EP0215820A1 - Bioreacteur fluidise et procede de culture de cellules - Google Patents

Bioreacteur fluidise et procede de culture de cellules

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Publication number
EP0215820A1
EP0215820A1 EP86901270A EP86901270A EP0215820A1 EP 0215820 A1 EP0215820 A1 EP 0215820A1 EP 86901270 A EP86901270 A EP 86901270A EP 86901270 A EP86901270 A EP 86901270A EP 0215820 A1 EP0215820 A1 EP 0215820A1
Authority
EP
European Patent Office
Prior art keywords
liquid
reaction zone
beads
nutrient medium
liquid nutrient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP86901270A
Other languages
German (de)
English (en)
Inventor
Robert C. Dean, Jr.
Peter V. RR 1 GRELA
Subhash B. Karkare
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Verax Corp
Original Assignee
Verax Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Verax Corp filed Critical Verax Corp
Publication of EP0215820A1 publication Critical patent/EP0215820A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • C12M25/20—Fluidized bed
    • 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
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2531/00—Microcarriers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • This invention relates to fluidized bed reactors for contacting fluids and solids, such as for carrying out chemical reactions, and par ⁇ ticularly relates to processes for cultivating cells, e.g., tissue cultures and fermentations, using such reactors.
  • Fluidized bed reactors are known in which the fluid is delivered upwardly from the bottom of the reactor through a distribution plate or other resistance which stabilizes the fluidized bed. Stabilization is achieved by virtue of the positive resistance to flow offered by the distribution plate.
  • the distribution plate tends to prevent gross distortion of the flow in the fluidized bed by offering lower resistance in regions having lower fluid velocity, and high resistance in regions of high velocity. Thus, the fluid flow tends to redistribute itself toward uniformity across the cross-section available for flow. A uniform fluid velocity profile is important to avoid channeling and other aberrant flow phenomenon whi ch prevent good solids suspension and good fluid/solid contact.
  • Typical examples of distribution plates known in ⁇ > the art are perforated metal plates, sintered materials, open-cell
  • Fluidized bed reactors provide a convenient way for conducting chemical processes which require mass and energy transport between a solid and a liquid or gas. Such reactors potentially offer the advantages of high mass and energy transfer rates over a wide range of throughputs, and have been used in many applications.
  • biocatalyst beads In the fermentation-related art, various methods have been devised for immobilizing bioactive materials such as enzymes and microorganisms on or in bead-like supports, referred to herein as biocatalyst beads. Although often quite fragile, these beads generally are suitable for fluidization and thus offer the potential for adaptating fluid bed technology to enzyme catalyzed processes and processes for cultivating cells. There are some problems, however.
  • Another object of this invention is to provide a fluidized bed reactor and method for continuously cultivating cells at high cell densities under optimum conditions while maintaining aseptic operation.
  • a method of continuously contacting a liquid with a bed of particulate solids comprising: fluidizing the bed of solids with the liquid in a reaction zone; separating the solids from the fluidizing liquid at one end of the reaction zone; treating a portion of the separated liquid in a treatment zone, separate from the reaction zone, so as to alter the temperature or composition of the separated liquid; recir ⁇ culating the treated liquid to the reaction zone as at least part of said liquid for fluidizing the bed of particulate solids; and recovering another portion of said separated liquid as product.
  • the method of the present invention has specific application in continuous aerobic cell culture processes wherein a bed of relatively fragile biocatalyst beads containing immobilized bioactive material is fluidized with a liquid nutrient medium in a reaction zone.
  • a liquid stream containing unconsumed nutrients and biochemical (metabolic) products is separated from the biocatalyst beads at one end of the reaction zone and a part of this stream is oxygenated in a separate treatment zone and is recirculated to the other end of the reaction zone for fluidizing the biocatalyst beads.
  • a portion of the liquid stream containing unconsumed nutrients is removed to recover the biochemical product.
  • the fluidizing of the bed of solids comprises simultaneously pumping and stabilizing the flow of the liquid upwardly through a vertical reaction zone with a pump impeller located at the bottom of the reaction zone.
  • the rotation of the impeller forces the liquid from below the impeller upwardly into the reaction zone to sus ⁇ pend the solids above the impeller, and the blades of the impeller are adapted to stabilize the velocity profile of the liquid above the bottom of the reaction zone without the need for any other stabilizing means above the impeller.
  • the diameter of the reaction zone may increase along the direction of upward flow of the liquid and this taper is such that when the liquid is in the fluidizing velocity range for the solids in the central portion of the reaction zone, the liquid in the bottom portion of the reaction zone is at a velocity above the fluidizing velocity range and liquid in the top portion of the reaction zone is at a velocity below the fluidizing velocity range.
  • One reactor for carrying out this particular process comprises a vertical reaction vessel and a bladed rotary pump impeller means at the bottom of the reaction vessel for simultaneously pumping the liquid nutrient medium and stabilizing the flow thereof upwardly through the reaction vessel to suspend the solids above the impeller means without the need for any other stabilizing means above the impeller means.
  • Means are provided for supplying fresh liquid nutrient medium to the reactor, for withdrawing at least a portion of the liquid nutrient medium that has passed upwardly through the reaction vessel, and for recirculating liquid nutrient medium exiting the top of the reaction vessel to the bottom of the reaction vessel.
  • impeller is an open axial propeller having a relatively flat angle of blade setting which moves liquid through the fluidized bed at typical fluidization velocities of from about 0.01 to about 0.5 meters per second and which, through its dynamic action, stabilizes the bed in order to counter velocity distribution distortion that leads to nonuniform fluidized bed operation.
  • a study of the velocity diagrams of the propeller shows that, where the axial (through flow) liquid velocity is low, the propeller adds more work because the effective angle of attack on the blade is increased. Conversely, where the axial velocity is high, the effective angle of attack decreases and so does the work input. Where the work input is high, there tends to be an increase of axial velocity countering the defect and vice-versa.
  • the propeller has an ⁇ open" design, i.e., with large spaces through which the biocatalyst beads can pass.
  • the probability of collision with the blades is therefore low and, because the blades move relatively slowly and with low power consumption, the incidence of damage to the beads and the bioreactive material, e.g., microorganisms, within them is low.
  • the bed of solids is fluidized in a stable fashion by pumping the liquid into the bed of solids in a vertical reaction zone through a distribution plate having one or more nozzles which horizontally direct the flow of liquid substantially parallel to the surface of the plate comprising the bottom of the fluidized bed.
  • biocatalyst bead is used to gen- erically catagorize supports containing immobilized bioactive materials such as enzymes, microorganisms and the cells of higher organisms, particularly microorganisms and cells requiring a constant supply of oxygen for proper development, including without limitation bacteria, fungi, plant cells and mammalian cells (e.g., hybridomas).
  • bioactive materials such as enzymes, microorganisms and the cells of higher organisms, particularly microorganisms and cells requiring a constant supply of oxygen for proper development, including without limitation bacteria, fungi, plant cells and mammalian cells (e.g., hybridomas).
  • Such beads can be used in connection with a wide variety of processes and the present invention is directed to a fluidized bed method and apparatus for carrying out such processes. Normally, suitable beads will have a porous structure and may be fibrous or sponge-like in appearance.
  • the beads can be prepared using a wide variety of materials including, inter alia, natural polymers such as polysaccarides and fibrous proteins, synthetic polym ers, such as polyamides (nylon), polyesters and polyurethanes, minerals including ceramics and metals.
  • natural polymers such as polysaccarides and fibrous proteins
  • synthetic polym ers such as polyamides (nylon), polyesters and polyurethanes
  • minerals including ceramics and metals.
  • phrases such as "process for cultivating cells,” “cell culture process” and the like are intended to embrace a wide variety of biochemical processes involving bioactive materials. These phrases embrace processes in which microorganisms or the cells of higher organisms are cultured, using an appropriate nutrient medium, to enhance the production of desired metabolic products. For instance, monoclonal antibody and other metabolite production by continuous culture of mammalian cells (e.g., hybridomas) is specifically included within the intended meaning of these phrases.
  • Figure 1 is a schematic vertical sectional view of a fluidized bed reactor in accordance with one embodiment of the invention
  • Figure 2 is a propeller characteristic plot of fluid pressure rise as a function of fluid flow
  • Figure 3 is a schematic view of a fluidized bed reactor in accordance with a another embodiment of the invention.
  • Figure 4 is a schematic view of a multi-stage fluidized bed reactor in accordance with the invention.
  • Figure 5 is a schematic view of a multistaged fluidized bed reactor having a conventional perforated distribution plate in accord ⁇ ance with another embodiment of this invention
  • Figure 6 is a schematic illustration of a nozzle which when used on the distribution plate of a vertical reactor substantially horizontally directs the flow of liquid parallel to the distribution plate at the bottom of a fluidized bed reactor;
  • F igure 7 is a schematic flow sheet of apparatus useful for practicing continuous aerobic cell cultivation in accordance with the invention.
  • a fluidized bed reactor 10 in accordance with one embodiment of the invention comprises a containment vessel 12 having within a stationary, tapered reaction vessel 14.
  • An annular recirculation channel 16 provides fluid communication between the top and bottom portions of reaction vessel 14.
  • a rotatable shaft 18 is journalled in bearings 20, 22 in the upper and lower portions of containment vessel 12.
  • a propeller 24 is fixed to and rotated by shaft 18, along with propeller tail cone 26. Tail cone 26 and the surrounding portion of reaction vessel 14 function as a diffuser to spread the liquid flow evenly across the entire cross-section of the reaction vessel.
  • Propeller 24 has blades 28 and is of a substantially open design as described below.
  • An inlet 30 is provided for supplying fresh liquid nutrient medium to the reactor.
  • Outlet 32 permits the withdrawal of at least a portion of the liquid nutrient medium or fermentation liquor that has passed upwardly through the reaction vessel 14.
  • reaction vessel 14 and the recirculation channel 16 are substantially filled with liquid nutrient medium, while a fluidized bed of biocatalyst beads, for example polysaccharide gel beads containing entrapped microorganisms, is maintained suspended in the central portion of reaction vessel 14 above the propeller.
  • the inner diameter of the reaction vessel 14 increases along the direction of upward flow of the liquid nutrient medium.
  • the taper of reaction vessel 14 is such that when the liquid nutrient' medium is in the fluidizing velocity range for the beads 36 in the central portion of the reaction vessel, the liquid nutrient medium in the bottom portion of the reaction vessel, just above propeller 24, is at a velocity above the fluidizing velocity range, and the liquid nutrient medium in the top portion of the reaction vessel is at a velocity below the fluidizing velocity range.
  • the fluidizing velocity range is, of course, that range of upward fluid velocity of liquid nutri ⁇ ent medium 34 which overcomes the gravitational force on the biocatalyst beads 36 and maintains them in suspension with substantially little or no net movement of the beads either upward or downward.
  • Typical fluidization velocities may be on the order of about 0.01 to about 0.5 meters per second for biocatalyst beads having a size of about 0.1 mm up to about 0.5 mm or more.
  • the tapered bed can be replaced with other forms so long as means are provided to separate the solid particles from the fluidizing medium, such as for example a straight wall reactor with a stepped expansion zone at its upper end or uti ⁇ lizing various known separation devices employing centrifugal, magnetic, electrostatic (electrical) or gravitational forces.
  • the separa ⁇ tion of solids particles from the fluidizing medium is very distinct and only a mini mal am ount of freeboard, wi thout necessi ty for precautionary designs such as tapered or stepped expansion zones, is needed to ensure satisfactory removal of the bead solids from the fluidizing liquid.
  • Propeller 24 is designed to effect suspension of the particulate bed and stabilization of the fluid velocity above the propeller without any other stabilization means and to avoid damage to any recirculating solids.
  • the propeller should have an open design having large spaces through which biocatalyst beads 36 can pass undamaged. More specifically the preferred propeller design has a solidity value of less than 1.0. Solidity is the ratio of the propeller blade chord to the blade spacing.
  • the blade angle should be set very flat, i.e., typi cally not more than about 15° off a tangent to the axis of rotation.
  • the propeller blade profile is designed to move high volumes of fluid with a small rise in pressure, as in the case of cooling tower fans and the like.
  • the impeller of the present invention should also be adapted to run at a slow speed with low power consumption.
  • the propeller blade tip speed preferrably varies in the range of from about 12 to 24 times the fluidizing velocity, which in turn ranges from about 0.01 to about 0.5 meters per second depending on the bead size, bead material specific gravity and fluidizing medium viscocity.
  • the tip speed preferrably ranges from about 1 to about 10 meters per second.
  • the propeller power requirements of this low speed propeller are very low e.g., less than about 0.25 kilowatt for a 1000 liter reactor, excluding losses for seals and bearings.
  • Figure 2 illustrates a typical propeller characteristic representing fluid pressure rise as a function of flow across the propeller.
  • the characteristic graphically illustrates the stabilization effect that the propeller has on the fluidize*- bed.
  • the propeller pump When the local flow up into the fluidized bed tends to decrease (represented by a movement of the operating point from desired point M to a lower flow point N), then the propeller pump generates a higher pressure as shown. This higher pressure tends to increase the fluid flow and move operation from point N back to the desired point M.
  • the fluidized bed is sta ⁇ bilized, that is, the velocity profile distortions tend to be eliminated by the propeller's action.
  • the "steeper" the propeller's characteristic i.e. the flatter the blade setting), the stronger the stabilizing action.
  • Bead-separating centrifuge 44 slings any beads 36 which rise within it along with upwardly flowing liquid nutrient medium 34 outwardly and back downwardly into reaction vessel 14.
  • Vaneless diffuser 42 recovers the kinetic energy of the flow and converts it to a pressure rise.
  • Figure 4 shows a multi-cell fluidized bed reactor made up of a serial arrangement of individual reactors such as that illustrated in Figure 1.
  • AU of the propellers 124 of the several individual reactors 100 are driven by a com mon shaft 118.
  • Treatment of the recirculating culture liquid to maintain opti ⁇ mum conditions in reactors 100 is accomplished by withdrawing side loops 160 from each reactor and effecting the necessary treatment in a separate treatment zone without injuring the fragile biocatalyst beads or disturbing conditions within the reactor itself.
  • Such treatments result in a change in the temperature and/or composition of the culture liquid.
  • the treated liquid then is recirculated to the associated reaction zone as at least part of the fluid for fluidizing the bed of solids.
  • the recirculated liquid comprises the major part of the fluidizing flow.
  • Product streams 170 can be taken off at any desirable stage in the process.
  • the reactor may be operated hyperbarically to ten or more at mospheres in order to proportionally enhance the oxygen carrying capacity of the recirculating liquor.
  • Figure 5 shows an alternative arrange m ent to Figure 4.
  • Figure 5 employs a serial or parallel arrangement of individual reactors for conducting a cell culture process in accordance with this invention. While both aerobic and anaerobic processes are contemplated, this embodiment is directed to the cultivation of cells and microorganisms which require the continuous supply of oxygen for proper development.
  • elements corresponding to elements in the Figure 4 arrangement are identified by reference numerals having the same last two digi ts.
  • Figure 5 differs from Figure 4 in that instead of employing a propeller, the bed of solids 236 in each reactor 200 is fluidized by pumping the fluid through a distribution plate 225 which stabilizes the fluidized bed.
  • the fluidized bed reactors may employ any of a wide variety of available distribution designs including, inter alia, a perforated plate or sieve tray, a slotted tray, a pebble bed, e.g., glass beads, a porous ceramic, an open cell foam and a sintered metal.
  • Figure 5 illustrates a conventional perforated distribution plate, in order to avoid plugging and back-flow of solids through the distribution means, for example, during inoperative periods, particularly in the case where fragile biocatalyst beads are being fluidized by and reacted with a liquid nutrient medium
  • the normal perforations in the conventional distribution plate can be replaced with one or more hori ⁇ zontal flow-directing nozzles in a suitable array.
  • a suitable nozzle design is illustrated in Figure 6.
  • the nozzle 400 consists of an enlarged head member 401 and a stem 402.
  • the head member has a top surface 405 and a generally vertical side wall 406.
  • the nozzle can be provided with any cross section although a cylindri ⁇ cal shape is convenient.
  • the stem 402 is sized for a friction fit with a perforation 403 in the distribution plate 425.
  • the stem has a cen ⁇ trally located bore 404 which extends into head member 401 (indicated by dotted outline).
  • the side wall of the head member is provided with substantially horizontal ports 407 which communicate with bore 404.
  • the ports are equally spaced around the circumfer ⁇ ence of the nozzle.
  • a normal 3/4 inch diameter nozzle may have twelve, approximately 1/8 inch diameter, ports equally spaced about its circumference.
  • liquid (and biocatalyst beads when occasionally recirculated) passes through the distribution plate by flowing through bore 404 and then radially outwardly (horizontally) through ports 407 into the fluidized bed reactor.
  • such nozzle designs also reduce the incidence of stagnant regions in lower corners of the reactors.
  • the number and arrangement of such nozzels in any application is, among other things, a function of the size of the reactor and the characteristics of the biocatalyst beads. For example, in a small reactor (diameter of about 2-4 inches) a single centrally located nozzle generally would be sufficient; while in a larger reactor (e.g., a diameter of about 8 inches) about 16 nozzles positioned in a symmetric pattern on the distribution plate should be employed. If advantageous, a bed of pebbles, e.g., glass beads, of an appropriate diameter, also can be supported by the distribution plate fitted with said nozzles to further stabilize performance. The number and arrangement of such nozzles for any particular application is within the skill of the art.
  • the nozzles are designed so that fluidization velocities in the range of about 0.001 to 0.01 m/sec are achieved at a pressure drop through the nozzle on the order of about 0.1 to about 1.0 psi using the heads of the desired characteristics.
  • the reactors 200 are designed and operated so that the biocatalyst beads readily separate from the upwardly flowing fluidization liquid in the upper region of each reactor. Any of the arrangements identified above in connection with Figures 1, 3 and 4 for effecting this separation could be employed. With a thick slurry of beads (e.g., 2596-60% solids), however, which is preferred, it is suf ⁇ ficient to rely on the force of gravity simply by providing a small dis ⁇ engagement zone (free-board) above the expected (design) level of the expanded bed. When operating at such high concentrations of the biocatalyst beads, the separation of solid particles from the fluidizing liquid is very distinct, eliminating the need for any precautionary designs such as a tapered disengagement zone or centrifugal separators.
  • Fresh nutrient medium is introduced into stage 1 of the reactor assembly of Figure 5 through line 210 and a portion of the unconsumed nutrient medium, discharged in lines 260 from stages 1 and 2 respectively, is passed to the succeeding stages 2 and 3 through lines 261 and 262 as feeds for these stages.
  • Product is removed from the reactor arrangement primarily through line 270 of stage 3.
  • Lines 271 can be used either for adding additional material to stages 2 or 3 or for withdrawing a portion of the unconsumed nutrient medium from stages 1 or 2, for example, for analysis or interim product removal.
  • each bed of biocatalyst beads 236 contains an immobilized bioactive material that requires oxygen to remain active.
  • Exemplary bioactive material includes aerobic microorganisms and cells such as aerobic bacteria, fungi and mammalian cells.
  • the beads may consist of a polysaccharide gel such as carrageenan or agarose gels entrapping the microorganisms and cells.
  • Other bead supports include natural and synthetic polymers, ceramics and metals.
  • the beads generally are porous and may be fibrous or sponge-like in appearance.
  • the beads comprise a fibrous polymer such as collagen in which the microorganisms are entrapped.
  • the beads will be treated to alter their specific gravity.
  • the beads may be weighed with an inert material, such as silica, to suitably increase their density above the density of the fluidizing medium. Alterna ⁇ tively, the beads may include entrapped gas to lower their density.
  • a particularly preferred fibrous polymer bead can be manufactured from collagen, e.g., using known procedures.
  • the bed of biocatalyst beads in each reactor stage is simultane ⁇ ously fluidized and oxygenated by recirculating a major portion of the unconsumed nutrient medium discharged from each reactor stage in lines 260 through a side loop.
  • the portion of the unconsumed nutrient medium discharged in line 260 and circulated for fluidizing the bed of biocatalyst beads is first passed through the oxygenators 230 where the dissolved oxygen content of the liquid is increased by contacting the liquid with an oxygen-containing gas.
  • oxygenators 230 While a wide variety of devices can be selected for oxygenating the recirculating fluid, including porous fine- bubble diffusers, mechanical aerators, or membrane oxygenators, membrane devices generally will be preferred for aerobic cell culture applications. Fine bubble diffusers and mechanical aerators tend to be plagued by foaming problems.
  • Membrane oxygenators transfer the oxygen directly into the liquid on a molecular level without any gas-liquid interface.
  • Membrane oxygenators suitable for fermentation applications include commercially available blood oxygenators such as available from Cobe, Denver, Colorado; A merican Bentley Corp., Irvine, California, and SciMed Co., Minneapolis, Minnesota.
  • Microporous filters such as the Gel man Acroflux cartridge Gelman Sciences, Inc., Ann Arbor, Michigan, and the Millipore Minidisk, Millipore Co., Milford, Massachusetts, may also be used in appropriate circumstances.
  • a particularly preferred oxygenator is a shell and tube oxygenator employing tube material having a suitable oxygen permeability.
  • Any of the well-known shell and tube-type designs used for example in the heat transfer art can be employed. See, for example, Perry, R.H. and Chilton, C.H., Chemical Engineers' Handbook.
  • a particularly useful design simply comprises a single helical strand of suitable tubing in a pressure vessel. Silicone tubing having an oxygen permeability on the order of about 1.3-1.4 X 10"*- 1 m mols O2 - mm per cm2-cm Hg per minute has proven to be particularly effective as tube material. Of course, other materials having different oxygen permeation characteris ⁇ tics can be employed. The selection of suitable materials and gas exchanger designs is within the skill of the art.
  • the liquid to be oxy ⁇ genated is flowed through the tubes of the gas membrane exchanger and the oxygen-containing gas, normally comprising air, enriched air or oxygen, is flowed through the exchanger on the shell side.
  • oxygen-containing gas normally comprising air, enriched air or oxygen
  • the liquid is recirculated using pumps 235 to the fluidized bed through heat exchangers 240, which adjust the temperature of the liquid to optimize conditions in reactors 200.
  • recirculation pumps such as blade-less centrifugal blood pumps, e.g., a Biomedicus pump, or flexible rotor vane pumps, e.g., Jabsco pumps
  • blade-less centrifugal blood pumps e.g., a Biomedicus pump
  • flexible rotor vane pumps e.g., Jabsco pumps
  • Enzyme released into the liquid as a consequence of cellular damage may degrade the desired product or other components of the culture liquid.
  • the recirculated liquid Prior to being introduced into the reactor, the recirculated liquid may be further treated (e.g., by adding reagents to control pH; by adding nutrients, drugs or ot her materials to influence the metabolism and/or growth of the cells or microorganisms in reactors 200; and by removing metabolic products and by-products, both desirable and undesirable, from the recirculating liquid by a variety of techniques including ultrafiltration, affinity absorption, adsorption and many others).
  • suitable reagents may be introduced into the reactors through lines 271 and 261.
  • the recirculation rate needed in any cell culture application is a function of many variables including, inter alia, the targeted solids (biocatalyst bead) concentration during fluidization; the density of the bioactive material, i.e., the concentration of organisms, e.g., mam malian cells such as hybridomas, immobilized on or inside of the biocatalyst beads the nature of the bioactive material (e.g., its oxygen demand), the morphology of immobilization (i.e., whether the bioactive material is contained on or in the bead matrix), the nature of the biocatalyst beads, e.g., the size of the beads and their specific gravity, the nature of the culture medium (e.g., its chemical characteristics and oxygen carrying capacity), and the nature of the treatment to be per ⁇ formed on the recirculating liquid.
  • the targeted solids (biocatalyst bead) concentration during fluidization the density of the bioactive material, i.e., the concentration of organisms, e.
  • the cell culture process in order to maximize efficiency the cell culture process is operated under fluidization conditions that yield a fluidized bed void volume within the range of about 60% to about 75%.
  • the void packing density of a packed bed of typical biocatalyst beads is about 50%-60%.
  • Operation at a fluidized bed void volume of lower than about 60% gives insufficient mixing between the biocatalyst beads and culture medium resulting in poor mass transfer, while operation at a fluidized bed void volume of above about 75% constitutes an inef ⁇ ficient usage of the reactor volume and impedes the natural dis ⁇ engagement of beads from the recirculating liquid at the effluent or outflow end of the fluidized bed.
  • the fluidization condi ⁇ tions are adjusted to give a fluidized bed void volume of about 60% to about 70%, depending upon the rate of mass and energy transfer needed, the bead attrition rate, etc.
  • the recirculation rate also is influenced by the concentration and nature of the cells or microorganisms in the biocatalyst beads. For example, for any specific oxygen-consuming microorganism, as the concentration (density) of the microorganisms inside the biocatalyst beads increases, higher recirculation rates are required in order to maintain adequate oxygen and/or nutrient transfer in the fluidized bed. Additionally, certain microorganisms, such as bacteria, generally have very high specific oxygen demands, and therefore normally will require higher recirculation rates and smaller, denser beads to maintain proper operating conditions than certain other organisms such as eukaryotes and mam malian cells which have much lower specific oxygen demands.
  • Penicillium chrysogenum generally requires only about 0.02 mol O2 per g-cell per minute and a typical hybridoma mammalian cell may require only about 5 x 10 ⁇ 7 mmol O2 per 10** cells per minute.
  • the properties of the biocatalyst beads also influence the necessary recirculation rate. While suitable bead sizes will be influenced by the particular cell culture process and reactor design, beads having a particle size within the range of about 100 um to about 1000 urn generally have proven to be suitable. Bead specific gravities between about 1.05 and 2.0 also have been investigated and found to be suitable depending upon the circumstances. Generally, beads having higher specific gravities within this range may be preferred since a higher specific gravity permits a higher recirculation rate and a higher reactor aspect ratio. At higher reacter aspect ratios (the aspect ratio is a quotient of the height of the reactor, to its diameter) the fluidized bed reactor is less likely to encounter fluid distribution problems such as short circuiting.
  • FIG. 7 a schematic flow diagram of a cell culture process and apparatus suitable, for example, for continuously manufacturing antibody in accordance with this invention is shown.
  • Biocatalyst beads containing immobilized hybridomas manufactured for example by the procedure in the above-noted copending application are particularly suited for this process.
  • a thick slurry of such biocatalyst beads is suspended and agitated in fluidized bed 300, for example, of the type illustrated above in Figure 5.
  • the cell concentration inside such biocatalyst beads typically is about 10*- 1 cells per ml of beads for hybridomas of a 14 um diameter.
  • the liquid surrounding the beads typically contains a concentration of organisms of about 1/10th that value.
  • the average cell concentration in the bioreactor volume is about 40% of the concentration in the beads themselves.
  • the actual reactor cell concentration (beads and liquor together) is approximately 4.6 X 10 7 cells per ml.
  • fresh nutrient medium is fed to a fluidized bed bioreactor 300 from a medium storage vessel 302 through pump 303 and valves 304 and 305.
  • a liquid stream 360 is separated from the top of the bioreactor 300 to form a recirculating culture liquor.
  • the culture liquor is circulated outside the fluidized bed through line 360 and valves 306 and 307 and pump 335 in order to appropriately treat the culture liquor in separate treatment means, which liquid then is returned to the bottom of the bioreactor through line 362 and valves 308 and 309 to produce the fluidization action in bioreactor 300.
  • a heat exchanger 340 also is inserted in the recirculation loop in order to adjust the temperature of the recir ⁇ culating liquor and thereby control the temperature in bioreactor 300 at its optimum condition.
  • Oxygen is transferred to this recirculated liquid and CO2 is removed therefrom by a membrane gas exchanger 330.
  • the culture liquid is flowed through the membrane gas exchanger 330 in counter- current flow with a stream of air or enriched oxygen, preferably pressurized, delivered from oxygen storage tank 310 through control valve 311 and filter 312. Waste gas containing CO2 is discharged through filter 313, valve 314 and flow meter 315.
  • an external heat exchanger 340 controls the tempera ⁇ ture of the recirculating culture liquor and hence the bioreactor tem ⁇ perature.
  • Instrumentation and sensors such as a pH probe 341, a dis ⁇ solved oxygen probe 342, temperature sensors 343 and 344, and a turbidity probe 345, are installed in the external recycle loop for easy access and calibration.
  • the recycle dilution rate can be adjusted independently of the. feed dilution rate in order to control each separately to achieve optimum performance in reactor 300.
  • the recycle dilution rate which normally is the major fluidizing flow, can be very high, e.g., up to about 1000 hr ⁇ l, while the fresh nutrient medium throughflow dilution rate can, at the same time, be very low, e.g., down to about 0.006 hr"" 1 .
  • Separation of the recir ⁇ culating and throughflow dilution is a very convenient and advantageous feature of the present invention.
  • a product strea m of har vest liquor is removed from the bioreactor through line 370 and valves 316 and 317, is passed through an in-line microporous filter 321 to remove cells and cell debris and is collected in storage vessel 318. Thereafter, the liquid is removed from vessel 318 and the antibodies can be separated and recovered by first removing additional cells and cell debris from the harvest liquor, with a microporous filter, filter 319, followed by removal of about 95% of the water by tangential-flow ultrafiltration. An ion-exchange chro otography column may then be employed to extract bovine serum albumin or other constituents of the recovered liquid if present in the culture medium. The column is followed by one or more steps of high pressure liquid chromotography for final purification before filter steri ⁇ lization and lyophilization, or the bottling of a sterile-liquid product containing the recovered antibody.
  • the product may also comprise the cel ⁇ lular material or biomass itself.
  • the product may also comprise the cel ⁇ lular material or biomass itself.
  • certain mate ⁇ rials for the biocatalyst beads e.g., porous or fibrous structures as described in the above-noted copending application, excess cells from an expanding cellular colony are expelled directly through the outer pores of the fibrous beads without rupturing the bead structure, thereby permitting the desired cell product to be recovered as an entrained component of the culture medium.
  • other methods for recovering the biomass directly from the biocatalyst beads themselves also can be employed and the present invention is not intended to be limited to any particular embodiment.
  • Example 1 is intended to more fully illustrate the invention without acting as a limitation on its scope.
  • a fluidizing bed reactor of the configuration shown in Figure 1 may have an internal base diameter of 0.86 meters and a height of 2.0 meters. The capacity of this reactor may be about 1000 liters.
  • the propeller should have a tip diameter of about 0.86 meters and a hub diameter to tip diameter ratio of about 0.5.
  • the propeller tip speed during fluidization should be between 1.2 and 2.4 m/sec.
  • the biocatalyst beads can be formed from a hydrocolloid matrix weighted with a mineral (i.e., kappa-carrageenan with silica powder) and can contain immobilized therein live yeast cells of Saccharomyces cerevisiae (from PEDCO International - M-4 molasses strain).
  • the nutrient medium (substrate) for feeding to the reactor can be predominantly glucose with trace amounts of other nutrients normally employed in this type of fermentation reaction (anaerobic).
  • the medium from the top of the reaction zone would be partially recovered continuously to give an average yield of about 50 kg/hr of ethanol.
  • a fluidized bed bioreactor was constructed having a diameter of 2 inches and a height of about 8 inches. The capacity of this reactor is about 400 ml.
  • Biocatalyst beads were formed from a hydrocolloid matrix weighted with a mineral, i.e., K-carrageeman weighted with sil ⁇ ica powder. The beads had live recombinant yeasts cells of S. cerevisiae, obtained from Integrated Genetics of Framingha m , Massashusetts, im mobilized therein.
  • a nutrient medium also obtained from Integrated Genetics, containing glucose and other nutrients nor ⁇ mally employed in this type of aerobic fermentation process, was pumped to the bioreactor at a rate sufficient to yield a residence time (based on the feed rate) of about 2 hours.
  • the bed of biocatalyst beads was fluidized by recirculating the fermentor (culture) liquor, using a magnetically coupled gear pump, at a rate sufficient to yield a residence time of less than about one minute in the reactor.
  • the recirculation liquid was passed through a silicone membrane oxygenator manufactured by SciMed Co., Minneapolis, Minnesota. High purity oxygen gas was used as the oxygen source. Carbon dioxide was removed through the silicone membrane simultaneously with oxygenation.
  • the recirculation liquid was introduced into the bioreactor through a nozzle similar in design to Figure 6.
  • the above-described bioreactor was operated continuously for more than 1000 hours producing a product stream containing approxi ⁇ mately 100 nanograms per milliliter of alpha human chorionic gonadatropin ( ⁇ -HCG).
  • a batch reactor operated under similar condi ⁇ tions produced product at a rate about 20 times lower.
  • a ten liter capacity fluidized bed bioreactor similar in construc ⁇ tion to one stage of the configuration shown in Figure 5 may be con ⁇ structed having an internal diameter of about 4 inches and a height from the distribution plate to the effluent line of about 5 feet.
  • the distributor plate will be provided with a horizontal flow-directing noz ⁇ zle of the design illustrated in Figure 6.
  • the nozzle will have twelve approximately one-eighth inch diameter holes equally spaced about its circumference located 0.5 inch above the surface of the distribution plate.
  • a bed of glass pebbles three inches deep also may be supported by the distribution plate.
  • the bioreactor can contain about four liters of biocatalyst beads formed from a weighted fibrous polymer (i.e., collagen with silica powder) containing immobilized mammalian cells (hybridoma VX-7).
  • the beads will have an average diameter of about lOOOu, a specific gravity of about 1.15 and the cell density within the beads can be about 7 x 10 7 cells/ ml.
  • the biocatalyst beads will be fluidized by recirculating the culture medium in the reactor at a flow rate of about 2 liters per minute. Fresh nutrient medium also will be introduced into the bioreactor at a rate of about 1 liter per hour and a product stream having an equivalent flow rate will be removed.
  • the bioreactor will be aerated by passing the recirculating culture medium through a gas membrane oxygenator.
  • the oxygenator will consist of a 3 liter vessel having 100 feet of a single, helically wound tube of silicon tubing (0.25 inch internal diameter) having an oxygen permeability of about 1.34 x 10 6 mmols 02-m per cm 2 Hg per minute. Air can be fed to the oxygenator at a rate of about 1 liter per minute and a carbon dioxide-containing gas will be discharged at a substantially equivalent rate.

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Abstract

Un réacteur (100) à lit fluidisé et un procédé de réaction particulièrement utiles pour cultiver des cellules, pour des procédés de fermentation et de culture de tissus par exemple, impliquent le traitement d'au moins une partie du fluide sortant du réacteur (100) à lit fluidisé dans une boucle latérale (160), de façon à modifier sa température ou sa composition, par exemple en l'oxygénant. Ce fluide traité est réintroduit dans le réacteur comme partie du fluide à l'origine de la fluidisation du lit.
EP86901270A 1985-02-28 1986-02-06 Bioreacteur fluidise et procede de culture de cellules Withdrawn EP0215820A1 (fr)

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US70687285A 1985-02-28 1985-02-28
US706872 2000-11-07

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CA1307225C (fr) * 1988-07-19 1992-09-08 David W. Armstrong Bioreacteur pour la culture de cellules
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US5102790A (en) * 1988-12-19 1992-04-07 Baxter International Inc. Method for culturing animal cells
CA2011653A1 (fr) * 1989-03-08 1990-09-08 Hiroshi Deno Methode de culture des tissus vegetaux, appareil utilise a cette fin et methode de production de metabolites
AT394576B (de) * 1991-01-16 1992-05-11 Vogelbusch Gmbh Reaktor zur durchfuehrung biologischer reaktionen mittels biokatalysatoren
JP3604715B2 (ja) * 1993-09-07 2004-12-22 サッポロホールディングス株式会社 酒類の製造法
DE19528871C2 (de) * 1994-08-24 1997-08-14 Forschungszentrum Juelich Gmbh Begasungs-Rohrmodul mit selektiv gasdurchlässiger Schlauchmembranen und damit versehene Reaktoren für Zellkulturtechnik sowie Wirbelschicht-Reaktoren für Zellkultivierung
EP0727480A3 (fr) * 1995-02-16 2000-04-12 Forschungszentrum Jülich Gmbh Procédé pour déterminer les pharmacocinétiques ou toxiqocinétiques des substances d'essais utilisant un système de culture de cellules in vitro et dispositif appropriés
DE59610339D1 (de) * 1995-02-16 2003-05-22 Forschungszentrum Juelich Gmbh Verfahren zur Kultivierung von Organfunktionszellen
EP0787803A4 (fr) * 1995-08-21 2002-01-30 Kyowa Hakko Kogyo Kk Procede de reaction enzymatique immobilisee
JP4231563B2 (ja) * 1997-09-02 2009-03-04 サッポロビール株式会社 発酵生産物の製造法
EP2290050B1 (fr) * 1999-09-08 2012-06-13 Levitronix Technologies, LLC Bioréacteur
DE10016877A1 (de) 2000-04-05 2001-10-18 Scintec Diagnostics Gmbh Zug (Glyko-)Proteine mit hoher Immunreaktivität sowie ein Verfahren zu ihrer Herstellung
DE10127869A1 (de) * 2001-06-08 2003-01-02 Hoefer Bioreact Gmbh Bioreaktor mit wenigstens zwei Reaktionskammern
DE102007020671A1 (de) * 2007-05-01 2008-11-06 Justus-Liebig-Universität Giessen Verfahren und Vorrichtung zum kontaktfreien Materialwachstum in einer strömenden Lösung (FLow suspended solution growth (FSSG))
GB0921836D0 (en) * 2009-12-14 2010-01-27 Prebble Andrew Anaerobic reactor
WO2011154886A1 (fr) 2010-06-07 2011-12-15 Jean-Louis Roux Dit Buisson Photobioréacteur à flux continu ou semi-continu et procédé d'utilisation
US20140024090A1 (en) * 2011-12-19 2014-01-23 Shell Oil Company Method and apparatus for converting a carbon source into a hydrocarbon or hydrocarbon precursor
FR3027678B1 (fr) * 2014-10-24 2016-12-30 Viovy Jean Louis Procede de detection d'organismes dans un echantillon dilue
KR102580754B1 (ko) 2016-08-21 2023-09-19 아드바 바이오테크놀로지 리미티드 생물반응기 및 이의 사용 방법
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CA1305681C (fr) 1992-07-28
WO1986005202A1 (fr) 1986-09-12

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