US20200139303A1 - Tangential flow filtration device for perfusion applications - Google Patents

Tangential flow filtration device for perfusion applications Download PDF

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US20200139303A1
US20200139303A1 US16/607,468 US201816607468A US2020139303A1 US 20200139303 A1 US20200139303 A1 US 20200139303A1 US 201816607468 A US201816607468 A US 201816607468A US 2020139303 A1 US2020139303 A1 US 2020139303A1
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filter element
feed
perfusion
retentate
pump
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Alison Dupont
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EMD Millipore Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/10—Separation or concentration of fermentation products
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/10—Spiral-wound membrane modules
    • B01D63/103—Details relating to membrane envelopes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/08—Flat membrane modules
    • B01D63/082—Flat membrane modules comprising a stack of flat membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/08—Flat membrane modules
    • B01D63/082—Flat membrane modules comprising a stack of flat membranes
    • B01D63/0822—Plate-and-frame devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/10—Spiral-wound membrane modules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/06—Specific process operations in the permeate stream
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/25—Recirculation, recycling or bypass, e.g. recirculation of concentrate into the feed
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2313/00—Details relating to membrane modules or apparatus
    • B01D2313/14—Specific spacers
    • B01D2313/143—Specific spacers on the feed side
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2313/00—Details relating to membrane modules or apparatus
    • B01D2313/24—Specific pressurizing or depressurizing means
    • B01D2313/243—Pumps
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/10—Cross-flow filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2325/00—Details relating to properties of membranes
    • B01D2325/02—Details relating to pores or porosity of the membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/18—Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D65/00—Accessories or auxiliary operations, in general, for separation processes or apparatus using semi-permeable membranes
    • B01D65/02—Membrane cleaning or sterilisation ; Membrane regeneration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D65/00—Accessories or auxiliary operations, in general, for separation processes or apparatus using semi-permeable membranes
    • B01D65/08—Prevention of membrane fouling or of concentration polarisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis

Definitions

  • Monoclonal antibodies are used as therapeutic agents for a variety of indications, including, for example, cancer, transplant rejections, and cardiovascular disease.
  • Various biopharmaceutical manufacturing techniques exist to produce and harvest mAbs from host cells, including, for example, fed-batch processes and perfusion processes.
  • fed-batch bioreactor systems cells are cultured in batches over a set period of time, for example, over about seven to about twenty-one days, after which point media nutrients have been consumed by the host cells and waste products have accumulated.
  • the batch undergoes a harvesting step in which the protein of interest (e.g., a product, such as a monoclonal antibody, or mAb) is separated from the cell mass.
  • the protein of interest e.g., a product, such as a monoclonal antibody, or mAb
  • perfusion bioreactors culture cells over longer periods of time, for example, over several weeks or months, while continuously feeding cells with fresh media, removing spent media, and harvesting product.
  • Perfusion systems offer several advantages over fed-batch systems. For example, as product is harvested and purified on a continuous basis, before the proteins of interest are exposed to high levels of waste, product degradation is reduced in perfusion systems.
  • a perfusion bioreactor can produce a similar product yield as a fed-batch bioreactor while occupying a significantly smaller space.
  • Perfusion is becoming a preferred manufacturing technique in the biopharmaceutical industry due to its advantages over batch-fed processes.
  • perfusion processes rely on a high density of host cells being maintained throughout each production process, and continuous harvesting, which involves several iterations of filtration, and can cause physical damage to the host cells.
  • Filter elements for perfusion systems and methods are provided that exhibit improved sieving and throughput of mAbs as compared with existing open-channel and hollow fiber devices.
  • the invention encompasses a filter element sheet that includes a microporous membrane having a mean pore size of at least about 0.65 ⁇ m and a feed spacer comprising woven fibers and having an open area of at least about 35%.
  • Feed spacers comprising woven fibers and having an open area of at least about 35% can provide for low shear rates within a feed channel of a filter element, such that host cells are not damaged during filtration.
  • the feed spacer can have an open area of, for example, about 35% to about 55%, and can comprise fibers having an average fiber diameter of at least about 270 ⁇ m, for example, about 300 ⁇ m to about 500 ⁇ m.
  • the pore size of a feed spacer can be, for example, about 0.8 ⁇ m to about 10 ⁇ m, or about 1.0 ⁇ m to about 5 ⁇ m.
  • a fiber density of the feed spacer can be of about 6 fibers/cm to about 13 fibers/cm.
  • the fibers of the feed spacer can be woven in a two-over-one twill pattern or a one-over-one weave pattern.
  • the invention provides for a filter element that includes at least one filter element sheet as described herein.
  • the filter element can be a spiral-wound filter element or a cassette filter element.
  • the invention encompasses a perfusion system that includes at least one filter element as described herein and a pump configured to control flow of a liquid feed through the at least one filter element.
  • Such perfusion systems can be configured to be operated in, for example, a tangential flow filtration (TFF) mode, a recirculation mode, and/or an alternating flow mode.
  • the pump can be, for example, a magnetic levitation pump, a peristaltic pump, or a diaphragm pump.
  • the invention in yet another embodiment, relates to a perfusion process that includes passing a liquid feed through a feed channel of at least one filter element and separating the liquid feed into permeate and retentate by tangential flow filtration (TFF) in the filter element.
  • the filter element includes a microfiltration membrane and a woven feed spacer located within the feed channel. Cells and a target protein can be present in the liquid feed.
  • perfusion processes of the present invention can also include recovering the target protein in the permeate and/or retaining the cells in the retentate.
  • at least a portion of the retentate can be recirculated through the at least one filter element. Additionally, or alternatively, flow of liquid through the at least one filter element can be alternated for self-cleaning of the filter element.
  • the perfusion process can also include supplying a volume of fresh media to the retentate and returning the retentate and fresh media to a bioreactor.
  • the perfusion process can be run on a continuous basis, with the retentate of an initial perfusion run being the liquid feed of a subsequent perfusion run.
  • the invention provides for a perfusion process for harvesting target proteins from a liquid feed containing host cells.
  • the process includes delivering a liquid feed containing target proteins and host cells to a feed channel of at least one filter element and separating the target proteins from the host cells in the at least one filter element.
  • the at least one filter element includes a microfiltration membrane and a woven feed spacer located within the feed channel.
  • the target proteins can be, for example, monoclonal antibodies, which can be separated from the host cells by TFF and recovered from the at least one filter element.
  • the perfusion process can further include recovering the host cells from the at least one filter element, supplying a volume of fresh media to the recovered host cells, and returning the recovered host cells to a bioreactor.
  • the perfusion process can be run on a continuous basis, with the recovered host cells of an initial perfusion run being the liquid feed of a subsequent perfusion run.
  • Perfusion processes of the present invention can provide for improved sieving over conventional perfusion processes involving open-channel filtration devices.
  • at least about 80%, at least about 90%, or at least about 95% of the target proteins can be recovered from the liquid feed at a harvest throughput of at least about 500 L/m 2 of the filter element.
  • at least about 80%, at least about 90%, or at least about 95% of the target proteins can be recovered from the liquid feed at a harvest throughput of at least about 1000 L/m 2 of the filter element.
  • FIG. 1 is a diagram illustrating an example of a spiral-wound filter element.
  • FIG. 2 is an exploded view of an example of a cassette filter element.
  • FIG. 3 is a schematic illustrating an example of a TFF system.
  • FIG. 4A is a simulated image of a woven-fiber feed spacer.
  • FIG. 4B is a three-dimensional graph of the woven-fiber feed spacer of FIG. 3A .
  • FIG. 4C is a three-dimensional graph of shear modeling results of the woven-fiber feed spacer of FIG. 4A .
  • FIG. 5 is a graph of modeled shear rates in an environment having a woven-fiber feed spacer.
  • FIG. 6 is a table of model results and experimental results for woven-fiber feed spacers.
  • FIG. 7 is a graph of experimental results of sieving versus harvest throughput of tangential flow filtration devices.
  • FIG. 8A is a graph of experimental results of membrane fouling in an open-channel filtration device.
  • FIG. 8B is a graph of experimental results of membrane fouling in a device with a ProstakTM Ultrafiltration (UF) screen.
  • FIG. 8C is a graph of experimental results of membrane fouling in a device with a D3 screen.
  • FIG. 8D is a graph of experimental results of membrane fouling in a device with a D screen.
  • FIG. 9 is a graph of experimental results of sieving versus harvest throughput of a tangential flow filtration device with 1 micron membrane and a D3 screen.
  • FIG. 10 is a graph of experimental results of sieving versus harvest throughput of a tangential flow filtration device with 1 micron membrane and a one-over-one woven screen.
  • spiral-wound filter element refers to a filtration membrane that is spirally wound about a core.
  • a spiral-wound filter element may be contained within a housing and may alternately be referred to as a spiral-wound filter module.
  • Pressure drop refers to the drop in pressure (e.g., psid) within a feed channel over the length of the filter element.
  • Frlux is the area-normalized flow rate.
  • Permeate flux is the area normalized flow rate of permeate in a permeate channel (e.g., Liters/hr/m2, lmh).
  • Cross-flow flux is the area normalized average flow rate of retentate in a feed channel (e.g., Liters/min/m2, LMM).
  • Cross-flow is the retentate flow rate between inlet and outlet of the feed channel in a filter or a series of filters. Unless otherwise stated, “cross-flow” refers to an average cross-flow.
  • shear refers to a strain in the structure of a substance that is produced by pressure.
  • shear rate refers to the rate at which a progressive shearing deformation is applied (e.g., s ⁇ 1 ).
  • feed refers to the solution being introduced into a filtration module for separation.
  • separation generally refers to the act of separating the feed sample into two streams, a permeate stream and a retentate stream.
  • permeate and permeate stream refer to that portion of the feed that has permeated through the membrane.
  • retentate and “retentate stream” refer to the portion of the solution that has been retained by the membrane, and the retentate is the stream enriched in a retained species.
  • Feed channel refers to a conduit in a filtration assembly, module or element for a feed.
  • Permeate channel refers to a conduit in a filtration assembly, module, or element for a permeate.
  • flow path refers to a channel comprising a filtration membrane (e.g., ultrafiltration membrane, microfiltration membrane) through which the solution being filtered passes (e.g., in a tangential flow mode).
  • the flow path can have any topology which supports tangential flow (e.g., straight, coiled, arranged in zigzag fashion).
  • a flow path can be open, as in an example of channels formed by hollow fiber membranes, or have one or more flow obstructions, as in the case, for example, of rectangular channels formed by flat-sheet membranes spaced apart by woven or non-woven spacers.
  • TFF assembly “TFF assembly,” “TFF system” and “TFF apparatus” are used interchangeably herein to refer to a tangential flow filtration system that is configured for operation in a single-pass mode and/or a recirculation mode (e.g., full or partial recirculation) and/or alternating flow mode.
  • a recirculation mode e.g., full or partial recirculation
  • Single leaf spirals are spiral-wound filter elements that can be formed with one continuous feed channel. They are generally made with one sheet of membrane.
  • Multi-leaf spirals are spiral-wound filter elements that have multiple feed channels. They are generally made with more than 1 sheet of membrane; but can be made with 1 membrane sheet also.
  • a “cassette holder” refers to a compression assembly for one or more cassettes. Typically, when a cassette holder contains more than one cassette, the cassettes are configured for parallel processing, although, in some embodiments, the cassettes can be configured for serial processing.
  • a “cassette” refers to a cartridge or flat plate module comprising filtration (e.g., ultrafiltration or microfiltration) membrane sheet(s) suitable for TFF processes.
  • Frtration membrane refers to a selectively permeable membrane capable of use in a filtration system, such as a TFF system.
  • microfiltration membranes and “MF membranes” are used herein to refer to membranes that have pore sizes in the range between about 0.1 micrometers to about 10 micrometers.
  • Fluidly connected refers to a plurality of spiral-wound membrane TFF modules that are connected to one another by one or more conduits for a liquid, such as, a feed channel, retentate channel and/or permeate channel.
  • Product refers to a target compound.
  • a product will be a biomolecule (e.g., protein) of interest, such as a monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • 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 a purified form).
  • the product can be recovered from the filtration system (e.g., a TFF assembly) in either the retentate stream or permeate stream depending on the product's size and the pore size of the filtration membrane.
  • parallel processing refers to processing a product in a TFF assembly that contains a plurality of processing units that are fluidly connected by distributing the feed directly from a feed channel or manifold to each of the processing units in the assembly.
  • serial processing refers to processing a product in a TFF assembly that contains a plurality of processing units that are fluidly connected by distributing the feed directly from the feed channel to only the first processing unit in the assembly.
  • each of the other, subsequent processing units in the assembly receives its feed from the retentate line of the preceding processing unit (e.g., the retentate from a first processing unit serves as the feed for a second, adjacent processing unit).
  • Perfusion systems and methods in contrast with fed-batch systems, involve continuous filtration of cell culture media.
  • target proteins such as mAbs, and optionally other soluble components, such as cellular waste products (e.g., lactic acid and ammonia)
  • cellular waste products e.g., lactic acid and ammonia
  • Perfusion systems (alternately referred to as cell-retention systems) present unique challenges over fed-batch systems as the cells contained in a perfusion system pass repeatedly through filtration equipment, which can cause physical damage to the cells and which, in turn, can reduce 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.
  • Tangential flow filtration 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.
  • 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.
  • 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.
  • the membranes are contained within filter elements that can be of a variety of configurations, such as spiral-wound filter elements ( FIG. 1 ) and cassette filter elements ( FIG. 2 ).
  • TFF devices used in perfusion systems include hollow fiber devices and open-channel cassette devices, also referred to as plate-and-frame devices.
  • Examples of currently-available filtration devices for perfusion systems include XCellTM ATF System (Repligen, Waltham, Mass.) and KrosFlo® Perfusion System (Spectrum Laboratories, Collinso Dominguez, Calif.), which are hollow fiber devices, and ProstakTM Microfiltration Modules (MilliporeSigma, Billerica, Mass.), which are cassette devices.
  • These devices contain open feed channels, so as to limit physical damage to cells in the feed stream, and both devices require high cross-flow rates to minimize fouling (i.e., the accumulation of particles along the wall of membrane).
  • Membrane fouling reduces product recovery because the passage of target proteins and waste materials through the membrane (i.e., sieving) is reduced. Eventually, membrane fouling can result in failure of the device, with product no longer being recovered during filtration.
  • perfusion filter element sheets that exhibit improved sieving and throughput of mAbs as compared with open-channel devices, while minimizing cell damage during filtration.
  • perfusion filter element sheets are provided that include a combination of open microporous membrane(s) and low-shear feed spacer(s).
  • the combination of an open membrane (e.g., membranes having a pore size greater than about 0.65 ⁇ m, greater than about 1.0 ⁇ m, or greater than about 3 ⁇ m) with a low-shear feed spacer promotes mixing in the feed channel to minimize fouling while also maintaining shear rates at the membrane and fiber surfaces that are within cell stability limits.
  • the invention encompasses a perfusion filter element sheet includes a microporous membrane and a woven-fiber feed spacer.
  • the microporous membrane can have a mean pore size of at least about 0.65 ⁇ m (e.g., 0.62 ⁇ m, 0.65 ⁇ m, 0.67 ⁇ m, 0.8 ⁇ m), at least about 1.0 ⁇ m (e.g., 0.95 ⁇ m, 1.0 ⁇ m, 1.2 ⁇ m), or at least about 3.0 ⁇ m (e.g., 2.9 ⁇ m, 3.0 ⁇ m, 5 ⁇ m).
  • the mean pore size can be of about 0.8 ⁇ m to about 10 ⁇ m (e.g., 0.77 ⁇ m, 0.8 ⁇ m, 0.9 ⁇ m, 2 ⁇ m, 4 ⁇ m, 6 ⁇ m, 8 ⁇ m, 10.3 ⁇ m), or of about 1.0 ⁇ m to about 5 ⁇ m (e.g., 0.97 ⁇ m, 1.2 ⁇ m, 3 ⁇ m, 5.3 ⁇ m).
  • the mean pore size can be selected to provide for sieving of target proteins and/or waste materials from a cell culture fluid, while retaining cells within the cell culture fluid. Examples of suitable microporous membranes include the membranes listed in Table 1, below.
  • Pore Size Permeability Membrane ( ⁇ m) (ml/min/cm2) Manufacturer Cellulose Acetate 0.8 81.3 Sterlitech Cellulose Acetate 1.2 180 Sterlitech Cellulose Acetate 3 500 Sterlitech Cellulose Acetate 5 375 Sterlitech Mixed Cellulose Ester 0.8 165 Sterlitech Mixed Cellulose Ester 1 220 Sterlitech Mixed Cellulose Ester 3 300 Sterlitech Mixed Cellulose Ester 5 400 Sterlitech Mixed Cellulose Acetate 1.2 270 MilliporeSigma Mixed Cellulose Acetate 3 320 MilliporeSigma Mixed Cellulose Acetate 5 580 MilliporeSigma Dupore (PVDF) 1 >100 MilliporeSigma Dupore (PVDF) 1.2 >100 MilliporeSigma Dupore (PVDF) 5 >208 MilliporeSigma Nylon 0.8 120 Sterlitech Nylon 1.2 190 Sterlitech Polycarbonate 0.8 90 Sterlitech Polycarbonate 0.8 90 Sterlitech Polycarbonate
  • the feed spacer can include woven fibers that are woven in a two-over-one twill pattern or a one-over-one weave pattern.
  • the feed spacer can have an open area of at least about 35%, or of about 35% to about 55% (e.g., 34.5%, 35%, 36%, 39%, 40%, 50%, 55%, 55.5%).
  • a fiber density of the feed spacer can be of about 6 fibers/cm to about 13 fibers/cm (e.g., 5.5 fibers/cm, 6 fibers/cm, 8 fibers/cm, 10.6 fibers/cm, 12.2 fibers cm, 13.5 fibers/cm).
  • the feed spacer fibers can have a mean fiber diameter of at least about 270 ⁇ m (e.g., 265 ⁇ m, 270 ⁇ m, 275 ⁇ m), or of about 300 ⁇ m to about 500 ⁇ m (e.g., 290 ⁇ m, 300 ⁇ m, 400 ⁇ m, 500 ⁇ m, 510 ⁇ m).
  • suitable feed spacers include D-screens (Propyltex® screens, product no. 05-500/36, Sefar, QC, Canada) having a two-over-one twill pattern, an open area of 36%, a fiber density of 12.2 fibers/cm, fiber diameter of 340 ⁇ m, and a thickness of 610 ⁇ m, and D3-screens (Propyltex® screens, product no. 05-590/39, Sefar, QC, Canada) having a two-over-one twill pattern, an open area of 39%, a fiber density of 10.6 fibers/cm, fiber diameter of 360 ⁇ m, and a thickness of 645 ⁇ m.
  • D-screens Propyltex® screens, product no. 05-500/36, Sefar, QC, Canada
  • D3-screens Propyltex® screens, product no. 05-590/39, Sefar, QC, Canada
  • a suitable feed spacer is an E screen (PETEX® screen, product no. 07-840/46, Sefar, QC, Canada) having a one-over-one weave pattern, an open area of 46%, a fiber density of 8 fibers/cm, fiber diameter of 400 microns, and a thickness of 785 microns.
  • E screen PETEX® screen, product no. 07-840/46, Sefar, QC, Canada
  • the invention provides a filter element that includes a filtration sheet as described above.
  • the filter element can be a spiral-wound filter element or a cassette filter element.
  • FIG. 1 An example of a spiral-wound filter element 100 is illustrated in FIG. 1 , with arrows indicating feed flow direction and arrows indicating permeate flow within a membrane envelope 115 .
  • the membrane envelope 115 includes a membrane 112 folded over an optional permeate spacer 117 .
  • One or more membrane envelopes 115 can be included in a spiral-wound filter element.
  • Membrane envelope(s) 115 are in planar contact with the outer surfaces of feed spacer(s) 120 .
  • the membrane envelope(s) 115 and feed spacer(s) 120 are wound about a perforated permeate collection pipe 130 .
  • cell culture media is introduced to a feed-side of the membrane 112 .
  • the liquid feed e.g., the cell culture media
  • the cell culture media travels across the surface of the membrane 112 and through and around the feed spacer 120 , it is separated into permeate and retentate.
  • target proteins pass through the membrane 112 and are recovered from the permeate exiting the filter through collection tube 130 .
  • Cells are retained and are recovered from the retentate exiting the filter.
  • the cell culture media in the retentate can then be returned to a bioreactor, and the target proteins contained in the permeate can be collected in a separate vessel for further processing.
  • the cassette filter 20 includes a cassette filter element 28 having at least one feed plate 25 and at least one membrane plate 27 .
  • the feed plate 25 can include or be partially formed of woven fiber feed-spacer.
  • the membrane plate 27 includes a membrane 63 .
  • the filter element 28 is positioned between a manifold 32 and a manifold 34 .
  • Manifold 32 includes a feed inlet 12 and a retentate outlet 36 .
  • Manifold 34 includes a permeate outlet 38 and a feed inlet 40 .
  • Holes 48 , 49 located on the feed and membrane plates 25 , 27 are sealed in a configuration such that liquid feed entering the filter 20 (e.g., through feed inlets 12 , 40 ) travels in a path represented sequentially by arrows 51 , 53 , 55 , and 57 and separates into permeate and retentate.
  • Retentate which does not pass through membrane 63 , travels to retentate conduits 44 and exits the filter through retentate outlet 36 (as shown by arrow 67 ).
  • Permeate which passes through membrane 63 then travels in a path represented sequentially by arrows 59 , 61 , and 65 , traveling through permeate conduit 42 and exiting the filter through permeate outlet 38 .
  • Filter elements of the present invention provide several advantages over existing hollow fiber and cassette devices used in perfusion systems.
  • Conventional filtration devices for the purification of monoclonal antibodies typically include membranes having a mean pore size of about 0.2 ⁇ m to about 0.5 ⁇ m.
  • microporous membranes included in embodiments of the present invention can be considered “open” microporous membranes, having a mean pore size of at least about 0.65 ⁇ m, for example, at least about 1.0 ⁇ m, or at least about 3 ⁇ m.
  • Examples 3 and 5 herein demonstrate that open microporous membranes provide improved sieving performance over conventional filtration devices that have membranes with pore sizes of 0.2 ⁇ m or 0.5 ⁇ m. As membrane pore size increases, it is likely that more cellular debris passes through the membrane. As understood by the inventor of the present invention, and without adhering to any particular theory, it is believed that cellular debris fragments interact with proteins and DNA through electrostatic and hydrophobic interaction to form a gel like layer on membrane surfaces in conventional perfusion filtration devices. Such interaction increases fouling and reduces sieving in devices having “tighter” membranes (e.g., membranes having pore sizes of 0.2 ⁇ m or 0.5 ⁇ m).
  • feed screens are not included in existing perfusion devices as they are believed to impose unacceptable shear stresses on cells travelling through the filter, which is undesirable in cell-retention systems.
  • the sieving performance of conventional filtration devices decreases sharply during operation, with sieving reduced to approximately 40% after a harvest throughput of only 200 L/m 2 or 400 L/m 2 , as shown in FIG. 7 .
  • example embodiments of the present invention which have open microporous membranes and woven-fiber feed spacer sheets, are able to achieve about 100% sieving over a harvest throughput of at least about 500 L/m 2 (e.g., with a membrane pore size of about 1 ⁇ m) or at least about 1000 L/m 2 (e.g., with a membrane pore size of about 3 ⁇ m or about 5 ⁇ m).
  • examples of filter elements of the present invention demonstrate improved sieving over longer periods of operation, as compared with existing perfusion filters.
  • the results of Examples 1-4 herein demonstrate that woven fiber spacer sheets having an open area of at least about 35% can promote sufficient turbulence in a feed channel to reduce fouling while resulting in acceptable shear rates for cells passing through the feed channel.
  • Filter elements of the present invention can be included a perfusion system.
  • the perfusion system can include a TFF system having one or more than one spiral-wound filter element or cassette filter element described herein.
  • the filter elements can be fluidly connected in series or in parallel, or both.
  • FIG. 3 An example TFF system 300 is shown in FIG. 3 .
  • Pressurized feed from a feed tank is connected to the feed port of the spiral-wound filter module or manifold of the cassette filter.
  • Feed flows through the membrane lined feed channel of the TFF device(s) under an applied trans-channel pressure drop, typically achieved by pressurizing the feed using a pump.
  • Some of the solvent from the feed stream flows through the face of the membrane into the permeate channel and carries with it a portion of the permeable species (e.g., target protein, waste products).
  • the remaining concentrated feed stream flows out of the module or manifold through the retentate port.
  • the permeate flowing from the module's permeate port is directed to a location that is dependent on the process, where it is either collected (e.g., as with target protein) or discarded (e.g., as with waste product).
  • TFF systems can be operated in a recirculation mode, where all or a portion of the retentate is returned to the filter element(s) for further filtration.
  • the retentate can be returned to a bioreactor where the cell culture media may be maintained for some period of time before being recirculated through the TFF system.
  • the TFF systems containing filter elements that are employed in recirculating TFF methods can include at least one pump or control valve for recirculating retentate through all or part of the system and at least one conduit for recirculating (e.g., carrying) retentate.
  • the amount of retentate that is recirculated can be controlled using, for example, a pump or a valve.
  • a flow meter can be used to provide a process value for the pump or valve to control the amount of retentate that is recirculated.
  • the TFF systems described can further comprise a valve or pump and/or a flow meter for controlling recirculation of retentate.
  • valve or pump and/or flow meter can be positioned on the retentate outlet or on the flow line carrying retentate out of the system to the retentate receptacle.
  • a valve or pump and/or flow meter can be positioned on the peremeate outlet or in the flow line carrying permeate out of the system to control or limit permeate flow.
  • TFF transmembrane pressure
  • attainment of the desired TMP is determined by measurement at the end of the module.
  • attainment of the desired TMP is determined 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 to discharge permeate from the permeate channel.
  • maximum achievable flux during a TFF system operation can be obtained by selection of an adequate permeate flow rate for permeate discharge.
  • the permeate flow rate can be controlled to a constant value by use of a permeate valve or pump.
  • a permeate valve or pump For perfusion applications, it can be desirable to maintain permeate flow rate at a lower level than would be possible with an uncontrolled permeate stream and to maintain a more stable flow.
  • TFF systems can also be operated in an alternating flow mode.
  • Alternating flow mode can be achieved by various methods.
  • a pump for example a diaphragm pump, is connected to the retentate port of the filter element. Feed is pulled into the feed port of the filter element by the pump and travels through the feed channel within the filter, out the retentate port, and into the pump. The pump is then reversed and the liquid media (formerly comprising the feed solution) is pushed out of the pump, through the retentate side of the filter element into the feed channel, out the feed port of the filter, and back to the bioreactor.
  • a pump alternately pushes and pulls liquid media through the filter element(s).
  • liquid feed is typically introduced to a feed-side of a filter element.
  • the feed is pulled from the bioreactor via a pump (e.g., a pump connected to a feed port of the filter element) and is pushed into the filter element through the feed port.
  • the liquid media then travels through the feed channel, out the retentate port, and back to the bioreactor.
  • the pump then reverses direction, pulling feed from the bioreactor in to the filter element through the retentate port.
  • the liquid media then travels through the feed channel, out the feed port, and through the pump, from which it is then pushed back to the bioreactor.
  • a TFF system can be operated in alternating flow mode by use of a pump and a valve block.
  • the pump continuously pulls a flow of liquid media from the bioreactor and pushes the media into the feed channel of the device.
  • valves are used to change a direction of the flow of the liquid media entering the filter.
  • the flow of liquid media initially passes through the pump and enters the feed port of the filter.
  • the media passes through the feed channel and exits the filter through the retentate port, returning to the bioreactor.
  • the valves switch position, causing a flow of liquid media from the pump to enter the retentate port of the filter, pass through the feed channel, and exit the filter through the feed port, then returning to the bioreactor.
  • Alternating flow can create a back flush of the filter membrane to self-clean the membrane and reduce fouling.
  • the feed pump as illustrated in FIG. 3 can be configured to operate in a recirculation mode and/or an alternating flow mode.
  • the feed pump can be a pump that is not damaging to cells, such as a magnetic levitation pump, a diaphragm pump, a peristaltic pump, or a rotary vane pump.
  • suitable magnetic levitation pumps include Levitronix® Puralev® Series pump (Levitronix Technologies, Framingham, Mass.).
  • suitable diaphragm pumps include Repligen XCellTM ATF pump (Repligen, Waltham, Mass.).
  • suitable peristaltic pumps include Watson Marlow Series 500 and Series 600 pumps (Watson Marlow, Wilmington, Mass.).
  • the invention relates to a method of passing a liquid feed through at least one filter element of the invention, separating the liquid feed into permeate and retentate in the filter element; and recovering the permeate and at least a portion of the retentate from the filter element.
  • the liquid feed can comprise a cell culture media, containing cells and a target protein.
  • the target protein can be recovered in the permeate and the cells can be retained in the retentate.
  • the process can include recirculating at least a portion of the retenate through the filter element(s). Recirculation can be performed on an ongoing basis or at regular intervals to continually harvest product from the cell culture media.
  • recirculating all or a portion of the retentate during start up provides a method by which to ensure that system has reached equilibrium and the retentate has achieved the desired concentration prior to collecting it into the product vessel. It also provides a convenient way to respond to system upsets during processing to provide a more robust process.
  • the fraction of retentate that is recirculated can be adjusted via modulation of the pump or control valve as a way to tune the system in order to assure consistent retentate concentration and/or consistent permeate flow rate to the product collection vessel every run even if cell concentration, new membrane permeability, membrane fouling, membrane permeability, or membrane mass transfer or pressure drop varies from batch to batch. This strategy has particular benefits in the context of continuous processing where the success of subsequent operations rely on the output of a previous operation. Recirculation of retentate can improve cleaning effectiveness through increased cross-flow velocity and reduce cleaning solution through recirculation.
  • the retentate that is being recirculated can be returned to any upstream location in or before the TFF system (e.g., a bioreactor located upstream of the TFF system).
  • the retentate is recirculated to the feed tank.
  • the retentate is recirculated to the feed line near the feed pump before the feed inlet on the TFF system.
  • the methods described herein comprise performing perfusion (e.g., to remove protein product and cellular waste components from the bioreactor and to supply the liquid feed with fresh media).
  • Perfusion is a type of diafiltration in which continuous bioprocessing occurs. As the perfusion cell culture media undergoes periodic filtration to remove target proteins and waste products, fresh media can be periodically or continuously resupplied.
  • perfusion is 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.
  • 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 diafiltration solution from the fresh media or diafiltration solution container to the bioreactor.
  • the methods described herein further comprise alternating flow of a liquid through the filter element(s).
  • the method can include reversing a direction of a feed pump, such that liquid feed enters the filter through the retentate-side and exits through the feed-side for a period of time to back flush the membrane.
  • the direction of flow can be reversed, for example, about every twelve seconds or longer.
  • the invention in yet another embodiment, relates to a perfusion process for harvesting target proteins from a liquid feed containing host cells.
  • the method includes delivering a liquid feed containing target proteins and host cells to a feed channel of at least one filter element of the invention and separating the target proteins from the host cells in the filter element(s).
  • the target proteins can be monoclonal antibodies, which are separated from the host cells by TFF and recovered from the permeate of the filter element(s).
  • perfusion processes of the present invention provide several advantages over perfusion processes involving the use of open-channel cassette filter elements and hollow fiber filter elements. Specifically, perfusion processes that include delivering a liquid feed containing host cells to a filter comprising an open microporous membrane and a low-shear feed spacer, provide for increased lifespan of the filter element. As shown, for example, in FIG. 7 and described further with regard to Example 4 herein, higher amounts of target proteins can be recovered from a cell culture solution over longer periods of time using perfusion processes of the present invention over conventional perfusion processes.
  • At least about 80%, at least about 90%, or at least about 95% of target proteins can be recovered from a cell culture solution at a harvest throughput of at least about 500 L/m 2 , at least about 800 L/m 2 , or at least about 1000 L/m 2 of the filter element.
  • the geometry of each of three feed screens was generated for the model using the parametric geometry function in Autodesk Inventor (Autodesk, Inc. Boston, Mass.).
  • the modeled feed screens included: (1) C-screens (Propyltex® screens, Sefar, QC, Canada) having a two-over-one twill pattern, an open area of 32%, a fiber density of 16.2 fibers/cm, fiber diameter of 270 ⁇ m, and a thickness of 515 ⁇ m; (2) D-screens (Propyltex® screens, Sefar, QC, Canada) having a two-over-one twill pattern, an open area of 36%, a fiber density of 12.2 fibers/cm, fiber diameter of 340 ⁇ m, and a thickness of 610 ⁇ m; and (3) D3-screens (Propyltex® screens, Sefar, QC, Canada) having a two-over-one twill pattern, an open area of
  • FIG. 4B illustrates a model of an environment containing the D3 woven fiber feed spacer of FIG. 4A .
  • the model was created using the Particle Tracing Module of the COMSOL software.
  • Boundary conditions for particle travel included a non-permeable upper plate, the geometry of the woven fiber feed spacer and a permeable membrane surface.
  • the membrane properties of the models were based on a MilliporeSigma 0.65 micron Durapore® membrane. The models were created based on the conditions summarized in Table 2.
  • FIG. 4C illustrates velocity magnitudes in the model of FIG. 4B with a 6 LMM cross-flow flux in the feed channel.
  • the maximum shear rates at both the membrane and fiber surfaces for each of the screens are shown in FIG. 5 .
  • a shear rate of approximately 3500 s ⁇ 1 is the accepted limit for Chinese Hamster Ovarian (CHO) cells, which are the predominant host cell-type used in the production of therapeutic proteins, such as mAbs.
  • CHO Chinese Hamster Ovarian
  • Two prototype filter elements were created, each including a single filter element sheet arranged in Pellicon® 3 micro plate and frame format. Both filter element sheets included a 0.65 micron Durapore® membrane. One filter element sheet contained a D-screen feed spacer and the other contained a D3-screen feed spacer.
  • the filter elements underwent testing in an AKTAcrossflowTM system (GE Healthcare Lifesciences, Marlborough, Mass.) with a cell culture solution containing CHO-S cells in a density of 30-60 million cells per milliliter in CHO CellventoTM 110 media. Feed, retentate and permeate flow were controlled during the experiments and feed pressure measurements were obtained, as shown in FIG. 6 .
  • Obtained experimental feed pressures were compared with those of modeled devices using corresponding retentate and permeate pressure values. As shown in FIG. 6 , the results of the modeled devices correlated well with feed pressure values as measured with the prototype devices. As such, the model is shown to provide accurate estimates of shear experienced by cells in the feed channels of the prototype devices.
  • Spiral-wound filter element including a 0.06 m 2 , 3 micron track-etched membrane (Sterlitech) and D3 feed spacer (Sefar), with peristaltic pump (Watson Marlow).
  • the system was operated in recirculation mode under recommended cross-flow rates.
  • Spiral-wound filter element including a 0.06 m 2 , 3 micron track-etched membrane (Sterlitech) and D3 feed spacer (Sefar), with Levitronix magnetic levitation pump.
  • the system was operated in recirculation mode under recommended cross-flow rates.
  • Spiral-wound filter element including a 0.06 m 2 , 3 micron track-etched membrane (Sterlitech) and D3 feed spacer (Sefar), with Repligen diaphragm pump.
  • the system was operated in alternating flow mode under recommended cross-flow rates.
  • Spiral-wound filter element including a 0.06 m 2 , 5 micron Durapore® membrane (MilliporeSigma) and D3 feed spacer (Sefar), with Repligen diaphragm pump.
  • the system was operated in alternating flow mode under recommended cross-flow rates.
  • test filter elements included:
  • test filter element ( 1 ) demonstrated the lowest flux values across the membrane surface, indicating that the highest amount of membrane fouling occurred in the open-channel device.
  • filter elements ( 3 ) and ( 4 ) demonstrated higher flux values than filter element ( 2 ), indicating that the D-screen and D3-screen each reduced particle accumulation on the membrane surface to a greater extent than the more tightly woven UF screen.
  • a spiral-wound filter element including a 0.06 m 2 , 1 micron Durapore® membrane (MilliporeSigma) and D3 feed spacer (Sefar), was tested with a Repligen diaphragm pump. The system was operated in alternating flow mode under recommended cross-flow rates. The system was tested with a feed comprising CHO cells.
  • MilliporeSigma Mobius® 3 L single use bioreactors were inoculated at 0.5 million cells per milliliter with a CHO-S cell line and CHO Cellvento 110 media. The cells were grown up to 4 million cells per milliliter by day 3, at which point a cell retention system was put online.
  • the perfusion rate in vessel volumes per day was increased from 1 to 3 over days 3-6 and then maintained at 3 for the remainder of the run.
  • FIG. 9 the 1 micron Durapore® membrane resulted in significantly improved sieving up to a throughput of approximately 900 L/m2.
  • a spiral-wound filter element including a 0.06 m 2 , 1 micron Durapore® membrane (MilliporeSigma) and 1 over 1 feed spacer (46% open area, 8.0 n/cm fiber density, 400 ⁇ m fiber diameter, 785 ⁇ m thickness, Sefar), with Repligen diaphragm pump.
  • the system was operated in alternating flow mode under recommended cross-flow rates.
  • the system was tested with a feed comprising CHO cells.
  • MilliporeSigma Mobius® 3 L single use bioreactors were inoculated at 0.5 million cells per milliliter with a CHO-S cell line and CHO Cellvento 110 media.
  • the cells were grown up to 4 million cells per milliliter by day 3, at which point a cell retention system was put online.
  • the perfusion rate in vessel volumes per day was increased from 1 to 3 over days 3-6 and then maintained at 3 for the remainder of the run.
  • Daily cell bleeds started on day 7 to maintain cell densities in the range of 30-60 million cells per milliliter.

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