WO2024092343A1 - Bioréacteur de culture cellulaire avec mélange rotatif - Google Patents

Bioréacteur de culture cellulaire avec mélange rotatif Download PDF

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Publication number
WO2024092343A1
WO2024092343A1 PCT/CA2023/051381 CA2023051381W WO2024092343A1 WO 2024092343 A1 WO2024092343 A1 WO 2024092343A1 CA 2023051381 W CA2023051381 W CA 2023051381W WO 2024092343 A1 WO2024092343 A1 WO 2024092343A1
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WIPO (PCT)
Prior art keywords
bioreactor
membranes
reactor
rotation
cells
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PCT/CA2023/051381
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English (en)
Inventor
Andrew Michael Pundsack
Rosane RECH
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Abec, Inc.
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Publication date
Application filed by Abec, Inc. filed Critical Abec, Inc.
Publication of WO2024092343A1 publication Critical patent/WO2024092343A1/fr

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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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/10—Apparatus for enzymology or microbiology rotatably mounted
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means

Definitions

  • This specification relates to methods for cell culture and cell culture bioreactors.
  • cell culture is sometimes used to refer to the culture of any cells and sometimes specifically to the culture of eukaryotes.
  • cell culture includes the culture of any cells including a) eukaryotes, for example animal cells such as mammalian cells, b) non-eukaryotes such as bacteria or eukaryotic organisms such as yeasts, fungi or protozoa (sometimes referred to as “microbial culture”) and c) plant cells (sometimes referred to as "plant cell culture” or “tissue plant cell culture”).
  • cell culture as used in this specification, unless stated otherwise, includes growing cells for the purpose of obtaining the cells themselves and growing cells for the purpose of obtaining a product produced by the cells, for example a genetic material, protein, peptide or enzyme. This is in contrast to growing cells primarily for the purpose of consuming a pollutant as in wastewater treatment.
  • a nutrient medium flows through the lumens of hollow fiber membranes to provide a perfusion culture mode wherein nutrients diffuse through pores of the membranes to cells growing within the bioreactor but outside of the membranes, optionally called the extra-capillary space (EOS).
  • EOS extra-capillary space
  • International Publication Number WO 2021/155469 A1 Cell Culture Bioreactor with Zone Control, describes a cell culture bioreactor having membranes divided into a plurality of zones.
  • the membranes include perfusion membranes carrying a liquid medium or gas transfer membranes or both.
  • the supply of one or more gaseous or liquid media to a selected zone may be controlled.
  • the bioreactor may be used to grow cells in suspension. Mixing within the ECS of the bioreactor is provided by a mixer, forced flow of liquid through the ECS or movement of the bioreactor.
  • the membrane bioreactor may be used to grow cells in suspension, for example to provide cells, or a substance produced by the cells, as a product.
  • the membrane bioreactor includes membranes within a plenum that defines outer surfaces of an extra-capillary space of the bioreactor.
  • the membranes may include liquid perfusion membranes or gas transfer membranes or both.
  • the method of mixing causes a liquid medium within the extra-capillary space of the bioreactor to move relative to the membranes.
  • the relative movement is provided by moving the bioreactor.
  • the movement of the bioreactor can include rotating the bioreactor in clockwise and counter-clockwise directions around an axis of rotation.
  • the axis of rotation may pass through the bioreactor.
  • the axis of rotation may be horizontal, pass through the center or axis (for example a cylindrical or longitudinal axis) of the bioreactor and/or be perpendicular to the membranes.
  • the bioreactor may have hollow fiber membranes extending in a plurality of directions perpendicular or oblique to the axis of rotation and/or the axis of the bioreactor.
  • the bioreactor may be rotated in a pattern including 5 or more reversals of direction and/or decelerations and accelerations, optionally stops and re-starts, in one direction per minute.
  • the bioreactor may be rotated through less than 360 degrees of rotation in one direction and then through less than 360 degrees in the opposite direction.
  • the rotation of the bioreactor may reach a speed in the range of 1 to 25 or 2 to 15 rpm or more.
  • the movement may include inverting and re-inverting the bioreactor.
  • This specification also describes a method of growing cells in a bioreactor including moving a liquid medium within a cell growing area of the bioreactor, optionally by moving the bioreactor, in a repeated pattern with a cycle time of 12 seconds or less.
  • This specification also descrbes a method of growing cells in a bioreactor including applying power to move a liquid medium within a cell growing area of the bioreactor, optionally by moving the bioreactor, wherein the applied power is above a mean power for a material part, i.e.
  • the bioreactor used in any of the methods described herein has an axis, for example a longitudinal axis, and hollow fiber membranes extending in a plurality of directions oblique or perpendicular to the axis.
  • the bioreactor may define a generally cylindrical plenum, wherein the axis is a cylindrical axis. Parts of the plenum may be defined by one or more interior surfaces of the bioreactor in the shape of segments of circles, cyolinders or cones centered on the axis.
  • Moving the bioreactor may include rotating the bioreactor around the axis.
  • the hollow fiber membranes may include gas transfer membranes.
  • the bioreactor may have membranes with an outside diameter of 0.3 mm or more.
  • the membranes may be arranged in layers with spaces between adjacent layers.
  • the membranes may be spaced apart by at least 0.2 mm in layers.
  • the membrane packing density may be 25% or less.
  • the system may include a bioreactor, a stand for supporting the bioreactor, and a motive device.
  • the bioreactor has an axis, for example a longitudinal axis, and hollow fiber membranes extending in a plurality of directions perpendicular or oblique to the axis.
  • the hollow fiber membranes may include gas transfer membranes.
  • the stand may support the bioreactor with the axis of the bioreactor generally horizonal.
  • the motive device is adapted to rotate the bioreactor around the axis.
  • the motive device may be, for example, a motor or an actuator, for example an electrical, hydraulic or pneumatic actuator.
  • the bioreactor provides a plenum for growing cells, wherein the plenum for growing cells has interior surfaces in the shape of segments of circles, cylinders or cones centered around the axis.
  • the interior surfaces may be provided by potting heads holding ends of the hollow fiber membranes.
  • the plenum may be generally cylindrical and the axis may be a cylindrical axis.
  • the system may also have a controller programmed or otherwise configured to produce, via the motive device, a pattern of movement of the bioreactor, for example any movement described herein.
  • the motive device may be connected to the controller.
  • the pattern of movement comprises one or more of a) rotating the bioreactor in clockwise and counter-clockwise directions around the axis, b) moving the bioreactor in a repeated pattern with a cycle time of 12 seconds or less, c) rotating the bioreactor in a pattern including 5 or more reversals of direction and/or decelerations and accelerations (optionally stops and re-starts) in one direction per minute, d) rotating the bioreactor through less than 360 degrees of rotation in one direction and then through less than 360 degrees in the opposite direction in a repeated pattern, e) rotating the bioreactor at a speed of rotation in the range of 1 to 25 or 2 to 15 rpm, f) moving the bioreactor according to any movement described herein or an analogous movement scaled to a reactor of
  • the bioreactor comprises a plurality of elements, each element containing a set of membranes potted separately from membranes in other elements.
  • the membranes have an outside diameter of 0.3 mm or more, the membranes are arranged in layers with spaces between adjacent layers, the membranes are spaced apart by at least 0.2 mm in layers, the membrane packing density is 25% or less or 20% or less or 15% or less or in the range of 8-12% and/or the volume of an extracapillary space of the bioreactor is in the range of 1-20, 2-20, or 2-10 mm 3 per mm 2 of surface area of the gas transfer membranes.
  • Figure 1A shows the outside of a second part of a mold.
  • Figure 1B shows the inside of the second part of the mold of Figure 1 A.
  • Figure 2A shows the inside of a first part of a mold.
  • Figure 2B shows the outside of the first part of the mold of Figure 18A.
  • Figures 3A, 3B and 3C show isometric, top and end views of a membrane plate assembly.
  • Figure 4 shows a mold being assembled with the first part of the mold of Figures 2A and 2B, the second part of the mold in Figures 1A and 1 B and membrane plate assemblies of Figures 3A-C.
  • Figures 5A and 5B show isometric and a vertical cross-sectional view of an assembled mold of Figure 4.
  • Figure 6 shows a horizontal cross section of a mold with potting material added to it.
  • Figure 7 shows an element made with part of the molded assembly of Figure 6 with some of the potting material, mold and membranes cut away and caps added.
  • Figure 8 shows a reactor with a magnetic base attached to a motor for rotating the reactor.
  • Figures 9A and 9B show alternative devices for rotating a compound reactor.
  • Figures 10A and 10B show a compound reactor with headers and manifolds connected to its elements.
  • Figure 11 shows a cell culture system
  • Figure 12 shows an alternative gas system for use with the cell culture system of Figure 11.
  • Figure 13 shows additional parts of the cell culture system of Figure 11.
  • Figure 14 shows the results of an experimental trial.
  • FIG. 10 The Figures show examples of a bioreactor element 402, a reactor 450 made from a single element 402, and a compound reactor 550 made from multiple elements 402.
  • bioreactor element 402 a reactor 450 made from a single element 402
  • FIG. 2021/155469 A1 Cell Culture Bioreactor with Zone Control, published on August 12, 2021 , which are both incorporated herein by reference.
  • the words “reactor” and “bioreactor” are used interchangeably herein unless noted otherwise.
  • a reactor 450 having a single element 402 or a compound reactor 550 having multiple elements 550 may be referred to generally as a reactor or a bioreactor.
  • One or more of the inventions described herein may be adapted to other bioreactors described in these publications or known in the art.
  • An element 402 has membranes 102.
  • the membranes 102 are hollow fiber membranes, but other types of membranes may be used.
  • the membranes 102 may be divided into multiple sets of membranes 102.
  • the membranes 102 include membranes oriented in two directions. In other examples, membranes 102 may be oriented in more or less directions.
  • an element may have, for example, only perfusion membranes 102a, only gas transfer membranes 102b, a mixture of different perfusion membranes 102a or a mixture of different gas transfer membranes 102b.
  • the perfusion membranes 102a are perpendicular to the gas transfer membranes 102b.
  • the membranes 102 may provide liquid medium perfusion or gas perfusion while retaining cells in the ECS.
  • the pore size or skin of the membranes 102 also retains selected medium components or cell culture products in the ECS.
  • a compound reactor 550 has three elements 402. However, an alternative compound reactor 550 might have more or less than three elements 402.
  • the extra-capillary spaces of the elements 402 in a compound reactor 550 are in liquid communication with each other and collectively form one continuous plenum inside the reactor 150.
  • the number of elements 402 is optionally sufficient so that the height of the plenum (measured perpendicular to the membranes 102 in the examples shown) is 50% or more, 100% or more or 200% or more of the (average) length of the hollow fiber membranes 102a and/or 102b (measured between the interior surfaces of potting material 428).
  • multiple reactors 450 may be attached to each other and move together but have separate extra-capillary spaces, but such assemblies are not considered to be a compound reactor 550.
  • Multiple reactors 450, 550 are optionally connected in parallel with shared perfusion medium and gas transfer systems. Parallel reactors may be used, for example, for autologous cell therapy manufacturing.
  • Multiple reactors 450, 550 may also be used sequentially, for example to expand a population of cells by growing cells first in a smaller reactor 450, 550 and then moving the cells to a larger reactor 450, 550.
  • a reactor 450 or compound reactor 550 also includes a top plate 436 and a base plate 438, which may be a magnetic base plate 444.
  • elements 402 can be sealed to each other or to the top plate 436 and the base plate 438, 444 by an adhesive.
  • Sensors or fittings may be located on a top plate 436, 444, a base plate 438 or on the sides of an element 402, or in multiple locations.
  • pieces of optical sensor foil are placed on the inside of translucent walls of an element 402. The sensor foil gives off a signal when interrogated with a detector unit through the transparent wall of the element 402.
  • the detector unit may have, for example, a source of light of one or more peak wavelengths, a camera (i.e. CMOS or CCD) chip and optionally one or more optical filters.
  • a camera i.e. CMOS or CCD
  • the VisiSens TDTM modular mapping system from PreSens may be used as the detector unit.
  • different sensors are provided in different locations of the reactor 450 or compound reactor 550.
  • a reactor 450 or compound reactor 550 may also have a harvest layer to enable removal of cells or cell products.
  • cells in suspension in a reactor 450 or compound reactor 550 receive nutrients through the perfusion membranes 102a and receive oxygen through the gas transfer membranes 102b.
  • soluble or dispersed waste products of the cells may be removed through the perfusion membranes 102a.
  • a first medium (alternatively called an ECS medium) is typically added to the extra-capillary space of the reactor 450, 550 during a set up phase, for example through a fitting 164.
  • a second liquid medium (alternatively called a perfusion medium) is circulated through the lumens 144 of the perfusion membranes 102a. The second medium may be the same as the first medium or a different medium.
  • Either of the first medium or the second medium may be a mixture of two or more media.
  • the composition of either the first medium or the second medium may change during use of the reactor 450, 550.
  • carbon dioxide released by the cells is removed from the extracapillary space of the bioreactor 150 through the gas transfer membranes 102b or in solution through the perfusion membranes 102a.
  • the gas transfer membranes 102b may have manufactured pores, for example of 30 Angstroms or less or 40 Angstroms or less in size, or may be dense walled. In other examples, the gas transfer membranes 102b have larger pores but are sufficiently hydrophobic to prevent the pores from being filled with water.
  • oxygen enriched air is supplied through the gas transfer membranes 102a, at some times or continuously.
  • the oxygen concentration of supplied air, the pressure of supplied air and/or the flow rate of supplied air may be varied over time to alter the amount of oxygen delivered to the extra-capillary space of the reactor 450, 550.
  • the amount of oxygen delivered may be increased over time to deliver oxygen at a higher rate when cells, or a population of cells, are maturing.
  • the reactor 450, 450 is used to grow cells in suspension.
  • Cells in suspension can move in the extra-capillary space, typically because they are entrained in a flow of a liquid moving within the extra-capillary space. The flow can be induced by moving the reactor 450, 550.
  • the cells can be suspended alone or in aggregates, or attached to carriers that are also in suspension.
  • the cells may be, for example, stem cells, CD34+ cells, HEK cells, CHO cells, RBCs or any other cells mentioned herein, including eukaryotic, microbial or plant cells.
  • the outside diameter of the membranes 102 may be 0.3 mm or more, 0.5 mm or more, 0.7 mm or more or 1.0 mm or more, up to for example 3 mm. Larger diameter membranes 102 are able to withstand more mechanical stress than smaller diameter membranes. Particularly for long membranes, for example 20 cm or more, larger diameter membranes 102 may also provide a more nearly even distribution of nutrients. [0039] The ability to grow and harvest cells, for example to keep at least a portion of the cells in suspension or to dislodge temporarily restrained cells, is also enhanced by having a controlled spacing between membranes 102 and/or a low packing density.
  • the packing density of membranes 102 may be 25% or less, 20% or less, or 15% or less.
  • gaps can be provided, as shown for example in Figure 1 , between sets of membranes 102 and/or between a set of membranes 102 and the inside surface of the reactor 450, 550.
  • the membranes 102 are laid out in a regular pattern with controlled spacing between adjacent membranes 102. In the examples shown, the membranes 102 are laid out in a stack of layers, optionally forming a rectilinear array.
  • adjacent membranes 102 are spaced apart from each other by gaps (measured between outer surfaces of adjacent membranes) for example of 0.2 mm or more, 0.5 mm or more, 0.7 mm or more or 1.0 mm or more.
  • Layers of membranes 102 are spaced apart from each other by gaps (measured between planes defining the opposed surfaces of adjacent layers) for example of 0.2 mm or more, 0.5 mm or more, 0.7 mm or more or 1.0 mm or more.
  • the gap between adjacent layers of membranes 102 may also be at least as large as the diameter of perpendicular membranes, if any.
  • the spacing between membranes 102 in a layer and/or the spacing between layers is optionally different for perfusion membranes 102b than for gas transfer membranes 102a.
  • the spacing between membranes 102 or between layers, potting densities and the arrangement of membranes 102 into layers or arrays are preferably determined and/or measured within the potting material.
  • a defined spacing is optionally maintained along the length of the membranes 102, for example by tension in the membranes 102 or weaving oblique or orthogonal membranes 102 together.
  • the diameter of an element 402, measured between inside surfaces of the potting material 428, may be for example in the range of 5 cm or more, 10 cm or more, 15 cm or more or 20 cm or more.
  • the diameter may be, for example, 40 cm or less or 30 cm or less.
  • the inside surfaces of the potting material define circles centered on an axis used for spin casting, which may be axis 165 of rotation or of the reactor 450, 550 as described further below.
  • the inside surfaces of the potting material may define segments of cylinders or cones, preferably mildly tapered cones. In the case of a conical element 402, the diameter of the element may be taken as the average diameter.
  • one or more dimensions between opposed interior surfaces of the element 140 may be within these ranges.
  • Longer membranes 102 may be strengthened if required by increasing their diameter, using multifilament yarns of membranes 102, weaving orthogonal membranes 102 together, or using braid supported or TIPS membranes 102.
  • an element 402 may have additional divisions among the membrane 102 to create more distinct sets of membranes 102.
  • the elements 402 shown have four sets of membranes 102, two sets of perfusion membranes 102a and two sets of gas transfer membranes 102b.
  • Other examples may have more or less sets of membranes 102 and there may be different numbers of sets for perfusion membranes than for gas transfer membranes. Creating more sets can assist with keeping at least some of the cells in suspension as the diameter of the element 402 increases.
  • an element 402 has most, i.e. 50% or more or 80% or more, or all of its membranes 102 within sets that are 10 cm or less, or 5 cm or less, wide.
  • the elements 402 may be used with an axis 165 perpendicular to the membranes 102 in a generally horizontal orientation, as shown for example in Figures 8, 9A and 9B.
  • the height of a reactor 450, 550, measured along the axis 165 (i.e. horizontally in Figures 8, 9A and 9B) within the ECS, may be more than the inside diameter of the elements 402.
  • the height of the reactor 450, 550 may be 2 times or more, 5 times or more or 10 times or more, than the inside diameter of the elements 402.
  • one or more harvest layers may be added between the elements 402.
  • the extra-capillary space of the bioreactor measured as the interior volume of the bioreactor but excluding the volume occupied by the membranes 102, may be 0.01 L or more, 0.1 L or more, 1L or more or 10L or more or 50L or more.
  • the extra-capillary space of the bioreactor may be 1000L or less, or 100L or less, 20L or less, or 10L or less.
  • Figures 5A and 5B show sets of membranes 102 in a mold 400.
  • the mold 400 is used to make an element 402, shown for example in Figure 7.
  • liquid potting material 428 for example an epoxy or polyurethane resin
  • the mold 400 is used for spin casting.
  • the mold 400 is spun as liquid potting material 428 is added to the mold to force the potting material 428 to the outside of the mold 400.
  • the mold 400 continues spinning until the liquid potting material 428 solidifies.
  • static potting may be used.
  • a part of the mold 400 that defines a potting cavity 416 is oriented at the bottom of the mold 400 while liquid potting material 428 is poured into it and then allowed to cure.
  • the mold 400 is then rotated to place another potting cavity 416 at the bottom of the mold and more potting material 428 is added.
  • the process of static potting is repeated until potting material 428 has been poured into each potting cavity 416.
  • Spin casting is preferred and may produce a generally cylindrical element 402.
  • static potting may be used and optionally produces a polygonal element 402, for example a square element or an octagonal element 402 if angled panels 408 are present in the mold 400.
  • the potting material 428 is surrounded by parts of the mold 400 except at an exterior face of the potting material 428 wherein the ends of the membranes 102 are open and the lumens of the membranes 102 are exposed. Interior surfaces of the potting material are within the mold 400 but not in direct contact with surfaces of the mold 400.
  • the mold 400 is made of a transparent plastic such as polycarbonate.
  • a portion of the mold 400 forms a panel 408.
  • the panel 408 allows for looking into the extra-capillary space or using a light-based analysis method to determine a property of first medium in the ECS.
  • the panels 408 optionally allow for taking measurements from one or more light activated sensor foils placed on the inside of the panel 408, in contact with first medium in the extra-capillary space.
  • the mold 400 may be made of opaque material and other forms of sensors may be placed on or through the panels 408.
  • the mold 400 may be reconfigured to produce a wider area for potting material 428 and to eliminate the panels 408 or reduce the size of the panels 408.
  • the mold 400 has a first part 404 and a second part 406.
  • the first part 404 and the second part 406 are assembled together, optionally with an adhesive, after the membranes 102 are inserted between them.
  • potting cavities 416 are created where potting material 428 will be added (as shown for example in Figure 6).
  • liquid potting material 428 flows into the potting cavities 416 of the mold 400 through one or more resin ports 412.
  • the element 402 has two apertures 410 on opposed sides of the second element 402.
  • one or both of the apertures 410 include an additional feature, for example a raised ring in the example shown.
  • an aperture 410 maybe a simple opening in the mold 400.
  • the outside diameter of one of the apertures 410 is generally the same as the inside diameter of the other aperture 410.
  • Multiple elements 402 can be stacked together by inserting the smaller aperture 410 of a second element 402 into the larger aperture of another second element 402.
  • the apertures 410 of two or more second elements 402 are connected by an adhesive or solvent bonded together.
  • the apertures 410 may be threaded such that two or more second elements 402 may be screwed together, or two apertures 410 may be press fit together.
  • Figures 1 A and 1 B show the second part 406 of the mold 400.
  • Figure 1A shows primarily the outside surfaces of the second part 406.
  • Figure 1 B shows primarily the inside surfaces of the second part 406.
  • the aperture 410 may have one or more registration areas 414. In the example shown, there are four registration areas 414 equally spaced around the aperture 410. Each registration area 414 is a flat spot on the otherwise round aperture 410. In combination with corresponding registration areas on the first part 404 of the mold 400, the registration areas cause the potting cavities 426 in a stack of second elements 402 to be aligned with each other.
  • Figures 2A and 2B show the first part 404 of the mold 400.
  • Figure 2A shows primarily the inside surfaces of the second part 406.
  • Figure 2B shows primarily the outside surfaces of the second part 406.
  • the aperture 410 may have one or more registration areas 414. In the example shown, there are four registration areas 414 equally spaced around the aperture 410. Each registration area 414 is a flat spot on the otherwise round aperture 410. As discussed above, in combination with corresponding registration areas 414 on the second part 406 of the mold 400, the registration areas 414 cause the potting cavities 416 in a stack of second elements 402 to be aligned with each other.
  • the mold 400 has four potting chambers 416 where potting material 428 will be added.
  • the potting material 428 (shown for example in Figure 6) does not extend radially inward beyond the potting chambers 416.
  • the potting material therefore does not flow through the inside of the mold 400 between potting chambers 416 and the inner surfaces of the potting material 428 may form segments of a cylinder or cone.
  • the potting material 428 may extend radially inward beyond the potting chambers and flow between potting chambers thereby forming a continuous cylinder or cone.
  • Each potting chamber 416 has one or more resin ports 412.
  • potting material 428 flows from a reservoir outside of the mold 400 through tubes connected to the resin ports 412 and into the potting chambers 416.
  • the potting material may flow by way of a pump or by centrifugal force generated by spinning the reservoir with the mold 400.
  • each potting chamber 416 also has one or more ribs 418.
  • the ribs 418 engage with notches 420 in plates 422 shown in Figures 3A, 3B and 3C.
  • Membranes 102 are attached to the plates 422, for example by an adhesive or welding, to form a membrane plate assembly 426.
  • the ends of the membranes 102 are typically closed before, or as a result of, being attached to the plates 422.
  • the ribs 418 locate the plates 422 when they are inserted into the potting chambers 416.
  • the mold 400 and or the membrane plate assemblies 416 may be used to hold the membrane plate assemblies 416 in a selected location in the mold 400.
  • the length of the membranes 102 may be selected, relative to the configuration of the mold 400 and the plates 422, such that the membranes 102 are taut when they are placed in the mold.
  • the length of the membranes 102 may be selected, relative to the configuration of the mold 400 and the plates 422, such that the membranes 102 have some slack when the membranes 102 are placed in the mold 400.
  • the membranes 102 may be divided into one or more sets of membranes 102 on a plate 424.
  • the membranes 102 of a set may be evenly spaced apart from each other.
  • An even spacing of the membranes 102 in combination with taut membranes 102 promotes a controlled and even spacing of membranes 102 within the extra-capillary space.
  • slackened membranes 102 move more in response to mixing which can inhibit cell attachment or help with cell harvesting for some cell types.
  • the thickness of the plates 422, including optional spacing blocks 424, can be varied to control the distance between the membranes 102 attached to one plate 422 and the membranes 102 attached to another plate 422.
  • the membrane plate assemblies 426 may be customized, for example, by having one or more of selected membrane 102 type or size, a selected spacing between membranes 102 in a membrane plate assembly 426, a selected spacing between membrane plate assemblies 126, a selected taut or slack mounting of the membranes 102, or selected treatments of membranes 102, for example to may them protein fouling resistant or environmentally (i.e. thermally) responsive.
  • an element 402, reactor 450 or compound reactor 550 may be produced that is suitable for use for growing a variety of cells or cell products.
  • the number of perfusion membranes 102a relative to the number of gas transfer membranes 102b is altered, only perfusion membranes 102a are provided, or only gas transfer membranes 102b are provided in an element 402, reactor 450 or compound reactor 550.
  • FIG. 4 shows a mold 400 being assembled.
  • Membrane plate assemblies 426 are inserted into the first part 404 of the mold 400.
  • Two membrane plate assemblies 426 are shown in Figure 4 but a mold 400 may contain multiple membrane plate assemblies 426, for example between 5 and 1000, or between 10 and 100, membrane plate assemblies 426.
  • a mold 400 may be loaded with only perfusion membranes 102a or only gas transfer membranes 102b.
  • alternating membrane plate assemblies 426 are oriented orthogonally to each other.
  • membrane plate assemblies 426 may form different patterns, for example two or three membrane plate assemblies 426 in one direction for every membrane plate assembly 426 in the orthogonal direction.
  • the membrane plate assemblies 426 in a mold 400 may all be oriented in the same direction.
  • the membranes 102 of one membrane plate assembly 426 may be woven with the membranes 102 of an orthogonal membrane plate assembly 426.
  • the membranes 102 of a membrane plate assembly 426 may be divided into two sets as shown, or into more than two sets.
  • the membranes 102 of a membrane plate assembly 426 may be spaced evenly across substantially the entire width of a membrane plate assembly 426 in a single set.
  • Figures 5A and 5B shows the mold 400 assembled and ready for spin casing.
  • Figure 6 shows a cross section of the mold 400 after spin casting. Potting material 428 has been added to each of the potting chambers 416 and enclosed the ends of the membranes 102. Once cured, the potting material 428 provides a seal to the outside surfaces of the membranes 102. Optionally, the potting material 428 also encloses the plates 422. In the example shown, the inside surfaces of the potting material 428 is withdrawn, i.e. radially displaced, from the panels 408.
  • the inside surfaces of the potting material 428 may be brought closer to, or substantially flush with, the edges of the panels 408.
  • the panels 408 may be flat as shown, rather than curved, such that the potting material 428 may encroach on the edge of a panel 408 without flowing across the entire panel 408.
  • some of the potting material 428 may be allowed to overflow the panels 408 from one potting chamber 416 to another.
  • the potting material 428 may be transparent, for example a clear epoxy. In this case, potting material 428 may cover the panels 408 and still allow light to travel through the panels 408.
  • the membranes 102 are inset from the sidewalls of the potting chamber 416. Alternatively, membranes 102 may be placed closer to the sidewalls of the potting chamber 416.
  • the size of the panels 408 (either their absolute size or their size relative to the size of the mold 400) may be varied, or the panels 408 may be removed.
  • the example shown is for a relatively small mold 400 with a roughly 10-15 cm outside diameter.
  • a larger mold 400 for example with an outside diameter up to 30 cm or more or up to 60 cm or more, panels 408 of essentially the same absolute size may be used but the panels 408 will be relatively smaller in the larger mold 400. Accordingly, a portion of the volume of extra-capillary space that is not crossed by membranes 102 can be reduced (or increased). However, it is not always necessary or desirable to have more of the extra-capillary space crossed by membranes 102.
  • Having multiple potting chambers 416 divides the amount of potting material 428 into smaller units, which can help with managing the heat generated when the potting material 428 cures, and also facilitates having the panels 408 not covered with potting material 428.
  • the mold 400 may be re-configured to provide one continuous potting chamber 416.
  • Figure 7 shows an element 402.
  • the potting material 428 and potting chambers 416 are cut to produce a cut face 432. Portions of the potting material 428 and the ends of the membranes 102 beyond the cut face 432 are removed. Optionally, the cut face 432 may be inward of the plates 422 and the plates 422 may also be removed. The lumens of the membranes 102 are open at the cut face 432. Caps 430 are sealed, for example by an adhesive or solvent, to the remaining parts of the potting chambers 416. The ends of the membranes 102 are in fluid communication with the insides of the caps 430.
  • Cap ports 434 allow a fluid to be added to, or withdrawn from, the caps 430, which in turn allows a fluid to be added to, or withdrawn from, a set of membranes 102.
  • each cap 430 has a single cap port 434.
  • a cap 430 may have two or more cap ports 434.
  • Figure 8 shows a side view of an element 402 used in a reactor 450.
  • a top plate 436 closes an upper aperture 410 at the top of the reactor 450.
  • a magnetic base plate 444 closes a lower aperture 410 at the bottom of the reactor 450.
  • multiple elements 402 for example between 2 and 100, or between 2 and 10, elements 402, may be assembled together into a compound reactor 550.
  • the aperture 410 of one element 402 may be attached to the aperture of another element 402 to create a stack of elements 402.
  • the top plate 436 may have one or more fittings 164 that provide access to the extra-capillary space.
  • a fitting 164 can be used to add first medium, or a particular substance such as a growth factor or nutrient, to the extra-capillary space.
  • a fitting 164 may be used to remove a substance from the extra-capillary space.
  • a fitting 164 is used to connect a sampler to the extra-capillary space.
  • the top plate 436 may have one or more adapters 442.
  • an adapter 442 is used to attach a sensor body connected to a fiber optic cable in a hole in the top plate 436.
  • the fiber optic cable is used to read a sensor dot attached to the inside of the sensor body in the adapter 442.
  • Sensor dots are made, for example, by PreSens and can be used to measure pH, dissolved oxygen concentration, dissolved carbon dioxide concentration or other aspects of the extra-capillary space.
  • an adapter 442 may be used to support another type of probe or sensor.
  • Figure 8 shows a reactor 450 with a magnetic base 444 in place of the base plate 438 of Figure 10A and 10B.
  • the magnetic base 444 has a ferromagnetic insert (not visible) held in a fixed position in the magnetic base 444.
  • the ferromagnetic insert allows the second reactor 450 to be coupled to a magnet 440 outside of the reactor 450.
  • the magnet 440 is attached to a motor 446, for example a stepper motor.
  • the motor 446 is supported on a stand 448.
  • the reactor 450 is thereby suspended from the stand 448.
  • the motor 446 may be attached or mechanically coupled (i.e. by way of gears or a drive belt) to the reactor 450 without a magnet.
  • the reactor 450 may also be supported directly on stand 448 (i.e. without relying on the motor 446 for support) or on a separate stand.
  • the reactor 450 rotates with the motor 446.
  • the motor 446 can be activated to move the reactor 450, for example to rotate or rock the second reactor 450 or to periodically invert the second reactor 450.
  • the second reactor 450 rotates the reactor 450 in one direction (i.e. clockwise) for 0.1-5 rotations, and then rotates the reactor 450 in the other direction (i.e. counter-clockwise) for 0.1-5 rotations, in a repeated pattern.
  • the potting material 428 may be optionally provided such than an inner surface of the potting material 428 is displaced radially outward from an inner surface of the panels 408.
  • Motor controller 640 (see Figure 13).
  • the motor controller 640 is attached to a power source 642 and to computer 630.
  • Computer 630 may be programmed to operate the motor 446 by way of motor controller 640 to rotate according to a predetermined pattern, for example any of the patterns that produce mixing as described herein, or a mixing pattern that varies in accordance with readings from one or more sensors, or as required to facilitate operations such as filling the ECS of the reactor 450 or harvesting cells.
  • a reactor 450 may have additional potting chambers 416, for example 6 or 8 potting chambers 416. In other examples, a reactor 450 may have between 2 and 20 potting chambers 416. In some examples, the potting chambers 416 are distributed radially around the reactor 450. In other examples, one or more sides of the reactor 450 have no potting chambers or 2 or more potting chambers 416.
  • a reactor 450 may have two different types of perfusion membranes 102a or two different types of gas transfer membranes 102b. The different types of membranes may differ, for example, in pore size, material or surface treatment.
  • Figures 9A, 9B, 10A and 10B show compound reactors 550.
  • the compound reactors 550 shown have three elements 402 stacked together. Alternatively, a different number, for example between 2 and 20, elements 402 may be stacked together.
  • the elements 402 may be all of the same size and configuration, or have two or more different sizes or two or more different configurations.
  • An aperture 410 on the bottom of a first element 402 fits into an aperture 410 on the top of another element 402 adjacent the first element 402.
  • the apertures 410 of two different elements 402 are optionally bonded together to enhance a seal between the apertures 410.
  • a base plate 438 or magnetic base plate 444 as is connected to an aperture 410 of an element 402 on the bottom of the compound reactor 550.
  • the extra-capillary spaces of the second elements 402 are in fluid communication with each other through the apertures 410 to form one larger extra-capillary space of the entire compound reactor 550.
  • the membranes 102 of each second element 402 can be individually accessed through the cap ports 434 on the caps 430 of the individual second elements 402.
  • a compound reactor 550 may have a harvest layer 300 as well as one or more elements 402.
  • a harvest layer resembles an element 402 but has exclusion membranes.
  • the harvest layer can be used to withdraw a portion of the first medium along with a product while selectively excluding at least some of the productive cells, which remain in the rest of the first medium.
  • a product may be, for example, enucleated red blood cells, a virus, a protein or another cell product.
  • the exclusion membranes may have pores of about 5 microns in diameter for retaining nucleated erythroid precursor cells, or a smaller diameter for selectively harvesting virus or proteins.
  • a harvest layer may have ports connected directly (i.e. not through membranes) to the ECS.
  • the harvest layer can be used to withdraw cells for transfer to another reactor or for collecting the cells as a product.
  • Figure 9A shows a compound reactor 550.
  • the compound reactor 550 has discs 512 inserted between pairs of second elements 402. Alternatively or additionally, discs 512 may be provided at the ends of the compound reactor 550.
  • the discs 512 rest on a pair of rollers 514.
  • the rollers 514 provide an alternative type of stand for holding the compound reactor 550.
  • the rollers 514, a disc 512 or the compound reactor 550 can be driven by a motor, directly or by way of a mechanical coupling, for example gears or a drive belt, (motor and mechanical coupling not shown) to rotate the compound reactor 550 to provide mixing in the extra-capillary space.
  • Tubing to carry a fluid to or from a second element 402 may pass through a hole or notch (not shown) in a disc 512.
  • Discs 512 are annular and surround the aperture 410 but do not block the apertures 410 such that the compound reactor 550 has one continuous ECS.
  • a motor 446 (not shown) may be connected to a roller 514 or directly to the compound reactor 550. The motor 446 may be connected to the computer 630 and operated as described for the motor 446 of Figure 8.
  • Figure 9B shows another compound reactor 550.
  • This compound reactor 550 is mounted at both ends on a stand 448.
  • One end of the stand 448 has a motor 446 that rotates a magnet 440.
  • the magnet 440 is connected to a magnetic base 444 attached to one end of the compound reactor 550.
  • the other end of the compound reactor 550 has a pinned top plate 516 supported in a bushing on the other end of the stand 448.
  • the motor 446 may be attached or mechanically coupled (i.e. by way of gears or a drive belt) to the reactor 450, 550 without a magnet.
  • the reactor 450 may also be supported directly on stand 448 (i.e. without relying on the motor 446 for support) or on a separate stand.
  • the motor 446 can be used to rotate the compound reactor 550 to provide mixing in the extracapillary space.
  • the motor 446 may be connected to the computer 630 and operated as described for the motor 446 of Figure 8.
  • another form of motive device may be used to move the reactor 450, 550 in place of a motor.
  • the reactor 450, 550 may be moved by an actuator, such as an electrical, pneumatic or hydraulic actuator.
  • Figures 8, 9A and 9B show an axis 165.
  • the axis 165 is an axis of rotation.
  • the axis 165 is also an axis of the reactor 450, 550.
  • the axis 165 may be considered a longitudinal axis of the reactor 450, 550 since material parts of the reactor, for example the inner surfaces of the potting material 428, are symmetrical through some planes that include axis 165 and/or the cross sectional shape of the reactor 450, 550 varies less along the axis 165 relative to variations in cross sectional shape along other lines that might be drawn through the reactor 450, 550.
  • the axis 165 also passes through the center of top plate 436 and the center of base plate 438 or magnetic base 444.
  • the inner surfaces of potting material 428 may define segments of circles, cylinders or cones centered on the axis 165.
  • the panels 408 might not form continuous circles, cylinders or cones with the inner surfaces of the potting material 428, and although the inner surfaces of potting material 428 may define segments of cones rather than cylinders, the reactor 450, 550 may still be considered to define a plenum that is generally cylindrical with the axis 165 as a cylindrical axis.
  • the membranes 102a, 102b may extend in a plurality of directions oblique to the axis 165.
  • FIG. 10A shows a compound reactor 550 with a liquid perfusion manifold 530.
  • the liquid perfusion manifold 530 connects a medium supply point AA to a set of cap ports 434.
  • these cap ports 434 are in fluid communication with the upstream ends of perfusion membranes 102a (not visible in Fig. 10A) in the compound reactor 550.
  • the branches of the liquid perfusion manifold 530 have control valves 538 that allow the flow of medium to be adjusted or stopped to a selected second element 402.
  • the compound reactor 550 also has a liquid perfusion header 532.
  • the liquid perfusion header 532 connects a set of cap ports 434 to a medium collection point BB.
  • these cap ports 434 are in fluid communication with the downstream ends of perfusion membranes 102a (not visible in Fig. 32A) in the compound reactor 550.
  • the branches of the liquid perfusion header 532 have control valves 538 that allow the flow of medium to be adjusted or stopped to a selected element 402.
  • the liquid perfusion manifold 530, liquid perfusion header 532 and control valves 538 allow the flow of medium to be adjusted to all of the elements 402 or to one set of the elements 402 relative to another set of second elements 402.
  • having control valves 538 both upstream and downstream of the compound reactor may provide additional control options. For example, selecting between an upstream control valve 538 and a downstream control valve 538 to adjust medium flow may affect the pressure inside of a second element 402.
  • one set of control valves 538 may be linked together (for example mechanically, electrically or in a control algorithm) to provide simultaneous adjustments in flow of medium to all of the second elements 402 while the other set of control valves 538 are controlled individually to make adjustments in the flow of medium to one second element 402 relative to another second element 402.
  • the liquid perfusion manifold 530 and liquid perfusion header 532 have three branches but in other examples the liquid perfusion manifold 530 and liquid perfusion header 532 may have a different number of branches corresponding to a different number of second elements 402 in a compound reactor 550.
  • second medium flows through the elements 402 of the compound reactor 550 in series.
  • medium supply point AA is connected to an inlet cap port 434 of a first element 402.
  • Outlet cap ports 434 of upstream elements 402 are attached to inlet caps ports 434 of downstream elements 402.
  • An outlet cap port 434 of the last element 402 is connected to medium collection point BB.
  • Figure 10B shows a compound reactor 550 with a gas perfusion manifold 534 connecting a gas supply point CC to a set of cap ports 434.
  • these cap ports 434 are in fluid communication with the upstream ends of gas transfer membranes 102b (not visible in Fig. 32B) in the compound reactor 550.
  • the branches of the gas perfusion manifold 533 have control valves 538 that allow the flow of gas to be adjusted or stopped to a selected second element 402.
  • the compound reactor 550 also has a gas perfusion header 536 connected to a set of cap ports 434 to a gas collection point DD.
  • these cap ports 434 are in fluid communication with the downstream ends of gas transfer membranes 102b (not visible in Fig. 32B) in the compound reactor 550.
  • the branches of the gas perfusion header 536 have control valves 538 that allow the flow of medium to be adjusted or stopped to a selected second element 402.
  • the gas perfusion manifold 534, gas perfusion header 536 and control valves 538 allow the flow of gas to be adjusted to all of the elements 402 or to one set of the elements 402 relative to another set of elements 402.
  • control valves 538 may be provided either upstream or downstream of a second element 402 or both upstream and downstream.
  • one set of control valves 538 may be linked together (for example mechanically, electrically or in a control algorithm) to provide simultaneous adjustments in flow of gas to all of the elements 402 while the other set of control valves 538 are controlled individually to make adjustments in the flow of gas to one element 402 relative to another element 402.
  • the gas perfusion manifold 534 and gas perfusion header 536 have three branches but in other examples they may have a different number of branches corresponding to a different number of second elements 402 in a compound reactor 550.
  • gas flows through the elements 402 of the compound reactor 550 in series.
  • gas supply point CC is connected to an inlet cap port 434 of a first element 402.
  • Outlet cap ports 434 of upstream elements 402 are attached to inlet caps ports 434 of downstream elements 402.
  • An outlet cap port 434 of the last element 402 is connected to gas collection point DD.
  • FIG 11 shows a cell culture system 600 including a reactor 450.
  • a compound reactor 550 optionally fitted with a liquid perfusion manifold 530, liquid perfusion header 532, gas perfusion manifold 534 and gas perfusion header 536, may be used in place of the reactor 450.
  • the extra-capillary space of the reactor 450 may be filled with a first medium through a fitting 164 while gas is released from another fitting 164, for example through a valve (i.e. a pinch valve or tubing clamp) and gas vent 612 (not shown).
  • the extra-capillary space may also be inoculated with cells that will be grown in the reactor 450 through a fitting 164 while gas is released through another fitting 164, for example through a valve (i.e. a pinch valve or tubing clamp) and gas vent 612 (not shown).
  • a valve i.e. a pinch valve or tubing clamp
  • gas vent 612 not shown.
  • the same two fittings 164 are used to fill the reactor 450 with inoculum and first media but additional dedicated fittings 164 may be provided for each task.
  • the fittings 164 are closed, for example using a tube sealer or aseptic disconnector, or used for other purposes.
  • the gas vents 612 include a membrane, for example with 0.22 micron pores, that allows gasses to pass through but prevents bacterial contamination of the second reactor 450.
  • the gas vent membrane is hydrophobic and retains liquids.
  • a fitting 164, or an array of fittings 164 may be connected, periodically or continuously, to a sampler (not shown). The sampler can be used to withdraw samples of first medium, including cells and compounds dissolved and/or suspended in the first medium, from the extra-capillary space.
  • one or more compounds may be added to the extracapillary space through a fitting 164 in addition to any compounds transferred to the ECS through the perfusion membranes 102a.
  • a growth factor may be added to the extra-capillary space, for example to make up for the decay or consumption of growth factors originally present in the first medium.
  • the reactor 450 has one or more sensors L, M, N in communication with the ECS through the top plate 436.
  • sensors L, M, N sense the pH, dissolved oxygen concentration and dissolved carbon dioxide concentration of the first medium in the extra-capillary space.
  • each sensor L, M, N is attached to an adapter 442 over a hole in the top plate 436.
  • the sensor L, M, N has a probe body that is sealed to the adapter 442.
  • the probe body has a sensor dot that is in liquid communication with the first medium in the ECS.
  • the adapter 442 also holds a fiber optic cable 628 (shown in Fig. 13) in a position suitable to probe the sensor dot.
  • the fiber optic cable 628 is connected to a fiber optic meter 634.
  • the fiber optic meter 634 is connected to a computer 630, optionally though a USB hub 632.
  • the computer 630 receives and optionally displays readings of pH, dissolved oxygen concentration and dissolved carbon dioxide concentration of the first medium in the extra-capillary space.
  • the computer 630 may control any of the controllable elements, for example pumps or valves, in the cell culture system 600.
  • a set of sensors similar to sensors L, M, N may be provided in the panels 408 of each second element 402.
  • One or more gasses are provided to the reactor 450 from one or more compressed gas tanks 602 or other gas supplies.
  • the gasses flow through tubing into a gas mixer 604, or optionally a mass flow controller, which produces a gas blend.
  • the gas blend passes through tubing, optionally past one or more inline sensors 606 and through an optional inline gas filter 608 to a cap 430 of the reactor 450.
  • inline sensors 606 I and J sense the oxygen concentration and temperature of the gas blend.
  • the gas blend flows from the cap 430, through gas transfer membranes 102b inside the second reactor 450, to an opposing cap 430.
  • the gas blend then flows through more tubing, optionally another in line gas filter 608 and one or more optional additional inline sensors 606 to an optional off-gas analyzer 610.
  • inline sensor K senses the temperature of the gas blend leaving the second reactor 450.
  • the off-gas analyzer measures the carbon dioxide concentration and the oxygen concentration of the gas blend.
  • a flow control valve may be provided before or after the reactor 450 to control the flow rate of gas through the second reactor 450.
  • the tubing to and from the compound reactor 550 may be connected to a gas perfusion manifold 534 and gas perfusion header 536, optionally with control valves 538, as described in relation to Fig. 10B to allow the flow of gas to be controlled for one or more individual elements 402.
  • a second medium is provided to the reactor 450 from a spinner flask 616 or another medium reservoir such as a bottle or bioprocess bag.
  • second medium may be added to the spinner flask 616 from a bottle 620 connected to a peristaltic pump 618.
  • second medium may also be removed from the spinner flask 616 by another peristaltic pump 618 connected to another bottle 620.
  • Another peristaltic pump 618 causes second medium to flow in a recirculation loop between the spinner flask 616 and the perfusion membranes 102a of the reactor 450.
  • the second medium flows from the spinner flask 616, to an upstream cap 430, through perfusion membranes 102a inside the reactor 450, to a downstream cap 430.
  • the second medium then flows from the downstream cap 430 through more tubing back to the spinner flask 616.
  • the second medium in the recirculation loop may also flow through one or more inline sensors 606 upstream and/or downstream of the reactor 450.
  • inline sensors 606 A, B, C and D sense the dissolved oxygen concentration, pH, dissolved carbon dioxide concentration and temperature of the second medium entering the reactor 450.
  • inline sensors 606 E, F, G, and H sense the dissolved oxygen concentration, pH, dissolved carbon dioxide concentration and temperature of the second medium leaving the reactor 450.
  • Filtered gas vents 612 are provided to allow air to move in and out of the second medium system while preventing contamination of the second medium.
  • flasks or bottles may be replaced with variable volume bioprocess bags.
  • filtered gas vents 612 are not required since air does not need to move in or out of the bioprocess bag as the volume of fluid in the bioprocess bag changes.
  • a flow control valve may be provided before or after the reactor 450 to help control the flow rate of second medium through the reactor 450 or the pressure of second medium in the perfusion membranes 102a.
  • the tubing to and from the compound reactor 550 may be connected to a liquid perfusion manifold 530 and gas perfusion header 532, optionally with control valves 538, as described in relation to Fig. 10A, to allow the flow or pressure of second medium to be controlled for one or more individual second elements 402.
  • pressure of the second medium in the perfusion membranes 102a (and the corresponding pressure of the first medium in the ECS) can be altered by raising or lowering the spinner flask 616 or other second medium reservoir relative to the reactor 450, 550.
  • the inline sensors 606 for pH, carbon dioxide concentration and oxygen concentration may be optical inline sensors.
  • optical inline sensors from PreSens have a fiber optic cable 628 connected to a fiber optic meter 634 as shown in Fig. 13.
  • the fiber optic meter 634 is connected to a computer 630 through a wire 635.
  • Other inline sensors 606, for example a temperature sensing thermocouple, may also be connected to the computer 630.
  • a wire 635 from a thermocouple may be joined to a fiber optic meter 634 before being connected to a USB port.
  • the cell culture system 600 shown is a small-scale system. Larger systems may be made, for example to operate a larger compound reactors 550, or to operate multiple reactors 450 or multiple compound reactors 550 in parallel. A larger or other system can use different sizes or types of equipment or different arrangements of conduits, valves, flow control devices, sensors, pumps, heaters or other equipment than the cell culture system 600 to achieve similar functions.
  • Computer 630 may be a general-purpose computer or any sort of programmable controller such as a programmable logic controller (PLC). The computer 630 may be a single free-standing computer located near the reactor 450, 550.
  • PLC programmable logic controller
  • the computer 630 may be made up of multiple elements, some of which are remotely located and connected to a local element through data cabling, wireless communication, or the Internet.
  • computer 630 may log data, communicate results, send error messages or alerts, or perform other functions.
  • the second medium circulates outside of the reactor 450, in contrast circulation of the first medium is preferably minimized. Optionally there is no circulation of first medium outside of the reactor 450.
  • the first medium differs from the second medium as determined by the pore size of the perfusion membranes 102a.
  • the perfusion membranes 102a may have a molecular weight cut off (MWCO) selected in the range of 5,000 to 250,000 Da or more.
  • MWCO molecular weight cut off
  • the perfusion membranes 102a may have pore sizes in the range of 0.04 to 0.5 microns, or in the range of about 0.1 to 0.2 microns.
  • Membranes with pore sizes in the range of 0.04 to 0.5 microns are useful for example when using second medium that contains large molecules, such as albumin or other proteins, that is intended to be delivered to the cells.
  • Cells, virus, and molecules above the MWCO are retained in the first medium.
  • Large molecules retained in the extra-capillary space can include, for example, growth factors and proteins.
  • the first medium contains large molecules, such as growth factors, that materially affect the cost of a cell culture process. By retaining these large molecules in the first medium, and minimizing or eliminating circulation of the first medium outside of the reactor 450, the amount of these large molecule that are required to operate a process is reduced.
  • the membranes 102 exclude cells, they also prevent any contamination, for example by bacteria, in the gas or second medium parts of the system from contaminating product cells or product producing cells in the extra-capillary space.
  • the second medium and gasses can influence the first medium.
  • the flow of a gas or second medium through the membranes 102 can be used to cool (or heat) the first medium.
  • the carbon dioxide concentration and pH of the first medium can be influenced, for example, by acids, bases or buffers provided to the first medium through the perfusion membranes 102a or by adding or removing carbon dioxide from the first medium through the gas transfer membranes 102b.
  • Some nutrients, with sizes small enough to pass through the pores of the perfusion membranes 102a can also be provided to the first medium from the second medium.
  • Small cell respiration products which may be inhibitory, can also be removed from the first medium by diffusion into the second medium through the perfusion membranes 102a.
  • Figure 12 shows an alternative gas system 620 for the cell culture system 600 of Figure 11.
  • a gas mixture travels from the gas mixer 604 to a pressure break 622.
  • the pressure break 622 may be a T-junction with a filtered gas vent 612 on one arm. Excess gas is vented to the atmosphere from the alternative gas system 620 through the pressure break 622.
  • the vented stream may be controlled with a valve.
  • a pressure regulator or other flow control valve may be used in place of, or in addition to, the pressure break 622.
  • a gas pump 624 downstream of the reactor 450 draws some of the gas mixture under vacuum through the gas transfer membranes 102b.
  • a rotameter 626 measures the flow of the gas mixture before it travels to an off-gas analyzer 610.
  • the rotameter 626 may be used to provide a signal for use in a feed back or other control loop connected to the gas pump 624 or the pressure break 622 or both.
  • the pressure break 622 and the gas pump 624 may be selectively configured such that the insides of the gas transfer membranes 102b are pressurized, under a partial vacuum, or under pressure at an upstream end and under a partial vacuum at a downstream end.
  • a gas for example oxygen
  • a gas can flow from the gas transfer membranes 102b to the first medium if there is sufficient partial pressure of the gas inside the gas transfer membranes 102b relative to the concentration of the gas in the first medium.
  • a gas can be delivered to the first medium even if the total pressure of the gas mixture inside the gas transfer membranes 102b is less than atmospheric, or less than the pressure of the first medium surrounding the gas transfer membranes 102b.
  • the total pressure of the gas mixture, or the partial pressure of one or more particular gasses, inside the gas transfer membranes 102b may cause undesirable consequences.
  • the pressure of the gas mixture inside of the gas transfer membranes 102b may be sufficient to cause one or more gasses to form bubbles in the first medium.
  • an undesirable pressure may be required to provide a desired flow rate of the gas mixture.
  • Changing to the alternative gas system 620 of Figure 12 may allow for a different combination of gas pressure and flow rate that better suits a particular process.
  • control valves 538 as shown in Figure 10B may also be used to control the flow of gas through individual second elements 402.
  • Erythroid precursor cells tend to expand faster and differentiate into red blood cells under low oxygen partial pressure and some other cells also prefer hypoxia.
  • a high oxygen transfer capacity (kLa) is needed to support a large and/or rapidly growing population of cells.
  • the oxygen transfer is preferably not driven by having local areas of high dissolved oxygen concentration (relative to the dissolved oxygen concentration in the reactor generally) in direct communication with cells in the ECS.
  • the gas transfer membranes 102b can assist in maintaining a hypoxic environment in the ECS that still supports a high cell density by providing oxygen transfer that is well controlled and generally homogenously distributed throughout the extra-capillary space.
  • the gas transfer membranes 102b may be used to provide a high dissolved oxygen concentration (i.e. 10% solubility or more) throughout most (i.e. 50% or more, 75% or more, or 90% or more) of the volume of the ECS.
  • Cells and other products may be harvested from the reactor 450, 550 through a fitting 164 on the top plate 436 or a panel 408.
  • a fitting 164 on the top plate 436 is used for harvesting, the fitting 164 is placed close to the perimeter of the top plate 436.
  • the reactor 450, 550 is rotated such that the fitting 164 is positioned at the bottom of the ECS.
  • An opposed fitting 164 at the top of the bioreactor 450, 550 is connected to a valve and an air filter. Opening the valve allows the ECS, including the cells or other products, to be drained from the reactor 450, 550.
  • the drainage fitting 164 may be attached to a tube to transport the contents of the ECS to a receiving vessel, for example a bioprocess bag or another reactor 450, 550. If the receiving vessel is not below the reactor 450, 550 a pump or compressed air may be used to convey the contents of the ECS to the receiving vessel. Optionally harvesting, which may include transferring cells from one reactor 450, 550 to another reactor 450, 550, may be triggered by a measured value, for example VCD or an indicator of cell differentiation or cell metabolism. [0086]
  • the ECS may be initially filled by adding an inoculum and first medium through the lower fitting 164 while venting air through an upper fitting 164 with the valve open. After a metered amount of inoculum and first medium substantially equal to the volume of the ECS is added, or when liquid is detected at the upper fitting 164, the valve is closed and filling stops.
  • a harvest layer can be added to the reactor 450, 550 and configured to provide one or more separation steps or allow harvesting of products other than the cells themselves.
  • a suspension of cells may be removed from a reactor and separated in a first step into a) nucleated cells and b) a mixture of enucleated cells and nuclei.
  • the mixture may be separated in a second step to extract the enucleated cells from the nuclei, which are waste.
  • the harvest layer is configured to perform part of the first step, i.e. nucleated cells are selectively retained in the reactor while enucleated cells and nuclei are selectively extracted from the reactor.
  • the degree of separation does not need to be a complete separation to be useful and a second separation of enucleated cells from nucleated cells may occur outside of the reactor.
  • the bioreactor 450, 550 is used to produce virus in cells
  • the produced virus may be extracted through the harvest layer while the cells are retained in the bioreactor.
  • the product may be extracted through the harvest layer while the cells are retained in the bioreactor 450, 550.
  • the perfusion membranes may be part of a recirculation loop including a bioprocess bag or other reservoir (for example a spinner flask 616 as shown in Figure 11) of second medium.
  • the reservoir preferably has a variable volume.
  • additional liquids for example a pH adjustment solution or a solution containing cytokines or other reactants, can be added as required without needing to simultaneously remove liquid from the recirculation loop.
  • the extra-capillary space (ECS) and the reservoir are filled with second medium before the start of a run and more second medium is not added during the run.
  • the ECS and the reservoir are filled to an initial volume before the start of the run but at one or more later times more second medium is added to the reservoir, for example from a second bioprocess bag or other vessel (for example bottle 620 as shown in Figure 11), without removing second medium.
  • a second bioprocess bag or other vessel for example bottle 620 as shown in Figure 11
  • the ECS and the reservoir are filled to an initial volume before the start of the run and second medium is a both added to the reservoir and removed from the reservoir during the run (which could include, for example, continuously, in pulses or at other discrete time intervals; at a constant rate or a rate the varies over time), for example as described in relation to Figure 11.
  • Second medium may be added to or removed from the reservoir directly or by adding medium to or removing medium from the recirculation loop.
  • the reservoir is preferably heated to the same temperature (i.e. 25-50 °C, or 37 °C) as the reactor 450, 550.
  • the reservoir may be located in an incubator with the reactor 450, 550 or in a separate heating area.
  • a second bioprocess bag or other vessel containing second medium for a fed batch or feed and bleed process may be kept at room temperature or refrigerated (i.e. 4-8 °C) during the run.
  • multiple reactors 450, 550 can be operated with distinct ECSs but with their membranes 102 linked.
  • the second medium perfusion and/or gas perfusion ports of multiple reactors 450, 550 can be connected in parallel. Because of the membranes 102, cells in the ECS of one reactor 450, 550 cannot enter the ECS of another reactor 450, 550. In that way cells of the same type but drawn from different donors or intended for different patients can be cultured simultaneously but separately. Although each cell population is different in some way, the cell population dynamics are sufficiently similar such that some second medium perfusion or gas supply system components can be shared.
  • the flow of second medium or gas to an individual reactor 450, 550 may be adjusted in a way analogous to the control of individual second elements 402 in a compound reactor 550 as described above.
  • the membranes 102 provide perfusion, i.e. delivery of a substance in a distributed manner.
  • the membranes 102 also provide retention, i.e. the cells are retained in the ECS.
  • the membranes 102 can also retain selected compounds in the ECS. This can decrease the amount of expensive elements, such as growth factors, that are required since the ECS is smaller than the entire bioreactor 450, 550 and its recirculation loops. As these selected compounds are consumed or degrade, more of the selected compound can be added directly to the ECS without the addition of whole medium. In this way, compounds that degrade at different rates can be added at appropriate rates.
  • a reactor 450, 550 can be cooled (or heated) by cooling the second medium or gas. This can make the reactor 450, 550 more accessible than, for example, wrapping a reactor in a cooling jacket. Further, with a cooling jacket as reactor diameter increases there may be a temperature difference between the core and the jacket. With cooling delivered through the membranes 102, heat is removed from the center of the reactor 450, 550.
  • the first medium moves, which helps to homogenize the delivery of gas and second medium components. Movement of the first medium can also be provided by moving the whole reactor 450, 550, for example by rotation about any axis, rocking back and forth, inversion, rotation on an incline, or another movement that changes the direction of the gravity vector relative to the reactor 450, 550.
  • the surface area of the gas transfer membranes may be reduced relative to a reactor without mixing in the ECS. Reducing the surface area of the gas transfer membranes 102b in turn facilitates culturing cells in suspension and harvesting cells and cell products.
  • the volume of the ECS may be in the range of 1-20, 2-20, or 2-10 mm 3 per mm 2 of surface area of the gas transfer membranes 102b (ratio of ECS volume to gas transfer surface area of 1-20 mm, 2- 20 mm or 2-10 mm). Length and surface area of the membranes 102 is measured within the ECS (i.e. only accounting for the length and surface area directly exposed to the ECS) unless stated otherwise.
  • Harvesting cells from the reactor 450, 550 may be done by opening one or more fittings 164 on the top plate 436 and/or on one or more panels 408, optionally attaching tubing to the one or more fittings 164, and draining the first medium out of the ECS. Draining the first medium may be done, for example, by orienting the reactor 450, 550 with the drainage fitting 164 near the bottom of the reactor 450, 550 and allowing the first medium to drain by gravity. In some examples, draining the ECS may recover 80% or more or 90% or more of the cells in the reactor 450, 550 before harvesting. Optionally, after draining the ECS, the ECS may be rinsed by adding a liquid (i.e.
  • Rinsing the reactor 450, 550 may recover additional cells, particularly in the case of semi-adherent cells such as HEK cells. Rinsing the reactor 450, 550 may also be useful to assist in transporting cells being harvested from one reactor 450, 550 directly into a larger reactor 450, 550.
  • a tube is used to connect the ECSs of two reactors 450, 550. Cells harvested from one reactor 450, 550 are used to inoculate the larger reactor 450, 550 for further cell expansion.
  • the rinsing agent may be a first medium that will be used in the larger reactor 450, 550.
  • movement of the first medium is provided by way of rotating the reactor 450, 550, optionally about an axis perpendicular to the membranes 102 and/or about a horizontal axis.
  • the axis of rotation may pass through the reactor 450, 550, optionally though the center of the reactor 450, 550.
  • a reactor 450, 550 may be rotated as shown in Figures 8, 9A and 9B.
  • the rotation is preferably in both directions (i.e. clockwise and counter-clockwise) rather than, for example, continuous or sustained (i.e. with more than 1, or more than 5, rotations in either direction).
  • the reactor 450, 550 does not complete more than a full rotation before the direction of rotation is reversed.
  • the reactor 450, 550 may rotate from a starting position by between 45-720 degrees, or between 90-360 degrees, or between 120 and 240 degrees, in one direction, then rotate back by between 45-720 degrees, or between 90-360 degrees, or between 120- 240 degrees in the opposite direction, optionally to the starting point.
  • the reactor 450, 550 is inverted (i.e. rotated by 180 degrees or more) or nearly inverted (i.e. rotated by 120-179 degrees) before the direction of rotation is reversed. Movement in either direction may be in one continuous movement or in a series of smaller movements.
  • a reactor 450, 550 may be rotated 180 degrees by six movements in one direction of 30 degrees each (or four movements of 45 degrees each, etc.) and then re-inverted by six movements of 30 degrees each (or four movements of 45 degrees each etc.) in the opposite direction.
  • the movements described herein are typically performed in a repeated pattern, optionally returning to the starting position at the conclusion of each pattern.
  • the axis of rotation may be vertical.
  • a horizontal axis of rotation, or at least an oblique axis of rotation may enhance, for example, the movement or suspension of cells or other solids in the first medium by way of inversion, partial inversion or other gravity-induced movement.
  • acceleration and deceleration and/or reversing the rotation of the reactor 450, 550 may help increase the movement of the first medium relative to the membranes 102. After a sustained period of rotation at a constant velocity in one direction, the first medium may tend to move in the same direction and at the same velocity as the membranes 102. Frequently accelerating and decelerating the reactor 450, 550 and/or reversing the direction of the reactor 450, 550, may help to provide and/or increase a difference in velocity of the first medium relative to the membranes 102.
  • the reactor 450, 550 typically operates substantially without a continuous gas phase or headspace (i.e. with less than 5% of the volume of the ECS filled with a continuous gas phase); the reactor 450, 550 does not have a length, measured in a horizontal direction perpendicular to the axis of rotation, that is significantly more than the depth of first medium; and/or, any free surface of liquid first medium may not extend along the entire length the reactor 450, 550.
  • the reactor 450, 550 may be round or a regular polygon (i.e.
  • the rotation of the reactor 450, 550 is also distinct, for example, from a flask on a shaker table wherein medium is made to rotate relative to the walls of the flask, but without rotation of the flask relative to a central vertical axis of the flask.
  • Such movement of a shaker flask typically requires generating velocities and forces that are undesirable in combination with membranes 102 and relies on producing a liquid film in contact with a large continuous gas phase. Movement of a roller bottle may be at a lower velocity but also relies on producing a large area film with a free surface, which does not occur in the reactor 450, 550 to a comparable degree due to the lack of headspace in reactor 450, 550.
  • the speed of rotation and/or rate or acceleration or deceleration of the reactor 450, 550 may be selected to provide a desired shear rate or tangential velocity at a selected location, for example at the periphery of the ECS. Tangential velocity varies with speed of rotation and diameter. For example, a 85 mm diameter reactor 450 having a 0.2L ECS was moved in a repeated pattern of rotation in one direction followed by rotation in the opposite direction back to the starting point. The maximum speed of rotation in the pattern was 5 rpm. Ramp up time and ramp down times (transition time between maximum speed of rotation and stop rotation) were minimal, i.e. about 0.4 seconds, and there was no dwell time between changes in direction.
  • a 200 mm diameter reactor 450 having a 1.2L ECS volume was moved according to the same pattern but with a maximum speed of rotation of 2.125 rpm.
  • movement of reactors can be compared by considering a rate of reversal.
  • a reactor rotated at 5 rpm back and forth through 180 degrees changes direction about once every 6 seconds, regardless of its diameter assuming that ramp up, ramp down and dwell times are minimal (i.e. ramp up and ramp down times or 0.1-0.5 seconds, dwell time of 0.0-0.5 seconds).
  • Another reactor rotated back and forth at 10 rpm through 360 degrees has the same rate of reversal of about once per 6 seconds.
  • a reactor rotated at 5 rpm through 360 degrees has a lower rate of reversal of about once per 12 seconds.
  • a pattern of movement may include movements back and forth through a first degree of rotation of less than 180 or 120 degrees to provide movement of the first medium relative to the membranes 102, periodically interrupted by movements back and forth through a second degree of rotation of at least 120 or 180 degrees to inhibit cell settling.
  • small movements i.e. movements of less than 180 or 120 degrees
  • a reactor 450, 550 may be rotated 90 degrees clockwise and 60 degrees counter-clockwise 6 times, and then 60 degrees clockwise and 90 degrees counter-clockwise, in a repeated pattern.
  • the movement in a direction may be broken into a series of smaller movements in the same direction as described further above.
  • the tangential velocity may be doubled relative to examples with frequent reversals of direction.
  • movement of the reactor 450, 550 includes 5-30, or 8-30, or 10-30, accelerations and decelerations, reversals of direction, or stop and re-starts in the same direction, per minute.
  • oxygen transfer from the gas transfer membranes 102b to the ECS is the limiting factor in increasing cell growth rate or density.
  • oxygen transfer from the gas transfer membranes was increased with rotation back and forth of the reactor 450, 550 as described above relative to operating an impeller inside the ECS of the reactor 450, 550.
  • the maximum speed is not always used since the maximum oxygen transfer rate is not required for all cell growth processes, cells may reach a maximum density for reasons other than oxygen transfer, or shear forces may be a limitation for some cells.
  • rotation back and forth through 360 degrees at a speed of 2.5 rpm provided adequate oxygen transfer.
  • T cells were grown to a cell density of 15 million cells per mL using speeds of 2.5 to 5 rpm with no additional oxygen needed in the gas stream.
  • Successful operation of a 200 mm diameter reactor similar to the reactor 450, oriented as in Figure 8, has been achieved with speeds of rotation of 2-7 rpm and movements back and forth through ranges of motion of 180-360 degrees.
  • the amount of movement between reversals is optionally 360 degrees or less, 270 degrees or less, or 180 degrees or less for reactors of similar sizes (i.e. diameters in the range of 50-300 mm or 75-250 mm).
  • appropriate parameters may be developed using the scaling guidelines described above.
  • Cells are typically grown in the ECS of the reactor 450, 550, outside of membranes 102.
  • the cells may be retained in the ECS of the reactor 450, 550, i.e. optionally without circulating through any components of the system external to the reactor 450, 550. Movement of the reactor 450, 550 helps to facilitate material transfer between the membranes 102 and the ECS, thereby allowing for a low packing density, for example 20% or less, 15% or less, or 10% or less, calculated on the basis of the volume of the membranes 102 divided by the interior volume of the reactor 450, 550 (i.e. volume of ECS added to volume of the membranes 102).
  • the reactor 450, 550 has an overall packing density (calculated as the volume the gas transfer membranes and the liquid transfer membranes, cumulatively, that passes through the ECS divided by the volume of the ECS) of about 8-12%.
  • the low packing density increases the volume available for cell growth (i.e. the ECS volume) for a given reactor size and/or helps with harvesting.
  • a large portion (i.e. 90% or more) of cells have been harvested from a reactor 450, 550 having a packing density of about 8% at viable cell density (VCD) of up to 58 million cells/mL.
  • VCD viable cell density
  • bioreactors with high packing densities i.e. 30-40% or more
  • the cells tend to become trapped between membranes, even when the cells are not adherent, and cannot be harvested effectively.
  • the pattern of movement causes membranes 102, for example gas transfer membranes 102b, to move through the top of the reactor 450, 550.
  • membranes 102 for example gas transfer membranes 102b
  • this allows any headspace to be minimized or removed.
  • an initial headspace may appear upon filling a reactor 450, 550.
  • Bulk phase gas may be removed from this initial headspace, thereby decreasing or removing the headspace, through the membranes 102.
  • gasses added to the reactor, or produced in the reactor, during operation are prevented from forming a significant headspace.
  • mixing by way of moving the reactor in combination with a low potting or packing density of membranes allows cells to be grown in suspension to high viable cell densities (VCD).
  • VCD viable cell densities
  • 90% or more of the cells can be harvested by simply draining the reactor.
  • mixing by way of moving the reactor in examples described below results in generally homogenous conditions throughout the reactor. A variety of different cell types can be grown.
  • movement of the reactor with frequent changes in velocity and/or direction may result in persistent transient flow conditions in the reactor.
  • the power required to move the bioreactor reaches a peak or maximum during or near periods of acceleration, deceleration or direction reversal of the bioreactor. Between these periods, or near the beginning of one of these periods, the power required may reach a minimum.
  • a mean power may be defined as the absolute value (i.e. ignoring direction or polarity) of peak power added to the absolute value of the minimum power and divided by two. Since liquid in the ECS may become entrained with the movement of the membranes, applied power may be above the mean power for longer than the duration of acceleration, deceleration or reversal portions of a movement cycle.
  • applied power may be above the mean power for a material part, i.e. 20% or more, 25% or more, or 33% or more, optionally up to 50%, of a movement cycle.
  • a relatively long duration of high power may increase transient flow or mixing in the ECS.
  • the movement cycle may not have sufficient periods of time with motion in one direction for the velocity of the liquid to reach the same velocity in the same direction as the membranes at least in part of the reactor, for example near the outer edge of the reactor. Accordingly, there may be a differential velocity between the membranes and the surrounding liquid for most (i.e. 50% or more or 70% or more or 90% or more) of the movement cycle at least in part of the reactor, for example near the outer edge of the reactor. Alternatively or additionally, differential velocity may be higher near the outer edge of the reactor than near the center of the reactor, which may cause shear (and therefore mixing) between the outer edge of the reactor and the center of the reactor.
  • ia is for oxygen unless indicated otherwise.
  • Reactors similar to the reactor 450 described herein and shown in Figure 8 were made with an ECS volume of approximately 200 mL, the ECS volume varying slightly between individual reactors.
  • the reactors had a generally cylindrical ECS with an inside diameter of about 85 mm.
  • the reactors had 15 layers of gas transfer membranes, with each layer having 40 membranes spaced apart at a center-to-center distance (pitch) of 0.8 mm (total 600 membranes).
  • the gas transfer membranes were OXYPLUS (TM) PMP skinned membranes by 3M with an outside diameter of about 0.38 mm. In use, the reactor was oriented with the membranes in vertical planes and rotated back and forth on a horizontal axis as shown in Figure 8.
  • the total surface area of the gas transfer membranes was 58,600 mm 2 .
  • the ECS volume to gas transfer membrane surface area ratio was 3.4 mm.
  • the reactors also had 8 layers of perfusion (liquid transfer) membranes, with each layer having 18 membranes spaced apart at a center to center distance (pitch) of 2 mm (total 144 membranes).
  • the perfusion membranes were PES membranes having a molecular weight cut off (MWCO) of about 10 kDa and an outside diameter of about 1.2 mm.
  • the perfusion membranes had a pore size of about 0.1 microns.
  • a reactor according to Example 1 has 10kDa perfusion membranes unless stated otherwise.
  • the membrane layers alternated with two layers of gas transfer membranes separating each layer of perfusion membranes.
  • Reactors similar to the reactors in Example 1 were made with 8 layers of gas transfer membranes, with each layer having 40 membranes spaced apart at a center-to- center distance (pitch) of 0.8 mm (total 320 membranes).
  • the gas transfer membranes were OXYPLUS(TM) PMP skinned membranes by 3M with an outside diameter of about 0.38 mm.
  • the reactor was oriented horizontally (with the membranes in vertical planes) and rotated back and forth on a horizontal axis as shown in Figure 8.
  • the total surface area of the gas transfer membranes was 31,200 mm 2 .
  • the ECS volume to gas transfer membrane surface area ratio was 6.4 mm.
  • the reactors also had 8 layers of perfusion (liquid transfer) membranes, with each layer having 18 membranes spaced apart at a center to center distance (pitch) of 2 mm (total 144 membranes).
  • the perfusion membranes were PES membranes having a molecular weight cut off (MWCO) of about 10 kDa and an outside diameter of about 1.2 mm.
  • MWCO molecular weight cut off
  • the perfusion membranes had a pore size of about 0.1 microns, with a different outside diameter and number of membranes but the same surface area as the 10 kDa membranes.
  • a reactor according to Example 2 has 10 kDa perfusion membranes unless stated otherwise.
  • the membrane layers alternated with a layer of gas transfer membranes separating each layer of perfusion membranes.
  • a reactor similar to the reactor 450 described herein and shown in Figure 8 was made with an ECS volume of approximately 1 ,200 mL.
  • the reactor had an inside diameter of the ECS of about 200 mm.
  • the reactor had 8 layers of gas transfer membranes, with each layer having 106 membranes spaced apart at a center to center distance (pitch) of 1.08 mm (total 848 membranes).
  • the reactor also had 8 layers of liquid transfer membranes, with each layer having 48 membranes spaced apart at a center to center distance (pitch) of 2.35 mm (total 384 membranes).
  • the liquid transfer membranes were PES membranes having a molecular weight cut off (MWCO) of about 10 kDa and an outside diameter of about 1.2 mm.
  • MWCO molecular weight cut off
  • the gas transfer membranes were OXYPLUS(TM) PMP skinned membranes by 3M with an outside diameter of about 0.38 mm.
  • the reactor was oriented with the membranes in vertical planes and rotated back and forth on a horizontal axis as shown in Figure 8.
  • the membrane layers alternated with a layer of gas transfer membranes separating each layer of perfusion membranes.
  • a reactor similar to the compound reactor 550 described herein and shown in Figure 9A or 9B was made with an ECS volume of approximately 1 L.
  • the reactor had a stack of 5 of the reactors of Example 2.
  • the reactor was oriented with the membranes in vertical planes and rotated back and forth on a horizontal axis 165 as shown in Figure 9A or 9B.
  • a reactor similar to the compound reactor 550 described herein and shown in Figure 9A or 9B was made with an ECS volume of approximately 5.5 L.
  • the reactor had a stack of 5 of the reactors of Example 3.
  • the reactor was oriented with the membranes in vertical planes and rotated back and forth on a horizontal axis 165 as shown in Figure 9A or 9B.
  • a reactor similar to the compound reactor 550 described herein and shown in Figure 9A or 9B was made with an ECS volume of approximately 10 L.
  • the reactor had a stack of 9 of the reactors of Example 3. In use, the reactor was oriented with the membranes in vertical planes and rotated back and forth on a horizontal axis 165 as shown in Figure 9A or 9B.
  • Reactors similar to the reactors in Example 1 were made with 14 layers of gas transfer membranes, with each layer having 40 membranes spaced apart at a center-to- center distance (pitch) of 0.8 mm (total 560 membranes).
  • the gas transfer membranes were OXYPLUS(TM) PMP skinned membranes by 3M with an outside diameter of about 0.38 mm.
  • the reactor was oriented horizontally (with the membranes in vertical planes) and rotated back and forth on a horizontal axis 165 as shown in Figure 8.
  • the total surface area of the gas transfer membranes was 54,600 mm 2 .
  • the ECS volume to gas transfer membrane surface area ratio was 11.2 mm.
  • the reactors also had 6 layers of perfusion (liquid transfer) membranes, with each layer having 7 membranes spaced apart at a center to center distance (pitch) of 6.5 mm (total 42 membranes).
  • the perfusion membranes were PES membranes having a pore size of about 0.1 pm and an outside diameter of about 2.6 mm.
  • the membrane layers alternated with two layers of gas transfer membranes separating each layer of perfusion membranes.
  • ki_a volumemetric oxygen transfer coefficient in initially de-oxygenated water was measured for reactors according to examples 2 and 3. The results are shown in Table 1 .
  • Linear velocity is calculated based on the rpm and the reactor diameter.
  • Rotation angle is the angle of back and forth rotation.
  • Reversals per minute is the number of changes in direction of rotation per minute estimated by the rotational speed and the rotation angle.
  • the column “Difference” is the difference between ki_a measured by a sensor near the inlet of the gas transfer membranes and ki_a measured by a sensor near the outlet of the gas transfer membranes, divided by the average of the two measurements.
  • increasing rotational speed provided an increase in ki_a and a reduction in difference between 5 and 7 rpm, but demonstrated smaller increases in ki_a and reductions in Difference thereafter.
  • Linear velocity is calculated based on the rpm and the reactor diameter.
  • Rotation angle is the angle of back and forth rotation.
  • Reversals per minute is the number of changes in direction of rotation per minute estimated by the rotational speed and the rotation angle.
  • the bioreactors were able to maintain an oxygen set point of 12.5% solubility (i.e. 50% of the oxygen concentration of water in equilibrium with the atmosphere) in the ECS throughout a batch process to the end of a period of glucose metabolism and to a viable cell density (VCD) of over 20 million cells per mL of ECS.
  • VCD viable cell density
  • Oxygen readings were compared between three sensors located in different parts of the headplate of a 1 ,2-L reactor according to example 3.
  • Sensor 1 is located near the inlet of the gas transfer membranes.
  • Sensor 2 is located near the inlet of the liquid transfer membranes.
  • Sensor 3 is located close to the center of the bioreactor in line with the gas transfer membranes.
  • the flow of oxygen is adjusted to provide 12.5% solubility at Sensor 1.
  • Results are shown in Table 3. At 2.5rpm/360° the difference in oxygen concentration at different locations is as high as 8.85% with an average noise of ⁇ 0.48% on each sensor. By increasing the rotation to 5rpm/360°, the difference among the sensors decreases to 7.6% and the noise to ⁇ 0.13%.
  • Reactors were made as described in Examples 2, 3, 4 and 5 (identified in Table 1 by their volume, which corresponds with volumes given in the description of examples 2, 3, 4 and 5 above), except that the reactor in run 9 had liquid perfusion membranes with a pore size of 0.1 microns rather than 10 kDa membranes.
  • the reactors were used to grow CHO cells in a batch process.
  • the term batch process indicates that the total volume of liquid medium used in the process was present at the start of the process.
  • the ECS was filled with liquid medium.
  • a perfusion system (including a circulating medium loop extending from a medium bag, through the liquid perfusion membranes, and back to the medium bag) was also filled with the same liquid medium.
  • the volume of the liquid medium in the reservoir was about twice the volume of the ECS. For example, for a trial with a reactor of example 2, about 600 mL of medium was provided, 200 mL in the ECS and 400 mL in the circulating medium loop.
  • the reactors were seeded with about 2 million cells/mL based on ECS volume and operated for over 3 days (about 78 hours). Glucose concentration of the medium declined from about 30 mM at the start of the process to near 0 over about 72 hours. Cell metabolism is believed to have been glucose metabolism for about 60-65 hours, followed by glucose and lactate co-metabolism and then by lactate metabolism for the remainder of the runs. Shaker flask cultures were run as controls (vented Erlenmeyer flask, orbital shaker, 0.03 L total medium volume). Speed and degree of rotation before reversing directions, and various other results, are shown in Table 4.
  • an indication of "kLa low” indicates that at some point in the run an oxygen sensor near the perimeter of the top plate used for controlling dissolved oxygen indicated that dissolved oxygen was dropping below the set point of 12.5% solubility.
  • An indication of "kLa high” indicates that the oxygen sensor used for controlling dissolved oxygen indicated 12.5% solubility throughout the run (though an oxygen sensor near the center of the reactor may have had a lower reading for part of the run). Cells continued to grow exponentially despite a "kLa low” condition in some runs. However, the oxygen reading at the inner periphery of the reactor may provide a qualitative indication of the effect of different patterns of rotation on oxygen transfer, "td" is cell VCD doubling time.
  • YVCD is the yield of cells from glucose during the exponential growth on glucose (determined between 0 and 60 hours) expressed in millions of cells per millimole of glucose consumed).
  • g G / U c is glucose consumption rate during the first 60 hours of the run (pmol/(cell day)).
  • gi_ac is the lactate production rate during the first 60 hours of the run. Ratio is the volume of the perfusion system divided by the volume of the bioreactor. Cell viabilities were above 90%, typically above 95%.
  • FIG. 14 shows VCD (measured based on the ECS volume) for the various runs.
  • a comparative control flask run reached a VCD of 13 million cells/mL at 72 hours.
  • Example 12
  • a reactor was made as described in example 2. The reactor was used to grow
  • the ECS was initially filled with about 200 mL of liquid medium.
  • a circulating liquid medium loop (perfusion system) extended from a medium bag, through the liquid perfusion membranes of the rector, and back to the medium bag.
  • the perfusion system was initially filled with about 200 mL of the same liquid medium.
  • fresh medium was added to the medium bag (and old medium was removed at the same rate) at a rate of 200 mL/day.
  • the rate of fresh medium addition and removal was increased in steps.
  • the rate of fresh medium addition and removal was maintained at 600 mL/day from about 96 hours of operation to the end of the run (about 140 hours). Over the entire run, 1.9 L of medium was added to, and removed from, the medium bag.
  • Total medium consumption was 2.3 L including the initial 200 mL of medium in the ECS and the initial 200 mL of medium in the medium bag.
  • the reactor was seeded with about 2 million cells/mL based on the ECS volume. Speed and degree of rotation before reversing directions were 11-13 rpm and 180 degrees respectively.
  • the gas transfer membranes were fed with compressed air for the first 24 hours, at some times with added carbon dioxide. Between 24 and 78 hours, pure oxygen was blended into the air at an amount that increased from 0 to 70% of the total gas flow rate as required to maintain an oxygen set point range of 10-15% solubility (i.e. 50% of the oxygen concentration of water in equilibrium with the atmosphere) in the ECS. After 78 hours, the pure oxygen flow was maintained at 70% of the total air flow rate.
  • VCD reached about 25 million cells/mL of ECS volume.
  • an oxygen sensor on the perimeter of the top plate indicated that oxygen concentration fell from 12.5% solubility to an undetectable amount.
  • cells continued to grow with an average doubling time of about 34.3 hours in the period between 70 and 120 hours to a maximum VCD of about 58.9 million cells/mL of ECS volume at 120 hours. Cell viability was over 90% throughout this time.
  • VCD remained constant at about 59 million cells/mL of ECS volume.
  • a reactor according to Example 3 was provided with two sampling ports on the head plate. One sampling port was close to the center (axis of rotation) of the reactor and one sampling port was near the perimeter of the head plate. The reactor was rotated back and forth at 5 rpm through movements of 180 degrees. The reactor was used to grow CHO cells to a final density of 27 million cells/mL. Samples were taken at 21 intervals during the growing period. The average difference in VCD readings between the two sampling locations was 0.9 million cells/mL.
  • Two reactors according to Example 4 were provided with two sampling ports. One sampling port was located on the head plate. The other sampling port was located between the two elements farthest from the head plate. One reactor was rotated back and forth at 5 rpm through movements of 360 degrees. This reactor was used to grow CHO cells to a final density of 21 million cells/mL. Samples were taken at 7 intervals during the growing period. The average difference in VCD readings between the two sampling locations was 1.4 million cells/mL. The second reactor was rotated back and forth at 11 rpm through movements of 180 degrees. This reactor was used to grow CHO cells to a final density of 36 million cells/mL. Samples were taken at 7 intervals during the growing period. The average difference in VCD readings between the two sampling locations was 1.4 million cells/mL. Differences in VCD readings for the reactor of Example 4 were about 8% between the two sampling locations for all readings at an average VCD of over 10 million cells/mL.
  • a reactor built according to example 6 was provided with a sample port in the inner part of the headplate and two sample ports at the side of the reactor, one located at element number 5, and another located at element number 9, the farthest element from the headplate.
  • the reactor was used to grow CHO cells, rotated back and forth at 5 rpm through 180 degrees. The final cell density was 35 million cells per mL. Samples were taken from the three sample ports along the culture. The maximum absolute difference in VCD was 2.6 million cells per mL, corresponding to 8.1% of the average reading.
  • cell density is generally homogenous both radially and axially in the reactors.
  • a bioreactor according to Example 1 was used to grow CHO cells in suspension.
  • a controller connected to a dissolved oxygen sensor was programmed to maintain an oxygen level in the ECS of 12.5%.
  • the bioreactor was rotated at 11 rpm back and forth through 180 degrees.
  • the ramp up and ramp down times between full velocity (11 rpm) and no velocity was 0.2 seconds. There was no pause (0 s pause delay) between changes in bioreactor direction.
  • the bioreactor was operated according to a feed and bleed operation similar to Example 12. Addition and removal of second medium started after 48 hours of operation at 200 mL/day with this rate increasing with VCD. A total of 2.5L of second medium was used. The run lasted about 160 hours. Oxygen concentration was maintained at the set point (12.5%) until the end of the run (in contrast to Example 10). Total gas flow was about 125 mL/min throughout the run. The composition of the gas varied from 95% ambient air and 5% carbon dioxide to a mixture of 75% oxygen and 25% ambient air over a period from about 30 hours to about 140 hours.
  • VCD reached a peak of about 70 million cells/mL at about 144 hours and was maintained above 60 million cells/mL until the end of the run.
  • the ability to grow cells to a higher VCD relative to the reactor of Example 2 while maintaining the oxygen setpoint is believed to be a result of the additional layers of oxygen transfer membranes.
  • Another bioreactor according to Example 1 was used to grow CHO cells in suspension.
  • a controller connected to a dissolved oxygen sensor was programmed to maintain an oxygen level in the ECS of 12.5%.
  • the bioreactor was rotated at 15 rpm back and forth through 180 degrees.
  • the ramp up and ramp down times between full velocity (15 rpm) and no velocity was 0.2 seconds. There was no pause (0 s pause delay) between changes in bioreactor direction.
  • the bioreactor was operated according to a feed and bleed operation similar to Example 12. Addition and removal of second medium started after 48 hours of operation at 200 mL/day with this rate increasing with VCD. A total of 5L of second medium was used. The run lasted about 192 hours. Oxygen concentration was maintained at the set point (12.5%) until reaching VCD of 75 million cells per mL. The total gas flow started at 100 mL/min and was increased to 150 mL at around 126 h of culture. The composition of the gas varied from 95% ambient air and 5% carbon dioxide to a mixture of 80% oxygen and 20% ambient air over a period from about 36 hours to about 144 hours.
  • VCD reached a peak of about 103 million cells/mL at about 168 hours and was maintained above 100 million cells/mL until the end of the run.
  • the ability to grow cells to a higher VCD relative to the reactor of Example 2 while maintaining the oxygen setpoint for a higher VCD is believed to be a result of the additional layers of oxygen transfer membranes.
  • a bioreactor according to example 5 was used to grow CHO cells in suspension. About 15.5 L of first medium was supplied in a batch mode, made up of 5.5 L in the ECS and 10 L in a bottle in a recirculation loop connected to the perfusion membranes. The bioreactor was rotated back and forth through 180 degrees. The ramp up and ramp down times between full velocity and no velocity was 0.2 seconds. There was no pause (0 s pause delay) between changes in bioreactor direction. The bioreactor was rotated at 5 rpm for a first part of the run and 7 rpm for a second part of the run
  • Glucose was substantially depleted after about 70 hours of operation. At this point, the VCD was about 25 million cells/mL. Cell growth continued until the end of the run in lactate or hybrid metabolism. The final VCD was about 32 million cells/mL.
  • Gas was supplied in parallel to the elements of the compound reactor. Total gas flow was 700 mL/min. Oxygen concentration as measured at edge of the bioreactor was maintained at the set point (12.5%) until the end of the approximately 96 hour run by varying the oxygen concentration of the gas. Oxygen concentration as measured on the headplate near the center of the bioreactor declined from near the set point at about 24 hours to near 0 at about 72 hours.
  • VCD was measured at various times throughout the run at four fittings located on the edge of the headplate, in the middle of the headplate, between elements in the middle of the reactor and in the element farthest from the headplate. VCD measurements were similar, i.e. with a variance of 2.5 million cells/mL or less between all four fittings, throughout the entire run. Metabolite measurements were also similar between the four sampling locations throughout the run.
  • a bioreactor according to example 6 was used to grow CHO cells in suspension. About 30 L of first medium was supplied in a batch mode, made up of 10 L in the ECS and 20 L in a bottle in a recirculation loop connected to the perfusion membranes. The bioreactor was rotated back and forth through 180 degrees. The ramp up and ramp down times between full velocity and no velocity was 0.2 seconds. There was no pause (0 s pause delay) between changes in bioreactor direction. The bioreactor was rotated at 5 rpm for a first part of the run and 7 rpm for a second part of the run.
  • VCD Glucose was substantially depleted after about 70 hours of operation. At this point, the VCD was about 32 million cells/mL. Cell growth continued until the end of the run in lactate or hybrid metabolism. The final VCD was about 35 million cells/mL.
  • oxygen concentration as measured at edge of the bioreactor increased back to the setpoint and remained there until the end of the run.
  • the oxygen concentration as measured on the headplate near the center of the bioreactor increased to about 3% and then steadily increased until the end of the run.
  • kLa measured at about 96 hours (at a 5 rpm rotation speed) was 10.2 IT 1 at edge of bioreactor and 12.0 IT 1 near the center of the bioreactor.
  • the gas mixture supplied at this time was about 60% oxygen and 40% air.
  • VCD was measured at various times throughout the run at three fittings located on the edge of the headplate, between elements in the middle of the reactor and in the element farthest from the headplate. VCD measurements were similar, i.e. with a variance of 3.5 million cells/mL or less between all three fittings, throughout the entire run. Metabolite measurements were also similar between the four sampling locations throughout the run.
  • a bioreactor built as described in example 7 was used to grow HEK293 cells.
  • the bioreactor was rotated back and forth at 11 rpm and 180 degrees.
  • the bioreactor was operated according to a feed and bleed operation similar to Example 12.
  • the feed and bleed started after 48 h of culture at 100 mL per day and was increased stepwise until 1200 mL per day at 120 h of culture.
  • HEK293 cells expanded at a doubling time of 28.9 h until a final VCD of 75 million cells per mL.
  • the specific glucose consumption observed during the feed phase was 4 pmol/(cell day).
  • the dissolved oxygen concentration reached the setpoint of 12.5% solubility around 48 h of culture and was controlled at the setpoint by the addition of pure oxygen to the gas stream.
  • VCD 40 million cells/mL
  • the percentage of oxygen in the gas stream reached its maximum value of 80%, resulting in the dissolved oxygen to drop from the setpoint of 12.5% to undetectable levels.
  • the inability to keep the oxygen at the setpoint for VCD higher than 40 million cells/mL is believed to be linked to the higher glucose consumption rate when compared to the bioreactor and process described in Example 12.

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Abstract

Bioréacteur de culture cellulaire présentant des membranes en fibres creuses à l'intérieur d'un espace extra-capillaire éventuellement utilisé pour cultiver des cellules en suspension. Les membranes peuvent comprendre des membranes de perfusion de liquide et/ou des membranes de transfert de gaz. Le milieu liquide se déplace à l'intérieur de l'espace extra-capillaire par rapport aux membranes. Le bioréacteur peut être tourné dans le sens des aiguilles d'une montre et dans le sens inverse des aiguilles d'une montre selon un schéma répété autour d'un axe de rotation passant par le bioréacteur. L'axe de rotation peut être horizontal, passer à travers le centre du bioréacteur et/ou être perpendiculaire aux membranes. Le schéma de rotation peut comprendre 5 inversions de sens ou plus et/ou des décélérations et des accélérations (éventuellement des arrêts et des redémarrages) dans une direction par minute. Le bioréacteur peut tourner sur moins de 360 degrés de rotation dans une direction puis à moins de 360 degrés dans la direction opposée. La rotation du bioréacteur peut atteindre une vitesse comprise entre 1 et 25 tr/min.
PCT/CA2023/051381 2022-10-31 2023-10-18 Bioréacteur de culture cellulaire avec mélange rotatif WO2024092343A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014132101A2 (fr) * 2012-10-18 2014-09-04 Yongxin Zhang Système de bioréacteur et procédés de culture cellulaire alternant entre un état statique et un état dynamique
WO2021155469A1 (fr) * 2020-02-05 2021-08-12 Membio Inc. Bioréacteur de culture cellulaire avec contrôle de zone
US20210380923A1 (en) * 2018-10-10 2021-12-09 Boehringer Ingelheim International Gmbh Method for membrane gas transfer in high density bioreactor culture

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014132101A2 (fr) * 2012-10-18 2014-09-04 Yongxin Zhang Système de bioréacteur et procédés de culture cellulaire alternant entre un état statique et un état dynamique
US20210380923A1 (en) * 2018-10-10 2021-12-09 Boehringer Ingelheim International Gmbh Method for membrane gas transfer in high density bioreactor culture
WO2021155469A1 (fr) * 2020-02-05 2021-08-12 Membio Inc. Bioréacteur de culture cellulaire avec contrôle de zone

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