianapolis, IN).

[0191] Yield is determined as a percent ratio of the cumulative product mass in the permeate to the summation of cumulative product mass in the permeate, cumulative product mass in the bleed, and daily product mass in the bioreactor.

[0192] Sieving perfonnance can be detennined as a percentage using the product titer in the permeate over the product titer in the bioreactor.

[0193] Comparable viable cell density (VCD) was seen for all conditions tested. Slightly higher viability and lower packed cell volume (PCV) was seen with the small-scale models. Titers were also slightly lower with the small-scale models.

[0194] Sieving decay and yields with the small-scale ATF models were comparable to the full-scale 2000L bioreactors. Sieving and yield were slightly lower for the small-scale RTF model.

[0195] To test an alternating reverse tangential flow (ARTF) strategy, the 0.9 working volume RTF model was employed with a reverse feed flow direction every 60 minutes. [0196] Increased sieving performance was seen with the ARTF strategy as shown in Figure 6A. This resulted in an increase in yield as shown in Figure 6B.

Example 2

[0197] The feasibility of ARTF at a 200L scale was tested compared to RTF and ATF. The three perfusion systems were connection to a bioreactor and perfusion was run concurrently for the duration of the process. The same hollow fiber membrane was used in each system (0.48sqm 30cm HFM with 520 lumen). The crossflow rate was the same for all systems (4.3 LPM) and at 3.8 LMH maximum permeate flux for used for all systems. For the ARTF system, the flow was reversed every two hours using an automated flow path control and pneumatic valves to redirect flow.

[0198] To stress the systems as much as possible, foam was allowed to build up in an effort to clog the perfusion systems, cell cultures were concentrated, and the cell culture duration was extended from 15 days to 20 days. Increased foaming inside the bioreactor can contribute to the formation of cellular debris, which once incorporated into cell culture, can enter perfusion systems and clog HFM lumen

[0199] Although none of the perfusion systems experienced failure, concentration of the cells increased the feed pressure for both the ARTF and RTF systems.

[0200] Table 1 : Feed Pressure

ARTF RTF

Initial 2.1 psig 0.8 psig

Final 4.5 psig 2.9 psig

[0201] Both ARTF and RTF systems showed an increase in feed pressure of 2. lx and 3.6x, respectively.

[0202] Permeable trend profdes for the ATF, ARTF and RTF systems showed similar pressure profiles. Under high percent PCV, ATF did show a drop in permeate pressure as the cell culture became more concentrated inside the ATF equipment indicating sonic filter fouling. The recirculating flow in ARTF and RTF systems prevents the cell culture from being concentrated inside the hollow fiber membrane.

[0203] Transmembrane pressure changes and differential pressure changes between ARTF and RTF were comparable.

[0204] As the pressure profiles suggested little impact of RTF on filter fouling, visual inspection of the filters in the ARTF and RTF systems was performed.

[0205] Figure 7 shows images of the filter lumen. The % lumen clogged was determined by visual inspection, counting the clogged lumen vs. unclogged lumen and then calculating the percentage of clogged lumen. In some cases, it can be difficult to see every lumen due to the presence of cellular debris. In such cases, the number of unclogged lumen are compared against the total number of lumen as provided by the manufacturer of the hollow fiber membrane. Table 2: Percentage of clogged lumen

[0206] The RTF inlet had almost 3/4 of its lumen clogged with cell culture debris. For the ARTF system, the inlet is initially the bottom side, but the system is reversed as provided for above. The ARTF system showed significantly less clogging at the inlet.

Claims

CLAIMS What is claimed is:

1. A system for perfusion culture of cells comprising: a) at least one bioreactor; b) at least one hollow fiber or flat sheet filter unit comprising a first end and a second end on opposite ends of the at least one filter unit: and c) at least one fluidic pump, wherein said fluidic pump is fhiidically connected to said at least one filter unit and said bioreactor such that fluid can be pumped from the bioreactor through the at least fluidic pump to the at least one filter back to the bioreactor and retentate return from the bioreactor, with the ability to collect membrane penneate to a separate receiver; and, wherein i) said fluidic pump is reversible or ii) said first end and second end of the filter unit are fhiidically connected to said at least one fluidic pump via a set of valves arranged to alternatively allow flow in the direction from the at least one bioreactor to the at least one fluidic pump to the first end of the at least one filter unit and back to the bioreactor while blocking flow in the reverse direction or allow flow in the direction from the at least one bioreactor to the at least one fluidic 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 fluidic pump is reversible.

3. The system of claim 1, wherein the at least one fluidic pump is unidirectional wherein said first end and second end of the filter unit are fhiidically connected to said at least one fluidic pump via a set of valves (and associated tubing connections) arranged to alternatively allow flow in the direction from the at least one bioreactor to the at least one fluidic pump to the first end of the at least one filter unit and back to the bioreactor while blocking flow in the reverse direction or allow flow in the direction from the at least one bioreactor to the at least one fluidic 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 fluidic pump is a centrifugal, peristaltic, rotary lobe, or reciprocating pump.

5. The system of claim 3, wherein each valve in the set of valves can be independently configurable in an open position or a closed position.

6. The system of any of claims 3 to 5, wherein the position of the valves is automatable.

7. The system of any 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 while two valves can be in the closed position.

9. The system of claim 8, wherein the two valves which are open are parallel, and the tw o valves which are closed are parallel.

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

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

12. The system of any of claims 1 to 1 1 , further comprising one or more pressure transmitters.

13. The system of claim 12, wherein the valves in the set of valves reverse position upon detection of high inlet pressure.

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

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

16. The system of claim 15, wherein the control unit is configured to control the at least one fluidic pump and the set of valves depending on pressure data received from said pressure transducer.

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

18. The system of any of claims 1 to 17, further comprising a wye.

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

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

21. The mediod according to claim 19, further comprising reversing the flow path which is triggered upon reaching a preset High Pf_eed value.

22. The method according to claim 21, wherein the preset Pf_eed value is 10 psig.

23. The method of any of claims 19 to 22. wherein the flow is reversed at a set interval or random interval every 1 to 4 hours.

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