Perfusion Bioreactor Tangential Flow Filtration

Abstract

Provided herein are systems and methods for improving product recovery using constant pressure and / or low flow rate tangential flow filtration.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 378,970, filed October 10, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Tangential flow filtration (also called cross-flow filtration or TFF) systems are widely used in the separation of particulates suspended in a liquid phase and have important bioprocessing applications. Tangential flow systems are characterized by a fluid feed flowing across the surface of a filter, separating the feed into two components: a permeate component that passes through the filter and a retentate component that does not. Compared to dead-end systems, TFF systems are less susceptible to fouling. Fouling in TFF systems can be further reduced by alternating the direction of the fluid feed across the filtration element, backwashing the permeate through the filter, and / or periodic cleaning.

[0003] Despite their advantages, TFF systems still suffer from drawbacks that result in costly product loss. Thus, there is a need to create a TFF system that reduces loss of the desired product. Summary of the Invention

[0004] The present disclosure provides a system for improving recovery of a product of interest during tangential flow filtration (TFF), the system comprising: (1) a feed reservoir; (2) a TFF unit comprising a pump in fluid contact with the fluid to be filtered and at least one filter element for separating the liquid feed into a permeate and a retentate; and (3) a second pump in fluid contact with the permeate from the first filter element.

[0005] In one embodiment, the system further comprises a second filter element in direct contact with the retentate and permeate from the first filter element. In another embodiment, the filter elements are stacked in series. In another embodiment, one or more of the filter elements comprises hollow fibers or cassettes. In another embodiment, the hollow fibers have a pore size of 0.2 to 0.65 μm.

[0006] In another embodiment, one or more of the pumps is a peristaltic pump, a diaphragm pump, or a magnetic levitation pump.

[0007] In another aspect, the system is configured to operate in a recirculation mode.

[0008] In another embodiment, the feed reservoir is a perfusion bioreactor. In another embodiment, the perfusion bioreactor is greater than 1000 liters in size. In another embodiment, the bioreactor is 5000 or 6000 liters in size. In another embodiment, the system is configured for large-scale processing of the product of interest.

[0009] The present disclosure also provides a method for minimizing retention of a product of interest in the permeate stream during TFF, comprising passing a liquid feed containing the product of interest through a system described herein. In another embodiment, the liquid feed comprises cells and a target product of interest. In some embodiments, the product of interest is an antibody or antigen-binding fragment thereof. In another embodiment, the product of interest is recovered in the permeate. In another embodiment, the liquid feed is passed through the system at a rate that minimizes cell shear. In another embodiment, the liquid feed is passed through the system at a rate of 1.8 to 8 mL / lumen / min. In another embodiment, the pressure differential between the retentate inlet and the permeate matches the system pressure after filtration.

[0010] The present disclosure also provides a method for minimizing retention of a product of interest in a permeate stream during TFF, comprising passing a liquid feed through a system described herein at a flow rate at least one-third slower than conventional TFF flow rates. In another embodiment, the flow rate is about 1800 s -1provides a shear rate of

[0011] In another embodiment, the method further comprises sparging air into the system. In another embodiment, the air comprises about 10-80% dissolved oxygen. In another embodiment, the liquid feed is sparged with air before contacting the filter element. In another embodiment, sparging is performed at a rate necessary to maintain greater than 10% dissolved oxygen throughout the TFF system. In some embodiments, sparging comprises introducing oxygen with a bubble diameter of about 1 μm to about 10 μm. In some embodiments, sparging comprises introducing oxygen with a bubble diameter of about 1 μm or about 10 μm. In another embodiment, the liquid feed comprises cells and a target product of interest. In another embodiment, sparging minimizes lactic acid production by the cells. In some embodiments, the product of interest is an antibody or antigen-binding fragment thereof. In another embodiment, the method further comprises recovering the product of interest in the permeate. In some embodiments, sparging increases the specific productivity of the cells compared to cells without sparging. In some embodiments, sparging increases the specific productivity of the cells by about 0.0034 g mL compared to cells without sparging. -1 day -1 In some embodiments, sparging increases the specific productivity of the cells by at least 0.0034 g mL -1 day -1 to approximately 0.0044 g mL -1 day -1 In another embodiment, the pressure differential between the retentate inlet and the permeate is matched to the system pressure after filtration. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram of a typical TFF system. [Figure 2A] Figure 2 shows product retention and corresponding yield loss during small-scale TFF. Figure 2A shows product titers in the permeate and retentate. Figure 2B shows percent yield of product in TFF. [Figure 2B]Figure 2 shows product retention and corresponding yield loss during small-scale TFF. Figure 2A shows product titers in the permeate and retentate. Figure 2B shows percent yield of product in TFF. [Figure 3] 1 is a schematic diagram of a high performance (HPTFF) system of the present disclosure. The TFF system uses a second pump in conjunction with the permeate. The TFF system can use one or more filter cassettes. [Figure 4A] Figure 4 shows the increased yield of AZ-1 (Figure 4A) and AZ-2 (Figure 4B) using HPTFF and low-flow TFF compared to conventional TFF. [Figure 4B] Figure 4 shows the increased yield of AZ-1 (Figure 4A) and AZ-2 (Figure 4B) using HPTFF and low-flow TFF compared to conventional TFF. [Figure 4C] Figure 4 shows the increased yield of AZ-1 (Figure 4A) and AZ-2 (Figure 4B) using HPTFF and low-flow TFF compared to conventional TFF. [Figure 4D] Figure 4 shows the increased yield of AZ-1 (Figure 4A) and AZ-2 (Figure 4B) using HPTFF and low-flow TFF compared to conventional TFF. [Figure 5A] Figure 5A shows the culture performance of AZ-1 using HPTFF at different viable cell density targets of 90 million and 120 million cells per mL (Figure 5A), viability (Figure 5B), glucose level (Figure 5C), pH (Figure 5D), osmolality (Figure 5E), and lactate level (Figure 5F). [Figure 5B] Figure 5A shows the culture performance of AZ-1 using HPTFF at different viable cell density targets of 90 million and 120 million cells per mL (Figure 5A), viability (Figure 5B), glucose level (Figure 5C), pH (Figure 5D), osmolality (Figure 5E), and lactate level (Figure 5F). [Figure 5C]Figure 5A shows the culture performance of AZ-1 using HPTFF at different viable cell density targets of 90 million and 120 million cells per mL (Figure 5A), viability (Figure 5B), glucose level (Figure 5C), pH (Figure 5D), osmolality (Figure 5E), and lactate level (Figure 5F). [Figure 5D] Figure 5A shows the culture performance of AZ-1 using HPTFF at different viable cell density targets of 90 million and 120 million cells per mL (Figure 5A), viability (Figure 5B), glucose level (Figure 5C), pH (Figure 5D), osmolality (Figure 5E), and lactate level (Figure 5F). [Figure 5E] Figure 5A shows the culture performance of AZ-1 using HPTFF at different viable cell density targets of 90 million and 120 million cells per mL (Figure 5A), viability (Figure 5B), glucose level (Figure 5C), pH (Figure 5D), osmolality (Figure 5E), and lactate level (Figure 5F). [Figure 5F] Figure 5A shows the culture performance of AZ-1 using HPTFF at different viable cell density targets of 90 million and 120 million cells per mL (Figure 5A), viability (Figure 5B), glucose level (Figure 5C), pH (Figure 5D), osmolality (Figure 5E), and lactate level (Figure 5F). [Figure 6A] FIG. 6 shows dissolved oxygen levels under typical TFF conditions (FIG. 6A) and with air sparging (FIG. 6B), as well as a schematic (FIG. 6C). [Figure 6B] FIG. 6 shows dissolved oxygen levels under typical TFF conditions (FIG. 6A) and with air sparging (FIG. 6B), as well as a schematic (FIG. 6C). [Figure 6C] FIG. 6 shows dissolved oxygen levels under typical TFF conditions (FIG. 6A) and with air sparging (FIG. 6B), as well as a schematic (FIG. 6C). [Figure 7A] Figure 7 shows the culture performance of AZ-3 for a typical TFF with and without air sparging in terms of viable cell density (Figure 7A), viability (Figure 7B), lactate level (Figure 7C), total product titer (Figure 7D), specific productivity (Figure 7E), and product retention (Figure 7F). [Figure 7B] Figure 7 shows the culture performance of AZ-3 for a typical TFF with and without air sparging in terms of viable cell density (Figure 7A), viability (Figure 7B), lactate level (Figure 7C), total product titer (Figure 7D), specific productivity (Figure 7E), and product retention (Figure 7F). [Figure 7C] Figure 7 shows the culture performance of AZ-3 for a typical TFF with and without air sparging in terms of viable cell density (Figure 7A), viability (Figure 7B), lactate level (Figure 7C), total product titer (Figure 7D), specific productivity (Figure 7E), and product retention (Figure 7F). [Figure 7D] Figure 7 shows the culture performance of AZ-3 for a typical TFF with and without air sparging in terms of viable cell density (Figure 7A), viability (Figure 7B), lactate level (Figure 7C), total product titer (Figure 7D), specific productivity (Figure 7E), and product retention (Figure 7F). [Figure 7E] Figure 7 shows the culture performance of AZ-3 for a typical TFF with and without air sparging in terms of viable cell density (Figure 7A), viability (Figure 7B), lactate level (Figure 7C), total product titer (Figure 7D), specific productivity (Figure 7E), and product retention (Figure 7F). [Figure 7F] Figure 7 shows the culture performance of AZ-3 for a typical TFF with and without air sparging in terms of viable cell density (Figure 7A), viability (Figure 7B), lactate level (Figure 7C), total product titer (Figure 7D), specific productivity (Figure 7E), and product retention (Figure 7F). DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure provides a highly effective approach to minimize the retention of a product of interest during TFF. In some embodiments, the present disclosure provides a method for minimizing product loss caused by pressure drop during large-scale processing of perfusion cell cultures. In other embodiments, the culture is larger than a 1000 L culture. In some embodiments, the present disclosure provides a method for using two or more pumps in a TFF system to minimize pressure drop. In some embodiments, the present disclosure provides a method for using low flow rates and air sparging to reduce the retention of a product of interest.

[0014] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used herein, unless expressly provided otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the specification.

[0015] It should be noted that terms such as "a" or "an" refer to one or more of that entity. For example, a "feed medium" is understood to represent one or more feed media. Thus, terms such as "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0016] The term "and / or," as used herein, should be construed as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" used herein in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0017] Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the words "consisting of" and / or "consisting essentially of" are also provided.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0019] Units, prefixes, and symbols are denoted in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of this disclosure, which may be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0020] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any list or listed members.

[0021] Terms such as "about" or "essentially comprising" refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within or more than one standard deviation, per the practice in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 20%. Moreover, particularly with respect to biological systems or processes, these terms can mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range for that particular value or composition.

[0022] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof (such as integer tenths and hundredths), where appropriate, unless otherwise indicated.

[0023] As used herein, the term "bioreactor" refers to any suitable vessel or other means for producing and maintaining a biological cell culture, including, but not limited to, a perfusion / perfusion bioreactor. The bioreactors of the present disclosure are used for large-scale production of a product of interest. In some embodiments, the bioreactor volume is greater than 1000 L. In some embodiments, the bioreactor volume is 1000 L, 1500 L, 2000 L, 2500 L, 3000 L, 3500 L, 4000 L, 4500 L, 5000 L, 5500 L, 6000 L, 6500 L, 7000 L, 7500 L, 8000 L, 8500 L, 9000 L, 9500 L, or 10,000 L.

[0024] As used herein, the term "perfusion" refers to a fermentation or cell culture process used to produce a target biological product, e.g., an antibody or recombinant protein, in which a high concentration of cells in a sterile chamber continuously receives fresh growth medium as spent medium, which may contain the target biological product to be harvested.

[0025] The term "cutoff size" or "molecular weight cutoff" as used herein with respect to ultrafiltration membranes refers to the molecular weight of molecules or particles at which 90% are retained by the membrane.

[0026] The term "spiral-wound filter element" refers to a filtration membrane that is spirally wound around a core. A spiral-wound filter element may be contained within a housing or may be called a spiral-wound filter module.

[0027] "Pressure drop" refers to the pressure drop (e.g., psid) from the retentate inlet and permeate.

[0028] "Flux" is the area-normalized flow velocity.

[0029] "Permeate flux" is the area-normalized flow rate of the permeate in the permeate channel (e.g., liters / hour / m 2 ,lmh).

[0030] "Crossflow flux" is the area-normalized average flow rate of the retentate in the feed channel (e.g., liters / minute / m 2 , LMM).

[0031] "Crossflow" is the retentate flow rate between the inlet and outlet of a feed channel in a filter or series of filters. Unless otherwise specified, "crossflow" refers to the average crossflow.

[0032] The term "shear" refers to the distortion in the structure of a material caused by pressure.

[0033] The term "shear rate" refers to the rate at which progressive shear deformation is applied (e.g., s-1).

[0034] The terms "feed," "feed sample," and "feed stream" refer to a solution introduced into a filtration module for separation.

[0035] The term "separation" generally refers to the act of separating a feed sample into two streams: a permeate stream and a retentate stream.

[0036] The terms "permeate" and "permeate stream" refer to the portion of the feed that has permeated the membrane.

[0037] The terms "retentate" and "retentate stream" refer to the portion of the solution that is retained by the membrane, the retentate being a stream concentrated in the retained species.

[0038] "Feed channel" refers to a conduit within a filtration assembly, module or element for feeding.

[0039] "Permeate channel" refers to a conduit within a filtration assembly, module, or element for permeate.

[0040] The term "flow path" refers to a channel containing a filtration membrane (e.g., an ultrafiltration membrane, a microfiltration membrane) through which a solution to be filtered passes (e.g., in a tangential flow mode). The flow path can have any topology that supports tangential flow (e.g., straight, coiled, zigzag-arranged). The flow path can be open, as in the example of a channel formed by a hollow fiber membrane, or it can have one or more flow obstacles, as in the case of a rectangular channel formed by flat sheet membranes separated by a woven or nonwoven spacer.

[0041] "TFF assembly," "TFF system," and "TFF device" are used interchangeably herein to refer to a tangential flow filtration system configured to operate in single-pass mode and / or recirculation mode (e.g., full recirculation or partial recirculation) and / or alternating-flow mode.

[0042] "Single leaf" spirals are spiral-wound filter elements that can be formed with one continuous feed channel. They are generally made from a single sheet of membrane.

[0043] "Multi-leaf" spirals are spiral-wound filter elements with multiple feed channels. They are generally made from two or more membrane sheets, but may also be made from a single membrane sheet.

[0044] "Cassette holder" refers to a compression assembly for one or more cassettes. Typically, when a cassette holder houses two or more cassettes, the cassettes are configured for parallel processing, although in some embodiments, the cassettes may be configured for serial processing.

[0045] "Cassette" refers to a cartridge or flat module containing filtration (e.g., ultrafiltration or microfiltration) membrane sheet(s) suitable for a TFF process.

[0046] "Filtration membrane" refers to a selectively permeable membrane that can be used in a filtration system, such as a TFF system.

[0047] The terms "microfiltration membrane" and "MF membrane" are used herein to refer to membranes having pore sizes ranging from about 0.1 micrometers to about 10 micrometers.

[0048] "Fluidly connected" refers to multiple spiral-wound membrane TFF modules connected to one another by one or more conduits for liquids, such as feed channels, retentate channels, and / or permeate channels.

[0049] "Product" refers to a target compound. In some embodiments, the product is a biomolecule (e.g., a protein) of interest, such as a monoclonal antibody (mAb).

[0050] "Processing" refers to the act of filtering a feed containing a desired product (e.g., by TFF) and then recovering the product (e.g., in purified form). The product can be recovered from the filtration system (e.g., TFF assembly) in either a retentate stream or a permeate stream, depending on the size of the product and the pore size of the filtration membrane.

[0051] The terms "parallel processing," "processing in parallel," "parallel operation," and "operation in parallel" refer to processing of products in a TFF assembly containing multiple processing units fluidly connected by direct distribution of feed from a feed channel or manifold to each of the processing units in the assembly.

[0052] The terms "serial processing," "processing in series," "serial operation," and "operation in series" refer to the processing of products in a TFF assembly containing multiple processing units fluidly connected by direct distribution of feed from a feed channel to only the first processing unit in the assembly. In serial processing, each subsequent processing unit in the assembly receives its feed from the retentate line of the preceding processing unit (e.g., the retentate from the first processing unit serves as the feed for the second adjacent processing unit).

[0053] As used herein, "perfusion" or "perfusion culture" or "perfusion culture process" refers to the continuous flow of a physiological nutrient solution at a constant rate through or over a population of cells. Because perfusion systems generally involve retaining cells within a culture unit, perfusion cultures characteristically have relatively high cell densities, but culture conditions are difficult to maintain and control. Additionally, because cells are grown at high density and then retained within the culture unit, the growth rate typically continuously decreases over time, leading to a late exponential or even stationary phase of cell growth. This continuous culture strategy generally involves culturing mammalian cells (e.g., non-anchorage-dependent cells) expressing a polypeptide and / or virus of interest during the production phase in a continuous cell culture system. In some embodiments, the perfusion culture is a large-scale culture. In some embodiments, the culture is greater than a 1000 liter culture. In another embodiment, the culture is a 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 liter culture.

[0054] Various aspects of the disclosure are described in further detail in the following subsections.

[0055] II. High performance tangential flow filtration (HPTFF) In contrast to fed-batch systems, perfusion systems and methods involve continuous filtration of cell culture medium. During filtration, the product of interest (e.g., a target protein such as a monoclonal antibody) and, optionally, other soluble components such as cellular waste products (e.g., lactate and ammonia) are removed from the cell culture medium. Perfusion systems present unique challenges relative to fed-batch systems because the cells contained in the perfusion system are repeatedly passed through a filtration device, which can cause physical damage to the cells and, in turn, reduce the productivity of the system. It is desirable to minimize cell damage during filtration in perfusion systems so as to retain as many cells as possible for ongoing production of the target protein.

[0056] Tangential flow filtration (TFF) is a separation process that uses membranes to separate components in a liquid solution or suspension based on size, molecular weight, or other differences. TFF is used in perfusion processes to remove a target product of interest (e.g., a protein) from cell culture media while retaining the cells within the media. In the TFF process, fluid is pumped tangentially along the membrane surface; particles, molecules, or cells too large to pass through the membrane are rejected and returned to the process tank. The TFF process may involve further passage 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 enabling high-volume processing per batch. The membrane is contained within a filter element, which can be in a variety of configurations, such as spiral-wound and cassette filter elements.

[0057] A typical TFF system is shown in Figure 1. A pressurized feed from a feed tank is connected to the feed port of a cassette filter spiral-wound filter module or manifold. The feed flows through the membrane-lined feed channels of the TFF device(s) under the control of a pump. A portion of the solvent from the feed stream flows through the face of the membrane into the permeate channel, carrying with it a portion of the permeate species (e.g., the desired product and waste). The remaining concentrated feed stream exits the module or manifold through the retentate port. The permeate flowing from the module's permeate port is directed to a process-dependent location where it is collected (e.g., as the desired product) or discarded (e.g., as the waste product).

[0058] However, under large-scale production conditions, for example, exceeding 1000 L of bioreactor capacity, the TFF system experiences a pressure drop between the retentate inlet and the permeate, which, due to the large processing volume, causes the desired product to remain in the retentate. This pressure drop leads to the desired product being retained in the retentate, which leads to a decrease in product recovery.

[0059] To overcome the pressure drop, the high performance TFF (HPTFF) systems described herein include two or more pumps for recirculating the retentate through all or a portion of the system and at least one conduit for recirculating (e.g., conveying) the retentate (FIG. 3). In one embodiment, one pump is located at the retentate inlet and one pump contacts the permeate. A flow meter can be used to provide a process value to the pump or valve to control the amount of retentate that is recirculated. Alternatively, or in addition, a valve or pump and / or flow meter can be located at the permeate outlet or in a flow line conveying the permeate flow from the system to control or limit the permeate flow.

[0060] The maximum achievable flux during TFF system operation can be obtained by selecting an appropriate transmembrane pressure (TMP) for the permeate discharge. This applies to pressure-dependent and mass-transfer-limited operating regions. In the case of a spiral-wound filter, achieving the desired TMP is determined by measurements at the end of the module. For example, for a cassette with two permeate outlets, achieving the desired TMP is determined by the average feed channel pressure. The transmembrane pressure must be sufficient to support both the pressure drop across the membrane and the maximum pressure required to discharge the permeate from the permeate channel. Alternatively, or in addition, the maximum achievable flux during TFF system operation can be obtained by selecting an appropriate permeate flow rate for the permeate discharge. The permeate flow rate can be controlled to a constant value using a permeate valve or pump.

[0061] Current TFF devices used in perfusion systems include hollow fiber devices and open-channel cassette devices (also called plate-and-frame devices). Examples of currently available filtration devices for perfusion systems include, but are not limited to, hollow fiber devices such as the XCell™ ATF System (Repligen, Waltham, Mass.) and the KrosFlo® Perfusion System (Spectrum Laboratories, Rancho Dominguez, Calif.), and cassette devices such as the Prostak™ Microfiltration Modules (MilliporeSigma, Billerica, Mass.). These devices have open feed channels to limit physical damage to cells in the feed stream, and both devices require high cross-flow velocities to minimize fouling (i.e., particle accumulation along the membrane walls). Membrane fouling and pressure drop in TFF systems reduce product recovery due to reduced membrane passage (i.e., sieving) of target proteins and waste products. In some embodiments, the hollow fibers have a pore size of 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 μM.

[0062] In a perfusion process, cell culture medium is introduced to the feed side of a membrane. As the liquid feed (e.g., cell culture medium) moves across the surface of the membrane, it is separated into a permeate and a retentate. Specifically, the target product of interest is collected from the permeate, which passes through the membrane and exits the filter through a collection tube. The cells are retained and collected from the retentate, which exits the filter. The cell culture medium in the retentate can then be returned to the bioreactor, and the product of interest contained in the permeate can be collected in a separate vessel for further processing.

[0063] The perfusion system can include a TFF system having one or more spiral-wound or cassette filter elements described herein. In systems having two or more filter elements, the filter elements can be fluidly connected in series or parallel, or both.

[0064] TFF systems can operate in a recirculation mode, where all or a portion of the retentate is returned to the filter element(s) for further filtration. In perfusion systems, following filtration, the retentate can be returned to the bioreactor, where the cell culture medium can be maintained for a period of time before being recirculated through the TFF system.

[0065] The feed pump shown in Figure 3 can be configured to operate in a recirculation mode. The feed pump can be a cell-safe pump, such as a magnetic levitation pump, a diaphragm pump, a peristaltic pump, or a rotary vane pump. Examples of suitable magnetic levitation pumps include, but are not limited to, the Levitronix® Puralev® Series pumps (Levitronix Technologies, Framingham, MA). Examples of suitable diaphragm pumps include the Repligen XCell™ ATF pump (Repligen, Waltham, MA). Examples of suitable peristaltic pumps include the Watson Marlow Series 500 and Series 600 pumps (Watson Marlow, Wilmington, MA).

[0066] In one aspect, the present disclosure relates to a method of passing a liquid feed through an HPTFF system described herein, comprising at least one filter element, separating the liquid feed into a permeate and a retentate within the filter element, and recovering at least a portion of the permeate and retentate from the filter element. The liquid feed can comprise a cell culture medium containing cells and a target product of interest. The target product of interest can be recovered in the permeate, and the cells can be retained in the retentate.

[0067] The method can include recirculating at least a portion of the retentate through the filter element, which can be done continuously or at periodic intervals to continuously harvest product from the cell culture medium.

[0068] The recycled retentate can be returned to any upstream location within or before the HPTFF system (e.g., a bioreactor located upstream of the HPTFF system). In one embodiment, the retentate is recycled to a feed tank. In another embodiment, the retentate is recycled to a feed line near the feed pump before the feed inlet on the HPTFF system.

[0069] In some embodiments, the methods described herein involve perfusion at a low flow rate to overcome pressure drops in the TFF system. A low flow rate correlates with improved product quality due to reduced shear rates. In one embodiment, the low flow rate is about one-third of the typical flow rate of a TFF system. In another embodiment, a conventional flow rate is about 1800 s -1 Thus, in another embodiment, a low flow rate is a flow rate of about 600 s -1 correlates with the shear rate.

[0070] In some embodiments, low flow TFF can cause oxygen deficiency in HPTFF systems due to the amount of time the feed stream spends outside the bioreactor. Therefore, in some embodiments, the feed stream is sparged with air. In some embodiments, the feed stream is sparged with 10-80% dissolved oxygen. In some embodiments, the feed stream is sparged with 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% dissolved oxygen. In some embodiments, oxygen is introduced with a bubble diameter of about 1 μm. In some embodiments, oxygen is introduced with a bubble diameter of about 10 μm. In some embodiments, oxygen is introduced with a bubble diameter of about 1 μm to about 10 μm.

[0071] In some embodiments, the present disclosure provides for the purification of any product of interest. In some embodiments, the product of interest is a protein. Thus, in some embodiments, the present disclosure relates to a perfusion process for recovering a target protein from a liquid feed containing host cells. The target protein can be a monoclonal antibody that is separated from host cells by TFF and recovered from the permeate of a filter element. [Example]

[0072] A bioreactor containing cells producing the monoclonal antibody AZ-1 was subjected to standard TFF as shown in Figure 1. The bioreactor volume was 2 liters, and TFF was performed using Repligen S04-P20U-10-N hollow fiber filters for 1360 seconds. -1 As shown in Figure 2, using standard TFF conditions, the titer in the TFF retentate increased relative to the target titer as the culture period and concomitant amount of protein produced increased (Figure 2A). This increase relative to the target titer caused a significant decrease in the yield of recovered protein (Figure 2B).

[0073] AZ-1 and AZ-2 (monoclonal antibodies) were then subjected to HPTFF and low flow TFF as described herein. The bioreactor feed from the 2 L reactor was filtered using Repligen S04-P20U-10-N hollow fiber filters for 1360 seconds. -1 For low-flow TFF, the flow rate was 600 s -1 This corresponds to a shear rate of 100 s. As shown in Figure 4, the percent yield of AZ-1 (Figure 4A) and AZ-2 (monoclonal antibody, Figure 4B) significantly increased when clarified using the HPTFF and low-flow systems described herein compared to standard TFF. Culture performance, as measured by viable cell density, viability, glucose levels, pH, osmolality, and lactate levels, was unaffected for AZ-1 (Figures 5A-5F).

[0074] To overcome product retention in the retentate, a low feed stream flow rate was also analyzed. A flow rate one-third lower than the typical TFF was analyzed. As shown in Figure 6A, dissolved oxygen levels were depleted during a typical TFF, but sparging the recirculation loop with air containing 21% dissolved oxygen restored normal levels. Low dissolved oxygen levels reduced specific productivity (Figure 7). Culture performance of a typical TFF for the bispecific antibody AZ-3 demonstrated that air sparging overcomes the negative effect of oxygen deficiency on specific productivity without affecting viable cell density and cell viability (Figures 7A-7D). Furthermore, specific productivity per cell volume was significantly higher than that of the control TFF (0.0034 g mL). -1 day -1 in the sparged TFF compared to approximately 0.0044 g mL -1 day -1 It was.

Claims

1. 1. A system for improving recovery of a product of interest during tangential flow filtration (TFF), comprising: (1) a feed reservoir; (2) a TFF unit comprising a pump in fluid contact with a fluid to be filtered and at least one filter element for separating a liquid feed into a permeate and a retentate; and (3) a second pump in fluid contact with the permeate from the first filter element.

2. 10. The system of claim 1, further comprising a second filter element in direct contact with the retentate and the permeate from the first filter element.

3. The system of claim 1 or 2, wherein one or more of the filter elements comprises a hollow fiber or a cassette.

4. The system of claim 3 , wherein the filter elements are stacked in series.

5. The system of any one of claims 1 to 4, wherein the hollow fibers have a pore size of 0.2 to 0.65 μM.

6. The system of any one of claims 1 to 5, wherein one or more of the pumps is a peristaltic pump, a diaphragm pump, or a magnetic levitation pump.

7. A system according to any one of claims 1 to 6, configured to operate in a recirculation mode.

8. The system of any one of claims 1 to 7, wherein the supply reservoir is a perfusion bioreactor.

9. 9. The system of any one of claims 1 to 8, wherein the perfusion bioreactor is greater than 1000 liters in size.

10. 10. The system of claim 9, wherein the bioreactor has a volume of 5000 or 6000 liters.

11. The system of any one of claims 1 to 10, configured for large-scale processing of a product of interest.

12. 12. A method for minimizing retention of a product of interest in a permeate stream during TFF, comprising passing a liquid feed containing said product of interest through the system of any one of claims 1 to 11.

13. 13. The method of claim 12, wherein the liquid feed comprises cells and a target product of interest.

14. 14. The method of claim 12 or 13, wherein the product of interest is an antibody or an antigen-binding fragment thereof.

15. 15. The method of any one of claims 12 to 14, further comprising recovering the product of interest in the permeate.

16. The method of any one of claims 12 to 15, wherein the liquid feed is passed through the system at a velocity that minimizes cell shear.

17. 17. The method of claim 16, wherein the flow rate is 1.8 to 8 mL / lumen / minute.

18. 18. The method of any one of claims 12 to 17, wherein the pressure difference between the retentate inlet and the permeate matches the system pressure after filtration.

19. 12. A method for minimizing retention of a product of interest in the permeate stream during TFF, comprising passing a liquid feed through the system of any one of claims 1 to 11 at a flow rate at least three times slower than conventional TFF flow rates.

20. The flow rate is about 1800 s －１ 20. The method of claim 19, wherein the shear rate corresponds to a shear rate of

21. 21. The method of claim 19 or 20, further comprising sparging air into the system.

22. 22. The method of claim 21, wherein the air contains about 10-80% dissolved oxygen.

23. The method of any one of claims 19 to 22, wherein the liquid feed is sparged with air before contacting the filter element.

24. 24. The method of any one of claims 21-23, wherein sparging is performed at a rate necessary to maintain greater than 10% dissolved oxygen throughout the TFF system.

25. 25. The method of any one of claims 21 to 24, wherein the sparging comprises introducing oxygen having a bubble diameter of about 1 μm to about 10 μm.

26. 26. The method of any one of claims 21 to 25, wherein the sparging comprises introducing oxygen with a bubble diameter of about 1 μm or about 10 μm.

27. 27. The method of any one of claims 19 to 26, wherein the liquid feed comprises cells and a target product of interest.

28. 26. The method of any one of claims 21 to 25, wherein the sparging minimizes lactic acid production by the cells.

29. The method of any one of claims 19 to 26, wherein the product of interest is an antibody or an antigen-binding fragment thereof.

30. 30. The method of any one of claims 21 to 29, wherein the sparging increases the specific productivity of the cells compared to cells without sparging.

31. The sparging increased the specific productivity of the cells by approximately 0.0034 g mL compared to cells without sparging. －１ day －１ The method according to any one of claims 21 to 30, wherein the concentration of hydroxybenzoates is increased to more than 100 ppm.

32. The sparging reduces the specific productivity of the cells to at least 0.0034 g mL －１ day －１ to about 0.0044 g mL －１ day －１ The method according to any one of claims 21 to 31, wherein the concentration of

33. 33. The method of any one of claims 19 to 32, further comprising recovering the product of interest in the permeate.

34. The method of any one of claims 19 to 33, wherein the system pressure before the filter element matches the system pressure after filtration.

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