Perfusion bioreactor tangential flow filtration

Abstract

Provided herein are systems and methods for enhancing recovery of products using constant pressure and / or low flow rate tangential flow filtration.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 378,970, filed on October 10, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Tangential flow filtration (also known as cross-flow filtration or TFF) systems are widely used to separate particles suspended in a liquid phase and have important bioprocessing applications. Tangential flow systems are characterized by the flow of a fluid feed across the surface of a filter, resulting in the separation of the feed into two components: a permeate component that has passed through the filter and a retentate component that has not passed through the filter. TFF systems are less prone to scaling than dead-end systems. Scaling of TFF systems can be further reduced by alternating the direction in which the fluid feed passes through the filter element, by backwashing the permeate through the filter, and / or by periodic washing.

[0004] Although TFF systems have advantages, they still have the disadvantage of resulting in expensive product losses. Therefore, there is a need to create TFF systems that reduce losses of products of interest. Summary of the invention

[0005] The present disclosure provides a system for enhancing 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 a 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.

[0006] In one aspect, the system further comprises a second filter element, which is in direct contact with the retentate and permeate from the first filter element. In another aspect, the filter elements are stacked in series. In another aspect, one or more of the filter elements in the filter element comprise a hollow fiber or a cartridge. In another aspect, the pore size of the hollow fiber is between 0.2 μM-0.65 μM.

[0007] In another aspect, one or more of the pumps is a peristaltic pump, a diaphragm pump, or a magnetic levitation pump.

[0008] In another aspect, the system is configured to operate in a recirculation mode.

[0009] In another aspect, the feed reservoir is a perfusion bioreactor. In another aspect, the size of the perfusion bioreactor exceeds 1000 liters. In another aspect, the size of the bioreactor is 5000 liters or 6000 liters. In another aspect, the system is configured for large-scale processing of the product of interest.

[0010] The present disclosure also provides a method for minimizing the retention of a product of interest in a permeate stream during TFF, the method comprising passing a liquid feed containing the product of interest through a system as described herein. In another aspect, the liquid feed comprises cells and a target product of interest. In another aspect, the product of interest is an antibody or an antigen-binding fragment thereof. In another aspect, the product of interest is recovered in the permeate. In another aspect, the liquid feed passes through the system at a rate that minimizes cell shear. In another aspect, the liquid feed passes through the system at a rate of 1.8 mL / chamber / minute-8 mL / chamber / minute. In another aspect, the pressure difference between the retentate inlet and the permeate is consistent with the system pressure after filtration.

[0011] The present disclosure also provides a method for minimizing retention of a product of interest in a permeate stream during TFF, the method comprising passing a liquid feed through a system described herein at a flow rate that is at least one-third slower than a conventional TFF flow rate. In another aspect, the flow rate provides about 1800 s -1 The shear rate.

[0012] In another aspect, the method further comprises sparging the system with air. In another aspect, the air comprises about 10%-80% dissolved oxygen. In another aspect, the liquid feed is injected with air before contacting the filter element. In another aspect, the injection is performed at a rate necessary to maintain greater than 10% dissolved oxygen throughout the TFF system. In some aspects, the injection comprises introducing oxygen with a bubble diameter of about 1 μm to about 10 μm. In some aspects, the injection comprises introducing oxygen with a bubble diameter of about 1 μm or about 10 μm. In another aspect, the liquid feed comprises cells and a target product of interest. In another aspect, the injection minimizes the lactic acid production of the cells. In another aspect, the product of interest is an antibody or an antigen-binding fragment thereof. In another aspect, the method further comprises recovering the product of interest in the permeate. In some aspects, the injection increases the unit productivity of the cells compared to cells without injection. In some aspects, the injection increases the unit productivity of the cells to greater than about 0.0034 gmL compared to cells without injection. -1 sky -1 In some aspects, injection increases the specific productivity of the cells from at least 0.0034 g mL -1 sky -1 Increased to about 0.0044 gmL -1 sky -1 In another aspect, the pressure differential between the retentate inlet and the permeate is consistent with the system pressure after filtration. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A schematic diagram of a typical TFF system is shown.

[0014] FIG2 shows product retention and corresponding yield loss during small-scale TFF. Figure 2A Product titers in the permeate and retentate are shown. Figure 2B The percent product yield in TFF is shown.

[0015] Figure 3 A schematic diagram of a high performance (HPTFF) system of the present disclosure is shown. The TFF system employs a second pump connected to the permeate. The TFF system may employ one or more filtration cassettes.

[0016] Figure 4 shows the results of HPTFF and low-flow TFF compared with conventional TFF. Figure 4A ) and AZ-2( Figure 4B ) of the increased yield.

[0017] Figure 5 shows the results for 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 ), osmotic concentration ( Figure 5E ) and lactate levels ( Fig. 5F ), Culture performance of AZ-1 using HPTFF.

[0018] Figure 6 shows that under typical TFF conditions ( Fig. 6A ) and using air injection ( Figure 6B ) of dissolved oxygen levels, and a schematic diagram ( Figure 6C ).

[0019] FIG. 7 shows the viable cell density ( Fig. 7A ), Viability( Figure 7B ), lactate level( Figure 7C ), total product titer ( Fig.7D ), unit productivity ( Fig. 7E ) and product retention ( Figure 7F ), Culture performance of AZ-3 against typical TFF with and without air injection. DETAILED DESCRIPTION

[0020] The present disclosure provides a kind of efficient method to minimize the retention of the product of interest during TFF. In some aspects, the present disclosure provides the method for minimizing the product loss caused by the pressure drop during the large-scale treatment of perfusion cell culture. In another aspect, the culture is greater than 1000L culture. In some aspects, the present disclosure provides the method for minimizing the pressure drop by adopting two or more pumps in the TFF system. In some aspects, the present disclosure provides the method for adopting low flow rate and air injection to reduce the retention of the product of interest.

[0021] I. definition

[0022] In order to more easily understand the present disclosure, some terms are first defined. As used in this specification, unless otherwise explicitly provided herein, each of the following terms should have the meaning set forth below. Additional definitions are set forth throughout the specification.

[0023] It should be noted that the term "a" or "an" refers to one or more of the entity; for example, "feed medium" should be understood to mean one or more feed media. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0024] As used herein, the term "and / or" should be understood as specifically disclosing each of the two specified features or components, whether or not the other is present. Thus, the term "and / or" as used herein in phrases 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" as used in phrases such as "A, B, and / or C" is intended to cover 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).

[0025] It should be understood that wherever herein aspects are described with the language "comprising," other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0026] 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 the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a general dictionary of many of the terms used in the present disclosure for the skilled person.

[0027] Units, prefixes and symbols are expressed in their SI recognized form. Numerical ranges include numerical values ​​that limit the range. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to the entire specification. Therefore, the terms defined immediately below are more fully defined by reference to the entire content of the specification.

[0028] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any listed or enumerated components.

[0029] The term "about" or "substantially comprising" means that a value or composition is within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially comprising" may mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. In addition, particularly with respect to biological systems or processes, these terms may mean values ​​of up to an order of magnitude or up to 5 times. When a specific value or composition is provided in the present application and claims, unless otherwise stated, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value or composition.

[0030] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range, and where appropriate, include fractions thereof (such as tenths and hundredths of integers).

[0031] As used herein, the term "bioreactor" refers to any suitable vessel or other member for producing and maintaining a biological cell culture, including but not limited to perfusion / perfusion bioreactors. The bioreactor of the present disclosure is used for large-scale production of products of interest. In some aspects, the bioreactor volume is greater than 1000L. In some aspects, the bioreactor volume is 1000L, 1500L, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L, 6000L, 6500L, 7000L, 7500L, 8000L, 8500L, 9000L, 9500L or 10,000L.

[0032] As used herein, the term "perfusion" refers to a fermentation or cell culture process for producing 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 that may contain the harvested target biological product.

[0033] As used herein, the term "cut-off size" or "molecular weight cut-off" with respect to ultrafiltration membranes refers to the molecular weight of molecules or particles that are 90% retained by the membrane.

[0034] The expression "spiral wound filter element" refers to a filter membrane that is spirally wound around a core. The spiral wound filter element may be contained in a housing and may alternatively be referred to as a spiral wound filter module.

[0035] "Pressure drop" refers to the drop in pressure (eg, psid) from the retentate inlet and the permeate.

[0036] "Flux" is the area-normalized flow rate.

[0037] "Permeate flux" is the area-normalized flow rate of permeate in the permeate channel (e.g., liters / hour / m 2 , lmh).

[0038] The “cross-flow rate” is the area-normalized average flow rate of the retentate in the feed channel (e.g., L / min / m 2 , LMM).

[0039] "Cross flow" is the retentate flow rate between the inlet and outlet of a feed channel in a filter or series of filters. Unless otherwise specified, "cross flow" refers to the average cross flow.

[0040] The term "shear" refers to the strain in the structure of a material caused by pressure.

[0041] The term "shear rate" refers to the rate at which progressive shear deformation is applied (eg, s-1).

[0042] The terms "feed," "feed sample," and "feed stream" refer to the solution introduced into a filtration module for separation.

[0043] The term "separation" generally refers to the act of separating a feed sample into two streams, a permeate stream and a retentate stream.

[0044] The terms "permeate" and "permeate stream" refer to the portion of the feed that has permeated through the membrane.

[0045] The terms "retentate" and "retentate stream" refer to the portion of the solution that has been retained by the membrane, with the retentate being the stream enriched in the retained material.

[0046] "Feed channel" refers to a conduit in a filtration assembly, module or element used for feeding.

[0047] "Permeate channel" refers to the conduit for permeate in a filtration assembly, module, or element.

[0048] The expression "flow path" refers to a channel including a filter membrane (e.g., an ultrafiltration membrane, a microfiltration membrane) through which the filtered solution (e.g., in a tangential flow mode) passes. The flow path can have any topological structure that supports tangential flow (e.g., straight, coiled, arranged in a zigzag manner). The flow path can be open, as in the example of a channel formed by a hollow fiber membrane, or have one or more flow obstacles, such as in the case of a rectangular channel formed by a flat sheet membrane separated by a woven or non-woven separator.

[0049] "TFF module," "TFF system," and "TFF apparatus" are used interchangeably herein to refer to a tangential flow filtration system configured to operate in a single-pass mode and / or a recirculation mode (eg, full or partial recirculation) and / or an alternating flow mode.

[0050] "Single-lobe" spirals are spiral-wound filter elements that form a continuous feed channel. They are usually made from one sheet of membrane.

[0051] "Multi-leaf" spirals are spiral-wound filter elements with multiple feed channels. They are usually made from more than one membrane sheet; but can also be made from 1 membrane sheet.

[0052] "Cartridge rack" refers to a compression assembly for one or more cartridges. Typically, when a cartridge rack contains more than one cartridge, the cartridges are configured for parallel processing, although in some embodiments, the cartridges may be configured for serial processing.

[0053] "Cassette" refers to a cassette or plate module containing a filtration (eg, ultrafiltration or microfiltration) membrane suitable for use in a TFF process.

[0054] "Filtration membrane" refers to a selectively permeable membrane that can be used in a filtration system, such as a TFF system.

[0055] The terms "microfiltration membrane" and "MF membrane" are used herein to refer to membranes having pore sizes ranging between about 0.1 microns to about 10 microns.

[0056] By "fluidically connected" is meant that a plurality of spiral wound membrane TFF modules are connected to each other by one or more conduits for liquids, such as feed channels, retentate channels and / or permeate channels.

[0057] "Product" refers to a target compound. In some aspects, the product will be a biomolecule of interest (eg, a protein), such as a monoclonal antibody (mAb).

[0058] "Processing" refers to the act of filtering (e.g., by TFF) a feed containing a product of interest and subsequently recovering the product (e.g., in purified form). The product can be recovered from the filtration system (e.g., TFF module) as a retentate stream or a permeate stream, depending on the size of the product and the pore size of the filtration membrane.

[0059] The expressions "parallel processing," "processing in parallel," "parallel operation," and "operation in parallel" refer to processing product in a TFF module comprising multiple processing units that are fluidly connected by distributing feed from a feed channel or manifold directly to each processing unit in the module.

[0060] The expressions "series processing", "processing in series", "series operation", and "operation in series" refer to processing a product in a TFF module comprising multiple processing units that are fluidly connected by distributing feed from a feed channel directly to only the first processing unit in the module. In series processing, each of the other subsequent processing units in the module receives its feed from the retentate line of the previous processing unit (e.g., the retentate from the first processing unit is used as feed to the second adjacent processing unit).

[0061] As used herein, "perfusion" or "perfusion culture" or "perfusion culture process" refers to the continuous flow of a physiological nutrient solution through or through a cell population at a steady rate. Since perfusion systems typically involve retaining cells in a culture unit, perfusion cultures characteristically have relatively high cell densities, but culture conditions are difficult to maintain and control. In addition, since cells are grown at high density and retained in a culture unit, the growth rate typically decreases over time, causing cell growth to enter an exponential late phase or even a stationary phase. This continuous culture strategy typically includes culturing mammalian cells, such as non-fixation-dependent cells, expressing polypeptides and / or viruses of interest during the production phase in a continuous cell culture system. In some aspects, perfusion culture is a large-scale culture. In some aspects, the culture is greater than 1000 liters of culture. In another aspect, the culture is 3,000 liters, 4,000 liters, 5,000 liters, 6,000 liters, 7,000 liters, 8,000 liters, 9,000 liters, or 10,000 liters of culture.

[0062] Various aspects of the disclosure are described in more detail in the following subsections.

[0063] II. High Performance Tangential Flow Filtration (HPTFF)

[0064] In contrast to fed-batch systems, perfusion systems and methods involve continuous filtration of cell culture media. During filtration, the product of interest (e.g., target protein, such as monoclonal antibody) and optionally other soluble components, such as cell waste products (e.g., lactic acid and ammonia) are removed from the cell culture media. Compared to fed-batch systems, perfusion systems present unique challenges because the cells contained in the perfusion system repeatedly pass through the filtration equipment, which may cause physical damage to the cells and thus may reduce the productivity of the system. It is desirable to minimize cell damage during filtration in a perfusion system so as to retain as many cells as possible for continued production of the target protein.

[0065] Tangential flow filtration (TFF) is a separation process that uses a membrane to separate components in a liquid solution or suspension based on size, molecular weight or other differences. TFF is used to remove a target product of interest (e.g., protein) from a cell culture medium during perfusion, while retaining the cells in the culture medium. During the TFF process, the 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 the treatment tank. The TFF process may involve fluid passing through the membrane additionally (e.g., recirculation) until the process fluid is fully clarified, concentrated or purified. The cross-flow properties of TFF minimize membrane fouling, therefore allowing each batch of high volume processing. The membrane is contained in a filter element, which can be a variety of configurations, such as a spiral wound filter element and a cassette filter element.

[0066] A typical TFF system is shown in Figure 1 In. The pressurized feed from the feed tank is connected to the feed port of the spiral wound filtration module or the manifold of the cassette filter. The feed flows through the membrane-lined feed channel of the TFF device under the control of a pump. Some solvent from the feed stream flows across the surface of the membrane into the permeate channel and carries a portion of the permeable material (e.g., product of interest and waste product). The remaining concentrated feed stream flows out of the module or manifold through the retentate port. The permeate flowing out of the permeate port of the module is directed to a location depending on the process, where it is collected (e.g., with the product of interest) or discarded (e.g., with the waste product).

[0067] However, under large-scale production conditions, e.g., bioreactor volumes greater than 1000 L, TFF systems experience a pressure drop between the retentate inlet and the permeate, which results in the product of interest being retained in the retentate due to the large processing volume. This pressure drop results in the product of interest being retained in the retentate, which in turn results in a loss of product recovery.

[0068] 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 part of the system and at least one conduit ( Figure 3 ). In one aspect, one pump is located at the retentate inlet and one pump is in contact with the permeate. A flow meter can be used to provide a process value for a pump or valve to control the amount of retentate that is recycled. Alternatively or in addition, a valve or pump and / or flow meter can be positioned on the permeate outlet or in a flow line that carries the permeate out of the system to control or limit the permeate flow.

[0069] By selecting appropriate transmembrane pressure (TMP) to discharge permeate, it is possible to obtain the maximum flux achievable during the TFF system operation. This is applicable to pressure-dependent and mass transfer-limited operating regions. For spiral wound filters, the desired TMP is determined to be reached by measuring at the module end. For boxes with, for example, two permeate outlets, the desired TMP is determined to be reached by the average feed channel pressure. The transmembrane pressure must be sufficient to support both the pressure drop through the membrane and the maximum pressure of the permeate discharged from the permeate channel. Alternatively or in addition, by selecting appropriate permeate flow rate to discharge permeate, it is possible to obtain the maximum flux achievable during the TFF system operation. The permeate flow rate can be controlled to a constant value by using a permeate valve or a pump.

[0070] TFF devices currently used in perfusion systems include hollow fiber devices and open channel cassette devices, also known as plate and frame devices. Examples of currently available filtration devices for perfusion systems include, but are not limited to, XCell TM ATF Systems (Repligen, Waltham, Mass.) and Perfusion Systems (Spectrum Laboratories, Rancho Dominguez, Calif.)—these are hollow fiber devices, and Prostak TM Microfiltration modules (MilliporeSigma, Billerica, Mass.)--they are cassette devices.These devices include open feed channels to limit the physical damage to the cells in the feed stream, and both devices require high cross-flow rates to minimize fouling (i.e., the accumulation of particles along the membrane wall).The membrane fouling and pressure drop in the TFF system reduce product recovery because the target protein and waste material are reduced by the passage of the membrane (i.e., sieving).In some aspects, the pore size of hollow fiber is 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 0.95 μM or 1.0 μM.

[0071] During the perfusion process, the cell culture medium is introduced into the feed side of the membrane. As the liquid feed (e.g., cell culture medium) travels across the surface of the membrane, it is separated into a permeate and a retentate. Specifically, the target product of interest passes through the membrane and is recovered from the permeate leaving the filter by a collection tube. The cells are retained and recovered from the retentate leaving 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.

[0072] The perfusion system may include a TFF system having one or more spiral wound filter elements or cassette filter elements as described herein. In systems having more than one filter element, the filter elements may be fluidically connected in series or in parallel or in series and in parallel.

[0073] TFF systems can be operated in a recirculation mode, where all or part of the retentate is returned to the filter element for further filtration. In a perfusion system, after 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.

[0074] like Figure 3 The feed pump shown can be configured to operate in a recirculation mode. The feed pump can be a non-cell-disrupting 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 Series pumps (Levitronix Technologies, Framingham, Mass.). Examples of suitable diaphragm pumps include the Repligen XCell TM ATF pump (Repligen, Waltham, Mass.). Examples of suitable peristaltic pumps include Watson Marlow Series 500 and Series 600 pumps (Watson Marlow, Wilmington, Mass.).

[0075] In one aspect, the present disclosure relates to a method of passing a liquid feed through a HPTFF system described herein, the HPTFF system comprising at least one filter element, the method separating the liquid feed into a permeate and a retentate in the filter element; and recovering the permeate and at least a portion of the retentate from the filter element. The liquid feed may comprise a cell culture medium containing cells and a target product of interest. The target product of interest may be recovered in the permeate, and the cells may be retained in the retentate.

[0076] The process may include recirculating at least a portion of the retentate through the filter element. The recirculation may be performed on a continuous basis or at regular intervals to continuously harvest product from the cell culture medium.

[0077] The recycled retentate can be returned to any upstream location in or before the HPTFF system (e.g., a bioreactor located upstream of the HPTFF system). In one aspect, the retentate is recycled to the feed tank. In another aspect, the retentate is recycled to the feed line near the feed pump before being recycled to the feed inlet on the HPTFF system.

[0078] In some aspects, the methods described herein include perfusing at low flow rates to overcome the pressure drop in the TFF system. Low flow rates are associated with improved product quality due to reduced shear rates. In one aspect, the low flow rate is about one-third of the typical rate of a TFF system. In another aspect, conventional flow rates are about 1800 s -1 Therefore, on the other hand, low flow rates are associated with shear rates of about 600 s -1 is related to the shear rate.

[0079] In some aspects, due to the longer time that feed stream stays outside the bioreactor, low flow TFF can cause hypoxia in the HPTFF system. Therefore, in some aspects, feed stream is injected with air. In some aspects, feed stream is injected with 10% to 80% dissolved oxygen. In some aspects, feed stream is injected with 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% dissolved oxygen. In some aspects, oxygen is introduced with a bubble diameter of about 1 μm. In some aspects, oxygen is introduced with a bubble diameter of about 10 μm. In some aspects, oxygen is introduced with a bubble diameter of about 10 μm to about 10 μm.

[0080] In some aspects, the disclosure provides purification of any product of interest. In some aspects, the product of interest is a protein. Therefore, in some aspects, the disclosure relates to a perfusion process for harvesting a target protein from a liquid feed containing a host cell. The target protein can be a monoclonal antibody, which is separated from the host cell by TFF and recovered from the permeate of the filter element.

[0081] Example

[0082] Bioreactors containing cells producing AZ-1 (a monoclonal antibody) were subjected to standard TFF, such as Figure 1 The bioreactor volume was 2 L and the TFF was performed at a rate corresponding to 1360 s -1 200 μL, using a Repligen S04-P20U-10-N hollow fiber filter. As shown in Figure 2, using standard TFF conditions, as the culture duration increases and the amount of protein produced increases, the titer in the TFF retentate increases relative to the target titer ( Figure 2A This increase above the target titer results in a significant decrease in the yield of recovered protein ( Figure 2B ).

[0083] AZ-1 and AZ-2 (a monoclonal antibody) were then subjected to HPTFF and low flow TFF as described herein. The bioreactor feed from the 2L reactor was at a rate corresponding to 1360 s -1 The flow rate was 600 s for low flow TFF, using a Repligen S04-P20U-10-N hollow fiber filter. -1 As shown in Figure 4, when the HPTFF and low flow system described herein were used for clarification, AZ-1 ( Figure 4A ) and AZ-2 (a monoclonal antibody, Figure 4B ) was significantly increased. The viable cell density, viability, glucose level, pH, osmotic concentration and lactate level ( FIG. 5A to FIG. 5F ) measurement, the culture performance of AZ-1 was not affected.

[0084] Low feed flow rates were also analyzed to overcome retention of product in the retentate. Flow rates as low as one-third of the typical rate of TFF were analyzed. Fig. 6A As shown, dissolved oxygen levels were depleted during typical TFF but returned to normal levels when the recirculation loop was injected with air containing 21% dissolved oxygen. Low dissolved oxygen levels reduced specific productivity (Figure 7). Typical TFF culture performance of AZ-3 (a bispecific antibody) showed that air injection overcame the negative effects of hypoxia on specific productivity without affecting viable cell density and cell viability. ( 7A to 7D ) In addition, compared with 0.0034 g mL in the control TFF -1 sky -1 In comparison, the specific productivity per cell volume in injected TFF was approximately 0.0044 g mL -1 sky -1 .

Claims

1. A system for enhancing recovery of a product of interest during tangential flow filtration (TFF), the system comprising: (1) 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.

2. The system of claim 1, further comprising a second filter element in direct contact with the retentate and 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 cartridge. The system of claim 3 , wherein the filter elements are stacked in series.

5. The system according to any one of claims 1 to 4, wherein the pore size of the hollow fiber is 0.2 μM-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. The system of any one of claims 1 to 6, wherein the system is configured to operate in a recirculation mode.

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

9. The system according to any one of claims 1 to 8, wherein the size of the perfusion bioreactor exceeds 1000 liters.

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

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

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

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

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

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 rate to minimize cell shear.

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

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

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

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

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

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

23. The method of any one of claims 19 to 22, wherein the liquid feed is injected with air prior to contact with the filter element.

24. A method according to any one of claims 21 to 23, wherein the injection is performed at a rate necessary to maintain more than 10% dissolved oxygen throughout the TFF system.

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

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

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

28. The method of any one of claims 21 to 25, wherein the injection minimizes lactate 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. The method of any one of claims 21 to 29, wherein the injection increases the specific productivity of the cells compared to non-injected cells.

31. The method of any one of claims 21 to 30, wherein the injection increases the specific productivity of the cells to greater than about 0.0034 gmL compared to cells not injected. -1 sky -1 .

32. The method of any one of claims 21 to 31, wherein the injection increases the specific productivity of the cells from at least 0.0034 gmL -1 sky -1 Increased to about 0.0044 gmL -1 sky -1 .

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 corresponds to the system pressure after filtration.

Images