WO1993003135A1 - Appareil et procede de culture et de fermentation - Google Patents

Appareil et procede de culture et de fermentation Download PDF

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Publication number
WO1993003135A1
WO1993003135A1 PCT/US1992/006640 US9206640W WO9303135A1 WO 1993003135 A1 WO1993003135 A1 WO 1993003135A1 US 9206640 W US9206640 W US 9206640W WO 9303135 A1 WO9303135 A1 WO 9303135A1
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WIPO (PCT)
Prior art keywords
medium
die
cells
cell
chamber
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PCT/US1992/006640
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English (en)
Inventor
Heath H. Herman
Kent M. Herman
Edward J. Pitt
Original Assignee
Biotec Research & Development, Inc.
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Application filed by Biotec Research & Development, Inc. filed Critical Biotec Research & Development, Inc.
Publication of WO1993003135A1 publication Critical patent/WO1993003135A1/fr

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    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • 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
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure

Definitions

  • the present invention relates to an improved method and apparatus for the continuous culture of biocatalysts. More particularly, the present invention relates to a method and apparatus for culturing biocatalysts, such as cells or enzyme systems, under high pressure conditions thereby allowing for the maintenance of biocatalysts at high density with significantly increased yields of cellular products.
  • culture means any of a group of chemical reactions induced by living or nonliving biocatalysts.
  • culture means the suspension of any such biocatalyst in a liquid medium for the purpose of maintaining chemical reactions.
  • biocatalysts as used herein, includes enzymes, vitamins, enzyme groups, immobilized enzymes, subcellular component, prokaryotic cells and eukaryotic cells.
  • hyperbaric pressure means any hydraulic pressure greater than atmospheric pressure.
  • the culture of microbial cells (fermentation) or animal and plant cells (tissue culture) are central to a multiplicity of commercially-important chemical and biochemical production processes.
  • Living cells are employed for such purposes as a result of the fact that living cells, using generally easily obtainable starting materials, can economically synthesize commercially- valuable chemicals.
  • yeast cells can produce large quantities of ethanol (useful for human consumption as beer, wine, or other spirits) if fed solutions of agriculturally-produced sugars under the appropriate conditions, while, in contrast, the de novo synthesis of ethanol by organic synthetic methods is quite expensive.
  • living cells can produce protein molecules of immense commercial value which could not be produced at all by synthetic methods.
  • Fermentation involves the growth or maintenance of living cells in a nutrient liquid media.
  • the desired microorganism or eukaryotic cell is placed in a defined medium composed of water (usually at least 1000 times the volume of the cells), nutrient chemicals and dissolved gases, and allowed to grow (or multiply) to a desired culture density.
  • the liquid medium must contain all the chemicals which the cells require for their life processes and also should provide the optimal environmental conditions for their continued growth and/or replication.
  • a representative microbial cell culture process might utilize either a continuous stirred-tank reactor or a gas-fluidized bed reactor in which the microbe population is suspended in circulating nutrient media.
  • in vitro mammalian cell culture employs a suspended culture of cells in roller flasks or, for cells requiring surface attachment, cultures grown to confluence in tissue culture flasks containing nutrient medium above the attached cells.
  • the living cells so maintained, then metabolically produce the desired product(s) from precursor chemicals introduced into the nutrient mixture.
  • the desired product(s) are either purified from the liquid medium or are extracted from the cells themselves.
  • the optimal conditions under which the desired cell type may be cultured is usually near the optimal conditions for the growth of many other undesirable cells or microorganisms. Extreme care and expense must be taken to initially sterilize and to subsequently exclude undesired cell types from gaining access to the culture medium.
  • fermentation methods particularly those employing aerobic organisms, are limited to low yields of product or low rates of product formation as a result of the inability to deliver adequate quantities of dissolved oxygen to the metabolizing organism.
  • batch processes can only be operated for a finite time period before the buildup of excreted wastes in the fermentation media require process shutdown followed by system cleanup and resterilization.
  • Another method for the immobilization of living cells or enzymes currently in use involves the use of packed-bed reactors.
  • free cells or cells bound to microcarrier beads are suspended in a rigid or semi-rigid matrix which is placed within a culture bioreactor.
  • the matrix possesses interstitial passages for the transport of liquid nutrient media into the reactor, similarly disposed passages for the outflow of liquid media and product chemicals, and similar interstitial passages through which input and output gases may flow.
  • Bioreactors of this type include the vat type, the packed- column type, and the porous ceramic-matrix type bioreactor. Such methods are taught, for example, in U.S. Patent Nos.
  • bioreactors are particularly susceptible to the "bleeding" of biocatalysts detached from the matrix (or released by cell division), with the result that output ports become clogged with cells and/or debris. The result is an unacceptable pressure drop across the bioreactor which causes further deterioration of production.
  • a final class of methods for cell immobilization involves the employment of capillary hollow fibers (usually configured in elongated bundles of many fibers) having micropores in the fiber walls.
  • capillary hollow fibers usually configured in elongated bundles of many fibers
  • cells are cultured in a closed chamber into which the fiber bundles are placed.
  • Nutrient aqueous solutions flow freely through the capillary lumena and the hydrostatic pressure of this flow results in an outward radial perfusion of the nutrient liquid into the extracapillary space in a gradient beginning at the entry port Similarly, this pressure differential drives an outward flow of "spent" media from the cell chamber back into the capillary lumena by which wastes are removed.
  • Cells grow in the extracapillary space either in free solution or by attachment to the extracapillary walls of the fibers.
  • Living cells are unable to derive any benefit from gaseous oxygen. Living cells derive benefit solely from oxygen dissolved within the aqueous media which surrounds the cells.
  • the sparging of air or oxygen-enriched gases through the aqueous nutrient media is intended to replace the dissolved oxygen consumed by the metabolizing cells. In this method, most of the gas exits unused while dissolved oxygen levels are maintained.
  • the sparging of air (or oxygen) into the nutrient media prior to its use in animal cell culture is intended to maintain a level of dissolved oxygen in the media.
  • U.S. Patent No.4,897,359 discloses a method for oxygenating animal cell culture media for subsequent introduction into cell culture vessels in which an oxygenated gas, at an indeterminate pressure, is passed through a multiplicity of gas permeable tubes surrounded by the liquid medium to be oxygenated. While the pressure of the input gas may be above atmospheric pressure, the pressure of the oxygenated exit liquid can be no more than atmospheric pressure. If the oxygenated exit liquid were above atmospheric pressure, it would result in outgassing of the liquid medium when the medium was introduced into the typical cell culture vessel. Such outgassing would also result in bubble formation within the media, which would be extremely deleterious to animal cell viability. Thus, the method of the invention of Oakley et al.
  • U.S. Patent No. 4,837,390 discloses a method of preservation of living organs (for subsequent transplant) in which hyperbaric conditions (2 to 15 bars or 29 to 218 pounds per square inch (psi)) are maintained.
  • psi pounds per square inch
  • a living organ is placed in a chamber capable of withstanding pressure and a perfusion liquid containing nutrients is pumped into and out of the chamber while a gaseous oxygen overpressure is also applied to the chamber.
  • the method does not discuss cell culture or fermentation.
  • U.S. Patent No. 4,833,089 discloses a cell culture method in which a gaseous overpressure of oxygen or air is applied over a stirred liquid media in which cells are cultured.
  • the pressure limitations of the apparatus which include peristaltic pumps, flexible low-pressure pump tubing, and low pressure filter apparati
  • the concentration of dissolved oxygen in the media bathing the cells is limited to values only slightly greater than that obtainable at atmospheric pressure.
  • U.S. Patent No. 4,774,187 discloses a method for the culture of microbial cells in which a gaseous overpressure is applied over stirred liquid media in which cells are cultured.
  • the gaseous overpressure makes it impossible to access the interior of the culture compartment without depressurization and cell destruction.
  • the inventor overcomes this problem by raising an overflow line from the media-containing bioreactor to a height such that the liquid pressure of this overflow line equals the gas overpressure.
  • a siphon originating in the elevated overflow vessel connected to the overflow line one may withdraw liquid or cells from the culture chamber without depressurizing the chamber.
  • the system pressure is limited to the height of a column of water which would balance the system pressure.
  • a column of water approximately 50 feet in height would be required.
  • the method from a practical standpoint is limited to dissolved oxygen levels obtainable at 1 - 2 atmospheres of overpressure.
  • U.S. Patent No. 4,169,010 discloses a method for improved oxygen utilization during the fermentation of single cell protein in which a gaseous overpressure above a stirred nutrient liquid in a bioreactor containing the growing cells is utilized to increase oxygen delivery to the growing cells.
  • a gaseous overpressure above a stirred nutrient liquid in a bioreactor containing the growing cells is utilized to increase oxygen delivery to the growing cells.
  • the recirculation of cell-free media (lean ferment) obtained by centrifugation of the bioreactor contents is passed back into the bioreactor through an absorber section containing a gas contacting zone.
  • the gaseous overpressure is maintained by a gas pressure regulator device which blocks pressure release or vents the gas in response to a desired dissolved oxygen sensor setting.
  • the patent discloses overpressures of about 0.1 to 100 atmospheres (approximately 16.2 to 1485 psi) (Col. 7, lines 28-30, of U.S. Patent No. 4,169,010).
  • the inventor states that a maximum desirable gaseous overpressure of 1 to 2 atmospheres is preferable.
  • 4,001,090 discloses a method for microbial cell culture which incorporates a process for improved oxygen utilization very similar to that outlined above for Marwil (U.S. Patent No. 4,169,010).
  • the method of Kalina directly addresses the problem of carbon dioxide removal mentioned earlier in connection with the method of Marwil. This problem is eliminated by the inclusion of a gas-liquid separator in the fermenter circuit.
  • an oxygenated gas at an unspecified pressure greater than atmospheric is released into the fermentation chamber at its bottom (common sparging).
  • the media is maintained at an overpressure of as much as 3 to 3.5 atmospheres (44.1 to 51.5 psi) to provide both a motive force for the media recirculation as well as to aid in the removal of excess gas distal to the fermentation zone (Col. 4, lines 35-37).
  • the Kalina process relies heavily on the presence of gas bubbles for the agitation of the media and is suitable solely for use in microbial cell fermentation; the method could not be applied to animal cell culture because such cells are extremely sensitive to hydraulic shear forces and are damaged or destroyed by contact with air-water interfaces such as those encountered in gas bubble-containing media,
  • U.S. Patent No. 3,968,035 discloses a method for the "super-oxygenation" of microbial fermentation media in which the common sparging of an oxygen-containing gas into the fermentation media is replaced by the introduction of this gas into an "oxidator" vessel in which high-shear agitation is used to reduce the average size of the gas bubbles, thus increasing the available surface area for gas-liquid contact with the result that maximal dissolved oxygen concentration is maintained.
  • the fermentation media which has thus been treated is pumped into the fermentation reactor while exhausted media from this same source provides the input to the "oxidator” vessel.
  • the process of this invention thus provides a combined liquid and oxygen-enriched gaseous mixture to the culture chamber, a situation which is inapplicable to animal cell culture for the previously-mentioned reasons.
  • these confinement chambers In all cases, the operating pressure of these confinement chambers is one atmosphere (or less) and thus these chambers are unsuitable for processes in which increased dissolved oxygen levels are desired and are necessarily limited to those dissolved oxygen levels obtainable at atmospheric pressure.
  • the present invention comprises a novel culture process in which cells or subcellular biocatalysts are confined within a chamber which is capable of being pressurized.
  • the cells are immersed in a nutrient medium with no gas phase in contact with the medium.
  • the chamber has an input port and an exit port through which the nutrient medium is circulated.
  • the exit port in the chamber is preferably blocked by a porous, liquid-permeable structure of defined pore size.
  • the pores of this structure are smaller than the physical dimensions of the cells or catalysts, yet are large enough to allow free passage of liquids and dissolved nutrient and product molecules.
  • the cells or biocatalysts are completely confined within this chamber with no gas phase above the medium.
  • the present invention can be used to produce high yields of industrial chemicals or pharmaceutical products from biocatalysts such as bacteria, yeasts, fungi, and eukaryotic cells or from subcellular organelles, such as mitochondria. These cells can be either naturally occurring or can be genetically manipulated to produce the desired product.
  • biocatalysts such as bacteria, yeasts, fungi, and eukaryotic cells or from subcellular organelles, such as mitochondria.
  • these cells can be either naturally occurring or can be genetically manipulated to produce the desired product
  • the present invention can be used to remove or destroy harmful toxic products via bioremediation in which the toxic chemical is converted into an environmentally benign product
  • biocatalysts are immobilized within a containment chamber under hyperbaric conditions while nutrient liquids are fed into the chamber and effluent liquids containing desired metabolic product(s) exit the chamber.
  • biocatalysts are immobilized within a containment chamber under hyperbaric conditions while media with toxic chemicals are fed into the chamber and the biocatalysts in the chamber neutralize the toxic chemicals thereby converting them into an environmentally benign product
  • biocatalysts including living cell populations, may be immobilized under hyperbaric conditions and either aerobic or anaerobic fermentations performed in which liquid nutrient and substrate nutrients are converted to product-containing output liquid streams.
  • Another object of the present invention is to provide a method and apparatus by which dissolved oxygen concentrations (or other dissolved gases) in the nutrient Hquid flow directed into a cell confinement chamber may be raised to any desired level, depending on die applied hydrostatic pressure.
  • Another object of the present invention is to provide a method and apparatus by .which either a nutrient gaseous substrate (such as oxygen) in the nutrient input liquid flow directed into a cell confinement chamber or an excreted respiratory gas (such as, for example, carbon dioxide) in the output liquid flow may be maintained in the dissolved state until liquid-gas disengagement is desired, generally far downstream of the cell immobihzation compartments).
  • a nutrient gaseous substrate such as oxygen
  • an excreted respiratory gas such as, for example, carbon dioxide
  • Yet another object of the present invention is to provide a method and apparatus by which the conversion of an available chemical substrate into a desired product may be effected by a series of stepwise biocatalyst-mediated conversions in which each chemical conversion step is effected by one of a series of hyperbaric immobilization chambers inserted serially or in parallel into the flow stream.
  • Mother object of the present invention is to provide a non ⁇ specific, general method and apparatus for cell culture or fermentation which can be applied to any cell type without significant variation.
  • Another object of the present invention is to provide a method and apparatus for cell culture or fermentation which significantly reduces both the capital and labor costs of production and production facilities.
  • Another object of the present invention is to provide a method and apparatus for cell culture or fermentation which is much less susceptible to contamination by opportunistic organisms.
  • other object of the present invention is to provide a method and apparams for cell culture or fermentation in which the liquid environment bathing the desired biocatalyst is essentially invariant in time, i.e., the pH, ionic strength, nutrient concentrations, waste concentrations, or temperature do not vary as a function of time in the biocatalyst's environment
  • Mother object of the present invention is to provide a continuous fermentative or cell culture method.
  • Mother object of the present invention is to provide a method and apparatus for culturing biocatalysts under hyperbaric conditions which thereby significantly increases the yield of products from the biocatalyst.
  • Mother object of the present invention is to provide a method and apparams for cell culture or fermentation which increases the conversion efficiency (of substrate to product) of the culture process.
  • Another object of the present invention is to provide a method and apparatus for cell culture or fermentation which significantly reduces the volume of water required to support the culture process.
  • Another object of the present invention is to provide a method and apparams for cell culture or fermentation which significantly reduces the cost of heating or cooling the aqueous media required to support the culture process.
  • Mother object of the present invention is to provide a method and apparams for cell culture or fermentation which results in higher yields of products such as antibiotics from bacterial fermentations.
  • Mother object of the present invention is to provide a method and apparams for cell culture or fermentation which results in higher yields of products such as enzymes or other proteins from fungal fermentations.
  • Mother object of the present invention is to provide a method and apparams for cell culture or fermentation which results in higher yields of products such as ethanol or other short-chain alcohols and acids from the fermentation of microorganisms.
  • Mother object of the present invention is to provide a method and apparams for cell culture or fermentation which results in higher yields of products such as protein hormones from genetically-transformed microorganisms.
  • Another object of the present invention is to provide a method and apparams for cell culture or fermentation which results in higher yields of products such as protein hormones from eukaryotic cells.
  • Mother object of the present invention is to provide a method and apparams for cell culture or fermentation which results in higher yields of products such as amino acids, nitrogenous bases, or alkaloids from the fermentation of microorganisms.
  • Mother object of the present invention is to provide a method and apparams for cell culture or fermentation which results in higher yields of products such as fuel-grade ethanol from the fermentation by yeasts of sugar- containing agricdtural material.
  • Mother object of the present invention is to provide a method and apparams which would reduce the fermentation time required to produce alcoholic beverages such as beer and wine.
  • Mother object of the present invention is to provide an easily scaled-up method and apparams for cell culture or fermentation which can be commercially employed.
  • Fig. 1 is an illustration in block form the basic hyperbaric process of the present invention.
  • Fig. 2 is an illustration in block form one embodiment of the basic aerobic hyperbaric process.
  • Fig. 3 is an illustration in pictorial form an embodiment of the basic aerobic hyperbaric process.
  • Fig. 4 is an illustration one embodiment of the hyperbaric confinement chamber.
  • Fig 5. is a view of the inside of the two halves of the hyperbaric confinement chamber shown in Fig.4.
  • Fig. 6 is an illustration of an alternative embodiment of the hyperbaric confinement chamber employing hollow-fiber technology.
  • Fig.7 illustrates in block form an alternative embodiment of the hyperbaric method which includes auxiliary control and sensing mechanisms as well as two serially-connected hyperbaric confinement chambers.
  • Fig. 8 is a representation of the data obtained in the experiments of Example I.
  • Fig. 9 is an illustration of one embodiment of the hyperbaric confinement chamber.
  • the present invention comprises a novel culture process comprising cells or subcellular biocatalysts which are confined within a chamber which is capable of being pressurized. According to the present invention, there is no gas phase in contact with the cells or biocatalysts.
  • the exit port of the chamber is blocked by a porous, liquid-permeable structure of defined pore size.
  • the input port may also be blocked by a porous, liquid-permeable structure of defined pore size.
  • the pores of this structure are smaller than the physical dimensions of the cells or catalysts, yet are large enough to allow free passage of liquids and dissolved nutrient and product molecules.
  • the cells or catalysts are completely confined within the pressurized chamber while liquids may be passed into and out of the chamber.
  • high pressure pumps are employed to force the nutrient liquid to flow through the chamber.
  • the confined cells or biocatalysts are unaffected by the resultant increase in hydrauhc pressure as long as high frequency pressure fluctuations are not present
  • fresh liquid medium with optimal nutrients is presented to the confined cells or biocatalysts at all times during the process flow while the desired cell products are immediately accessible at the output of the confinement chamber.
  • the fermentation and culture apparams of the present invention is significantly simpler and less expensive than that of the prior art
  • the ability to confine cells or other biocatalysts to a chamber in which virtually all of the available space is occupied by the cells or biocatalysts results in a considerable economic advantage.
  • the volumes of hquid required are reduced to as little as 1% of the water required in prior art fermentation apparati.
  • the overall cost of the fermentation process machinery is greatly reduced because the process is essentially a pumped liquid line with the biocatalyst chamber interposed in the flow stream.
  • the production costs associated with the normal heating and cooling of the large volumes of water used in typical fermentation processes (often 50,000 gallons or more) are not required in the process and apparams according to the present invention. All that is necessary in the method of the present invention is to maintain temperature control of the immobilization chamber itself .
  • the process system is a closed liquid flow line under pressure, there is no place in the process stream where a contaminant could be introduced.
  • the process system can be easily sterilized by conventional means and thus the only source of contamination is the input hquid reservoir.
  • the input liquid reservoir can be sterilized by conventional means. Such a reduction in the possible sources of contamination conveys a large economic advantage.
  • the process of the present invention reduces the costs associated with the maintenance of system variables such as pH, nutrient concentration, temperature control, and the buildup of waste products in the environment of the working biocatalysts.
  • system variables such as pH, nutrient concentration, temperature control, and the buildup of waste products in the environment of the working biocatalysts.
  • the confined biocatalysts "see" only an optimal hquid environment introduced via the input pump.
  • the working cells quite often are limited to short batch run times because of the inability to remove waste products which cause changes in the system variables.
  • the method and apparams according to the present invention is a continuous fermentation or culture process. That is, once the process is begun, there is never a need to shut down the process until the practical lifespan of the immobilized microorganism or cell is exceeded.
  • changes in the input nutrient flow composition can be used to trigger alternate cycles of growth or production, thereby reducing the need to shut down the process flow.
  • the process flow can continue for the lifetime of the immobihzed cells.
  • Prior art methods of fermentation are believed to have hfe spans which are considerably shorter than the present invention. It is to be understood that the present invention also includes the
  • the amount of dissolved oxygen (or any other gas) which can be delivered to the cells is a function of the applied pressure.
  • the carbon dioxide (or other excretory gases) liberated by die respiring cells remains in solution under hyperbaric conditions and is removed r om the environment of the immobihzed biocatalysts by the process liquid flow.
  • gases that may be used in accordance with the present invention include, but are not limited to, air, 02, NH3, NO2, He, Ar, N2 and H2 or a mixture thereof.
  • the gases helium, argon and nitrogen are particularly important in anaerobic systems in that these gases are generally inert and do not effect cell function and can be used to replace oxygen in the medium.
  • the selection of the gas to be used in accordance with the present invention will depend upon the particular biocatalyst being used in the chamber.
  • the present invention significantly increases the product yield of microbial fermentations or cell cultures. As noted earlier, because the method is a continuous one, production can continue for the effective lifetime of the immobilized biocatalyst This fact alone accounts for a sizable increase in productivity.
  • an increase of more than 100% in the rate of product formation may be observed when dissolved oxygen levels are raised above normal. This effect can be observed even when there is no apparent direct chemical connection between oxygen availabiHty and product formation. It is believed that this effect is related to basic metabolic rate increases.
  • cell or tissue culture are quite similar.
  • the desired organism or cell is placed in a defined medium composed of water (usually approximately 1000 times the volume of the cells) and nutrient chemicals, and allowed to grow (or multiply) to some culture density.
  • the living cells then produce d e desired product from precursor chemicals introduced into die nutrient mixture.
  • the desired product is then purified from of the hquid medium.
  • the present invention is a novel cell immobilization meti od which is named for me purposes herein "hyperbaric fermentation".
  • me process according to die present invention typically cells are confined in a chamber which is capable of being pressurized.
  • the chamber with the cells is gradually pressurized d ereby raising the hydrostatic pressure witiiin the cell bed until an optimal pressure level is reached. Because hydrostatic pressures are rapidly transmitted tiirough cell membranes, die internal cell pressure equals the externally-applied pressure, and die cells "see" no pressure gradient across tiieir boundary membranes.
  • the cell confinement chamber possesses an exit port which is blocked by a porous, liquid-permeable structure of defined pore size.
  • the porous, hquid-permeable structure of defined pore size is called the exit frit, although it is to be understood that in some embodiments it incorporates a membrane above a support grid.
  • the pores of this structure are necessarily smaller than the dimensions of the confined cells, yet are large enough to allow free passage of liquids and dissolved nutrient and product molecules.
  • the cell chamber is placed under a positive hydrauhc pressure of 10 to 20,000 psi or more.
  • the preferred hydraulic pressure is between 100 to 5000 psi with die most preferred hydrauhc pressure of approximately 1500 psi.
  • Nutrient liquids are pumped tiirough the chamber at constant flow rates, and products are immediately accessible at the output of me cell chamber. It is also contemplated as part of die present invention tiiat die flow rates of liquid medium can vary according to die needs of die particular biocatalysts.
  • the hyperbaric fermentation process of the present invention begins witii the supply of a cell culture medium from a medium resevoir to the input of a high pressure pump as illustrated by die flow chart in Fig. 1.
  • the media reservoir is maintained at ambient room pressure although it can be heated to any desired temperature.
  • this high pressure pump is a high pressure hquid chromatography (HPLC) pump capable of delivering constant flow rates of 0.1 to 10 mL min of aqueous media at pressures from approximately 100 to 5000 psi.
  • HPLC high pressure hquid chromatography
  • Otiier embodiments of the present invention may employ different high pressure pumps with different pressure and flow rate characteristics. The type of pump used is not critical to the present invention.
  • the liquid flow is next optionally passed tiirough a pulse dampening device similar to those found in many high pressure pumping systems to minimize pressure fluctuations downstream. Then the hquid flow is passed tiirough a flow-tiirough pressure sensor.
  • a pulse dampening device similar to those found in many high pressure pumping systems to minimize pressure fluctuations downstream.
  • the hquid flow is passed tiirough a flow-tiirough pressure sensor.
  • air may be excluded from the media reservoir by normal methods and the maintenance of anaerobic conditions throughout die process is easily accomplished because the pressure- resistant connecting piping precludes the entrance of oxygen into the process flow at any point downstream of die media reservoir.
  • a two-position high pressure switching valve 60 (Rheodyne, Inc., Cotati, CA) This valve is suitable for flow rates of 0 to 10 mL/min.
  • the hquid flow applied at port number 1 can be switched internally to connect to either ports number 2 or 6.
  • port number 1 is connected to port number. 6 (dotted path)
  • me hquid flow padi leads from port number 6 to port number 5 via a stainless steel bypass loop.
  • port number 5 is in internal communication witii port number 4.
  • the hquid flow passes from port number 1 to port number 4, the final output port.
  • a ball valve attached to port number 4 is normally closed and is only open during cell chamber pressurization. The hquid flow from port number 4 is directed into the system pressure regulator (see Fig. 1).
  • the system pressure regulator 12 is, in one embodiment, a flow controller such as tiiose available from Varian Associates (Sunnyvale, CA), which maintains die hydraulic pressure of the entire liquid system at a preset value.
  • the whole system may be maintained at pressures of approximately 0 to 5000 psi, aldiough higher pressures can be maintained.
  • gas remains dissolved in die liquid until die pressure drops to ambient values after exit of the fluid from the system pressure regulator.
  • the process flow is strictly a liquid flow. There is no gas phase in the system.
  • tiiat thermostatting of die system pressure regulator may be required as a result of the endodiermic nature of the liquid-gas disengagement process.
  • the cell chamber can then be opened and loaded witii die desired cells or biocatalysts.
  • the cell chamber 100 can be gradually pressurized by opening the ball valve connected to port number 4 (Fig. 1).
  • die pressure equalizing valve By gradually opening die pressure equalizing valve, pressure is allowed to build on botii the input and output sides of die cell chamber.
  • the pressure inside die loaded cell chamber can be monitored by d e sensor attached to d e chamber output. The chamber is thereby brought to a pressure equaling the system pressure.
  • the cell chamber 100 can be gradually brought from ambient pressure to the desired system pressure without applying a large pressure pulse to the cell compartment and its contents.
  • the ball valve connected to port number 4 is closed, tiius isolating tiie cell chamber.
  • the switching valve position in which the flow is directed to bypass die cell chamber, as oudined above, is employed when loading and pressurizing the confinement chamber and is also employed when calibrating the system as to gas and hquid composition, flow rate, etc. prior to actual use (see Example 1).
  • switch port number 1 In the case when switch port number 1 is internally connected to port number 2 (as shown in Fig. 1 witii solid lines), the liquid flow patii leads from port number 2 to port number 3 via a loop in which are mounted die cell confinement chamber and a pressure sensor. Normally, the ball valve connected to port number 4 is closed, allowing hquid flow only tiirough the ceU confinement chamber 100. In this switching valve position, port number 3 is in internal communication with port number 4. Thus, the hquid flow passes from port number 1 through the cell chamber loop and exits tiirough ports number 3 and 4. This is the normal operating flow path in which the hquid nutrient medium is directed to flow tiirough d e cell confinement chamber 100.
  • the embodiment of the present invention takes a different form (see Figs. 2 and 3).
  • a desired gas or gases typically oxygen or air
  • a volume of die aqueous media is pumped from the nutrient feed pump 10 into an absorption reservoir 20 wherein the gas and hquid medium are mixed and die gas is dissolved in the hquid medium.
  • a source of gas is also apphed to the absorption reservoir 20 through a line equipped witii a pressure regulator 23 and a check valve 17.
  • die concentration of gas which is dissolvable in the hquid witii which the gas comes in contact is directly related to die pressure of the gas-liquid system, it is possible to establish a desired dissolved gas concentration in the nutrient hquid simply by varying the gas pressure.
  • the driving force for the entrance of the applied gas(es) into the aqueous hquid flow is the pressure differential between the hquid and gas streams. Dissolving the gas in d e hquid medium is accomplished by adjusting the gas pressure to values greater tiian that of the liquid pressure.
  • the concentration of dissolved gas(es) in the net hquid flow is a function of: (1) the pressure differential between the liquid and gas flows; (2) die flow rates of the gas(es) and hquids; and (3) die kinetics of d e gas dissolution process.
  • a dissolved oxygen concentration of 11.3 mM may be achieved in an aqueous solution at 30°C by the application of approximately 200 psi of oxygen gas pressure, whereas the apphcation of approximately 1550 psi of oxygen gas pressure to a slightly different aqueous solution at 25°C results in a dissolved oxygen concentration of approximately 250 mM.
  • an absorber recirculation pump 30 (high pressure pump number 2 in Fig. 2) is used to recirculate the gas-hquid mixture from the bottom of the absorption reservoir 20 to the top.
  • both of these high pressure pumps are internally equipped witii output check valves to be sure the hquid only flows in the direction indicated by die arrows.
  • the absorption reservoir 20 in one embodiment of the present invention is a stainless steel container whose volume is a function of the desired flow rates of gas and hquid.
  • the absorption reservoir volume 20 must be adjusted such tiiat die physically-dictated time required for dissolution of the gas into a liquid state can be accomphshed. In die case of liquid flows of less tiian
  • this volume can be as little as lOOmL; in the case of larger flows a gas absorption reservoir of 1 to 2 L or more may be required.
  • the aqueous nutrient media feed circuit and die absorption recirculation pump circuit can be separated from the rest of the process by means of the isolation valve 40 connected to the input of the cell chamber feed pump 50 (high pressure pump number 3 in Fig. 2).
  • the isolation valve 40 connected to the input of the cell chamber feed pump 50 (high pressure pump number 3 in Fig. 2).
  • the fluid circulating in the recirculation pump circuit is composed of both liquid containing the desired concentration of dissolved gas, as well as excess undissolved gas.
  • the balance of the aerobic high pressure process depicted in Figs. 2 or 3 downstream of the gas-liquid mixing circuits is identical to the basic process stream of Fig. 1, and is operated in an identical fashion. Note that because it is necessary to establish a system hydrauhc pressure greater than that in the gas-hquid mixing circuits prior to opening the isolation valve which connects these circuits to the cell chamber feed circuit die apparatus is equipped with a priming line input 70 to which an aqueous hquid reservoir is apphed at system startup. This is used to establish a suitable system hydraulic pressure by adjustment of the system pressure regulator. As depicted in Fig. 3, the overall process system may be optionally equipped widi pressure gauges and/or microprocessor control loops monitoring flow indicating controllers (FIC) as well as pressure indicators (PI) and the like common to a person of ordinary skill in the art.
  • FIC flow indicating controller
  • PI pressure indicators
  • M alternative method for the mixing of hquid nutrient media widi a desired gas involves mixing of these two components in a hquid pump headspace.
  • Varian Instruments Model 5020 HPLC which possesses solenoid-operated inlet valves, can be used to accomphsh mixing of hquid media with gas in this manner.
  • die supply gas pressure must be carefully maintained at input pressures greater than the pump backpressure. Gas dissolution into the liquid flow and the mixing of the two liquid components then occurs in the pump head oudet chamber.
  • Another alternative method for the precise mixing of liquid nutrient media with a desired gas can be accomphshed by applying the gas supply to die input side of a high pressure positive displacement pump at pressures close to tiiat required for dissolution of the gas into the pressurized liquid flow and utilizing this high pressure pump to meter die gas flow delivery rate to the gas liquid absorption reservoir. Note that gas delivery by this metiiod eliminates the need for gas flow restrictors and check valves in the process flow system.
  • Figs. 4 and 5 show one embodiment of the cell confinement chamber.
  • the chamber is manufactured of stainless steel which is machined in two halves which are bolted togetiier with six machine bolts.
  • the upper half-chamber 105 is internally machined to provide an input to the chamber interior 110, flow distribution channels 115, an inner O-ring channel 120, and an internal biocatalyst confinement chamber 125 of approximately 3.0 mL volume.
  • the bottom half-chamber 130 is machined to allow die insetting of a support lip for a porous metal frit (the exit frit) 135 as well as an outer O-ring channel 140 and an solvent output channel 145.
  • the cell chamber forms a pressure- resistant vessel 100 with a single input port 110 above the cell chamber and a single exit port 145 beneath the exit frit 135.
  • die metal exit frit 135 widi either bead- immobihzed enzymes or microbial, plant, or animal cells is accomphshed by addition of a hquid or slurry suspension of the beads or cells prior to connection of the upper chamber-half input port 110 to the process flow system.
  • the choice of the porosity of die metal exit frit 135 is dictated by the size of die cell or enzyme, or cell immobilization bead one desires to retain within the compartment
  • a 0.2 micron ( ⁇ M) frit porosity is optimal for tiiose fermentations in which bacterial cells are to be confined witiiin the chamber, while a frit porosity of 5 ⁇ m is adequate for die retention of yeast cells.
  • the porous metal exit frit may be replaced by a honeycombed ceramic frit of defined porosity.
  • the exit frit may be a coarser metal frit which is overlaid widi a filter membrane of defined pore size (Micro Filtration Systems, Dublin, CA).
  • tiiat die geometry of the cell chamber is not critical except for its ability to withstand die apphed internal pressure.
  • CeU chambers of cylindrical, spherical, or otiier geometrical configurations may replace the embodiment specified in Figs.4 and 5.
  • Another embodiment of die present invention utilizes porous fiber bundles to deliver nutrients to the biocatalysts in die pressurized chamber.
  • the chamber 200 is manufactured of stainless steel.
  • the chamber has a high pressure fiber input end cap 205 into which the nutrient medium is fed under high pressure and a fiber output end cap 220.
  • the chamber 200 also has an inner fiber capillary bundle 210 in the cell culture compartment 215 as shown in the cutaway view of the chamber.
  • the inner fiber bundle 210 such as those available from .Miicon Inc., Beverly, MA, is connected to die input end cap 205 and output cap 220 so that the flow of nutrient medium flows through the interior of the fibers in the fiber capillary bundle 210.
  • Medium is circulated into the input flow cap 225 through the culture compartment 215 and out the output flow cap 230. In this way, the inner fiber bundle 210 is bathed by the medium that is circulated in the compartment 215.
  • the appropriate cells suspended in media are loaded into the culture compartment 215 via media input flow port 225.
  • Fresh media is circulated tiirough the fibers 210 under hyperbaric pressure. This allows the cells access to nutrients which diffuse through the fiber walls.
  • the cell medium is injected tiirough media input flow port 225 and circulated through the culture compartment 215 and out through cell media output flow port 230. Products can also be collected in the medium that is collected tiirough the cell media output flow port 230.
  • N Note that in the cell chamber embodiment of Fig. 6, a high pressure flow of liquid tiirough and around die fragile hollow fiber capillaries will not cause fiber rupture as long as the pressure differential across the fiber wall is held near zero.
  • the biocatalyst cell chamber may take the form of an elongated cylinder in which tubular frit "fingers" of the appropriate porosity are mounted in an axial configuration within die columnar body of die chamber to increase die available exit frit surface area without appreciably increasing the physical size of the biocatalyst cell chamber.
  • tubular frit "fingers" of the appropriate porosity are mounted in an axial configuration within die columnar body of die chamber to increase die available exit frit surface area without appreciably increasing the physical size of the biocatalyst cell chamber.
  • a chamber size of 1 to 10 mL is sufficient for larger scale commercial apphcations, chamber sizes of 1 to 10 L or more may be required, witii a similar scale-up of pump capacity to assure adequate perfusion of the chamber contents.
  • a disc-shaped cell bed is a preferred configuration in which the problems of concentration gradients, pH changes, and anoxic pockets within the cell bed can be eliminated with the appropriate choice of flow rate and disc thickness.
  • the present invention comprises optional features including, but not limited to, sensing electrodes for dissolved nutrients and gases, sampling ports in various configurations, in-line gas and/or air filtering apparati, and die like features common to microbial fermentations and/or cell culture.
  • the cell confinement chambers can optionally be thermostatted to provide optimal cell growth or productivity conditions, can be serially connected into a common flow process configuration to provide for the step-wise conversion of a substrate material into a final desired product by several different microorganisms or cell types.
  • various outputs can be entrained with analytical instruments such as HPLC separation columns, spectrophotometers, etc.
  • the general sequence of operations in the preferred embodiment of me present invention in the case of an aerobic fermentation is as follows: (1) the system is configured for aerobic operation by making the appropriate connections between the gas and hquid supply systems (see Fig. 2 and 3); (2) d e gas-liquid mixing circuits are disengaged from the cell chamber feed circuit by closing die isolation valve.
  • the cell chamber system priming valve is opened and a sterile aqueous reservoir apphed at this input; (4) die high pressure switching valve is turned to die confinement chamber bypass position, the cell chamber feed pump is started at die desired flow rate, and die desired system pressure set by the adjustment of die system pressure regulator, (5) the appropriate nutrient media for the organism to be cultured is prepared, sterilized, and apphed at die low pressure inlet to the nutrient feed pump and approximately 100 mL of nutrient is pumped into die absorption reservoir whereupon this pump is stopped; (6) the gas supply is opened and set at the desired operating pressure and the recirculation pump is flowed at an appropriate rate for approximately 30 minutes; (7) the cell chamber system priming valve is closed, die isolation valve is opened, die nutrient feed pump is started at the same flow rate as the cell chamber feed pump, and die flow of hquid is calibrated as to system pressure, system temperature, hquid flow rate, and die concentration of liquids and gas(es), pH, etc. in the output
  • die ball valve connected between switch port number 1 and the pressure equalizing valve (Fig. 3) is opened so tiiat the cell chamber is pressurized to the process system pressure; (10) a ball valve is closed and die switching valve is turned to direct die hquid flow tiirough the cell chamber loop.
  • Fig. 9 Mother embodiment of the present invention is shown in Fig. 9.
  • medium is introduced from eitiier end of the chamber at 320 or 325. How into and out of the chamber can be easily reversed.
  • the biocatalyst can be introduced via die inoculation port at 305 and die medium is introduced under pressure from ether end of die chamber.
  • the walls of mbes 320 are manufacture from either metallic or ceramic frit material which is permeable to the hquid medium but is impermeable to the biocatalysts. If cells are the biocatalyst a pore size of approximately 0.2 ⁇ is adequate.
  • the mbes 320 and 325 are shown in Fig. 9 as straight mbes but they can be any shape in any configuration.
  • the chamber 300 has a blind flange 330 and a screw-on flange 335 at both ends of die chamber.
  • the medium passes through the walls of the mbes 320 and batiies the biocatalyst. the medium then passes through the walls of the exit tube and is removed from the chamber.
  • the flow can be easily reversed if the exit tube becomes obstructed witii cells or other debris. By reversing the flow, the exit tube becomes die inlet tube and die debris is forced away from die tube.
  • System shut down (when it is desired tiiat the confined cells be recoverable) requires that first the gas supply is closed; die absorption reservoir relief valve 15 (Fig. 3) is opened to release any gaseous overpressure; and, d e system is allowed to flow until no gas is present in the output liquid, an indication that the only dissolved gases remaining in the system are at atmospheric pressure. Whereupon the remainder of d e system can be shut down by a reversal of die startup procedure. System sterilization between runs 5 may be accomplished by either passage of steam or a sterilizing solution through the process system.
  • the microbial organisms which may be used in the present invention include, but are not limited to, dried cells, wet cells harvested from broth by centrifugation or filtration, or from the cultured broth itself.
  • 10 microbial cells are classified into die following groups: bacteria, actinomycetes, fungi, yeast, and algae.
  • Bacteria of the first group belonging to Class Shizomycetes taxonomically, are Genera Pseudomonas, Acetobacter, Gluconobacter, Bacillus, Corynebacterium, Lactobacillus, Leuconostoc, Streptococcus, Clostridium, Brevibacterium, Arthrobacter, or Erwinia, etc.
  • Actinomycetes of the second group belonging to Class Shizomycetes taxonomically, are Genera Streptomyces, Nocardia, or Mycobacterium, etc. (see R.E. Buchran and N.E. Gibbons, Bergev's Manual of Determinative Bacteriology. 8th ed., (1974),
  • Yeasts of the fourth group belonging to Class Ascomycetes taxonomically, are Genera Saccharomyces, Zygosaccharomyces, Pichia, Hansenula, Candida, Torulopsis, Rhodotorula, Kloechera, etc. (see J. Lodder, The Yeasts: A Taxonomic Study. 2nd ed., (1970), North-Holland).
  • Algae of the fifth group i- include green algae belonging to Genera Chlorella and Scedesmus and blue- green algae belonging to Genus Spirulina (see H. Ta iya, Studies on Mieroalgae and Photosvnthetic Bacteria. (1963) Univ. Tokyo Press). It is to be understood that the foregoing listing of microorganisms is meant to be merely representative of the types of microorganisms that can be used in the
  • the culture process of the present invention is also adaptable to eukaryotic plant or animal cells which can be grown either in monolayers or in suspension culture.
  • the ceh types include, but are not limited to, primary and secondary cell cultures, and diploid or heteroploid cell lines. Otiier cells which can be employed for the purpose of virus propagation and harvest are also suitable. CeUs such as hybridomas, neoplastic cells, and transformed and untransformed cell lines are also suitable. Primary cultures taken from embryonic, adult, or tumorous tissues, as well as cells of established cell lines can be employed. Examples of typical such cells include, but are not limited to, primary rhesus monkey kidney cells (MK-2), baby hamster kidney cells
  • BHK21 pig kidney cells
  • IBRS2 pig kidney cells
  • RAG mouse renal adenocarcinoma cells
  • MPC-11 mouse medullary tumor cells
  • FS-4 or AG 1523 human diploid fibroblast cells
  • SK-HEP-1 human hver adenocarcinoma cells
  • HEL 299 normal human lymphocytic cells
  • WI 38 or WI 26 human embryonic lung fibroblasts HEP No.
  • the products tiiat can be obtained by practicing the present invention are any metabolic product that is the result of the culturin of a cell, either eukaryotic or prokaryotic; a ceh subcellular organelle or component, such as mitochondria, nuclei, lysozomes, endoplasmic reticulum, golgi bodies, peroxisomes, or plasma membranes or combinations thereof; or an enzyme complex, either a natural complex or a synthetic complex, i.e., a plurality of enzymes complexed togetiier to obtain a desired product
  • a natural complex or a synthetic complex i.e., a plurality of enzymes complexed togetiier to obtain a desired product
  • a mammalian cell that is known to produce a desired chemical can be directly cultured according to die present invention to produce large quantities of the desired chemical
  • Products tiiat can be produced according the present invention include, but are not limited to, immunomodulators, such as interferons, interleukins, growth factors, such as erythropoietin; monoclonal antibodies; antibiotics from microorganisms; coagulation proteins, such as Factor VHI; fibrinolytic proteins, such as tissue plasminogen activator and plasminogen activator inhibitors; angiogenic proteins; and hormones, such as growth hormone, prolactin, glucagon, and insulin.
  • immunomodulators such as interferons, interleukins, growth factors, such as erythropoietin
  • monoclonal antibodies antibiotics from microorganisms
  • coagulation proteins such as Factor VHI
  • fibrinolytic proteins such as tissue plasminogen activator and plasminogen activator inhibitors
  • culture medium includes any medium for die optimal growth of microbial, plant or animal cells or any medium for enzyme reactions including, but not limited to, enzyme substrates, cof actors, buffers, and die like necessary for the optimal reaction of the enzyme or enzyme system of choice.
  • Suitable culmre media for cell growth will contain assimilable sources of nitrogen, carbon, and inorganic salts, and may also contain buffers, indicators, or antibiotics. Any culture medium known to be optimal for the culture of microorganisms, cells, or biocatalysts may be used in the present invention.
  • While such media are generally aqueous in nature for the culture of living organisms, organic solvents or miscible combinations of water and organic solvents, such as dimethylformamide, methanol, dietiiyl ether and die like, may be employed in those processes for which they are proved efficacious, such as those bioconversions in which immobihzed biocatalysts are employed.
  • Passage of the liquid media through the process system may be either one-pass or the liquid flow may be recycled tiirough the system for higher efficiency of conversion of substrate to product Desired nutrients and stimulatory chemicals may be introduced into die process flow, eitiier via the low pressure nutrient supply or via an injection valve in the process flow upstream of the cell chamber.
  • tissue culture media including, but not limited to, Basal Medium Eagle's (BME), Eagle's Minimum Essential Medium (MEM),
  • DMEM Dulbecco's Modified Eagle's Medium
  • RPMI 1640 Roswell Park Medium
  • Medium 199 Ham's F-10
  • Iscove's Modified Dulbecco Medium phosphate buffered salts medium (PBS), and Earle's or Hank's Balanced Salt Solution (BSS) fortified with various nutrients.
  • PBS phosphate buffered salts medium
  • BSS Hank's Balanced Salt Solution
  • inoculation of the ceh chamber with a small starter population of ceUs can be followed by an aerobic fermentation regime in which glucose depletion, dissolved oxygen depletion, and carbon dioxide production across the cell confinement chamber are measured either chemically or via appropriate sensing electrodes.
  • cell replication can be allowed to proceed until an optimal cell bed size is reached. Withdrawal of dissolved oxygen input at this time causes the immobilized yeast cells to shift into anaerobic fermentation of glucose with a resultant production of ethanol, a process which can likewise be monitored chemically (see Example IH).
  • the process of the present invention can be utilized as a bioreactor for immobihzed chemical catalysts, enzymes or enzyme systems.
  • a catalyst, an enzyme or an enzyme system is chemically immobilized on a sohd support including, but not hmited to, diatomaceous earth, silica, alumina, ceramic beads, charcoal, or polymeric or glass beads which are then introduced into the cell chamber into which has been mounted an exit frit of a pore size larger than the solid support.
  • the reaction medium either aqueous, organic, or mixed aqueous and organic solvents, flows tiirough the process system and tiirough the packed bed within die cell chamber.
  • the catalyst, enzyme, or enzyme system converts a reactant in the process flow medium into die desired product or products.
  • cells or ceh components including, but not limited to, vectors, plasmids, or nucleic acid sequences
  • RNA or DNA may be immobilized on a sohd support matrix and confined under hyperbaric conditions for similar utilization in converting an introduced reactant into a desired product
  • kits for treating and/or diseases including, but not limited to, anti-tumor factors, hormones, therapeutic enzymes, viral antigens, antibiotics and interferons.
  • product molecules which might be advantageously prepared using the method of the present invention include, but are not limited to, bovine growth hormone, prolactin, and human growtii hormone from pituitary ceUs, plasminogen activator from kidney ceUs, hepatitis-A antigen from cultured hver cells, viral vaccines and antibodies from hybridoma cells, insulin, angiogenisis factors, fibronectin, HCG, lymphokines, IgG, etc. Otiier products will be apparent to a person of ordinary skill in the art.
  • Example I To demonstrate me capability of the hyperbaric culture process to markedly increase die concentration of dissolved oxygen in a typical aerobic microbial ceU culture medium, an apparams was set up which comprised all d e portions of the present invention iUustrated in Figs. 2 or 3.
  • the nutrient media was composed of: yeast extract (6 gm/L), peptone (6 gm. ), glucose (10 gm L), ethanol (4 % [v/v]), and acetic acid (2 % [v/v]).
  • the ceU chamber feed pump circuit was isolated from the gas mixing circuits by closing die isolation valve connecting this circuit to the gas mixing circuits and opening the cell chamber priming line valve to which a media reservoir was attached. The switching valve in the ceU chamber circuit was placed in die position in which the cell chamber would be bypassed, since no ceU inoculation was to be performed.
  • the cell chamber feed pump was started at a flow rate of 1.0 mL/min and a hydrauhc system pressure of 2000 psi was set for the liquid media flow. A system temperature of 25°C was established. A 200 mL stainless steel cylinder was used as die gas-liquid mixing/absorption chamber.
  • the nutrient feed pump also equipped with a media reservoir, was started after opening the absorption reservoir relief valve (Fig. 3) and 100 mL of media was pumped into the absorption reservoir at 10 mL/min, whereupon the nutrient pump was stopped and die relief valve closed.
  • a standard bottle of oxygen gas obtained commerciaUy at a tank pressure of 2200 psi, was equipped with a tank regulator set for 1550 psi, and die gas line connected to the absorption reservoir via a check valve (17 in Fig. 3).
  • the recirculation pump was started at a flow rate of 15 mL min and absorption of gas by the recirculated media aUowed to proceed for 30 minutes.
  • the ceU chamber priming line valve was closed and die adjacent circuit isolation valve opened to connect die gas absorption circuits to the chamber feed circuit and die nutrient feed pump was restarted at a flow rate of 1.0 mL/min.
  • the process was run for 30 minutes to allow steady-state conditions to be established through the process.
  • die output stream was a milky-white fluid composed of both gas and liquid which had begun to disengage in the output tubing distal to die system pressure regulator.
  • Fig. 8 Aliquots of the input and output liquid media were sampled for dissolved oxygen content. The results are presented in Fig. 8.
  • the input nutrient media was found to have a dissolved oxygen content of 0.284 mM, a value which is typical for continuous stirred tank reactors (CSTR). This value is displayed as the leftmost bar of Fig. 8.
  • the concentration of oxygen found in the output stream for this experiment was determined to be approximately 250 mM (rightmost bar, Fig. 8).
  • the two middle bars in Fig. 8 are representative dissolved oxygen values obtained from the literature for typical continuous stirred tank reactors utilizing either one atmosphere (14.7 psi) or two atmospheres of oxygen gas overpressure above hquid fermentation media.
  • the dissolved oxygen content of die ferment employing the method of the present invention in this example was approximately 160 mM, while the dissolved oxygen content for die comparative data (marked wid an asterisk) was in the range of 20-30% (v/v), equivalent to approximately 0.350 mM dissolved oxygen.
  • the fermentation according to the present invention resulted in the production of acetic acid at a rate approximately twice as large as that of the comparative prior art method.
  • an apparams was set up according to Figs.2 or 3.
  • a system temperature of 30°C was set a flow rate of 0.3 ⁇ L/min and a system pressure of 1500 psi was chosen for the hquid media flow.
  • the nutrient media was composed of: yeast extract (3 gm L), peptone (3 gm/L), and glucose (1 % [w v]).
  • a 200 mL stainless steel cylinder was used as the gas-hquid mixing absorption chamber.
  • a standard bottle of oxygen obtained commercially at a tank pressure of 2200 psi, was equipped witii a tank regulator set for 1000 psi.
  • the high pressure switching valve shown in Fig. 2 was set in an intermediate position, the absorption reservoir relief valve opened, and 100 mL of media was pumped into the absorption reservoir at 10 mL min, whereupon die nutrient feed pump was stopped, die absorption reservoir relief valve closed, die gas line opened to pressurize the absorption reservoir, and die switching valve was set in the position in which the ceU chamber would be bypassed.
  • Both the nutrient feed pump and die ceU chamber feed pumps were set at flow rates of 0.3 rcd_/min, while die recirculation pump was set at 15 mL/min.
  • the cell chamber was opened and a 1.0 mL slurry of Saccharomyces cerevisiae (ATCC 4126) suspended in the above medium was loaded into the chamber over a 5 ⁇ M metal exit frit and die chamber closed.
  • the isolation valve between ports number 1 and 2 of the switching valve (see Fig. 3) was opened and die pressure equalizing valve in this circuit gradually opened until the pressure in the cell chamber equaled die system hydraulic pressure, whereupon the shutoff valve was closed.
  • the switching valve was then set into the normal operating position (see Figs.2 or 3). The process was aUowed to run for approximately 15 hours, whereupon the supply of oxygen to the process system was shut off and die absorption reservoir rehef valve opened to remove any oxygen overpressure.
  • Example HI demonstrate several unique aspects of die fermentation process according to d e present invention.
  • First of ah the shift from a proliferative growth phase to an anaerobic production phase was accomplished simply by withdrawing the gaseous oxygen input; there was no additional change in the process flow.
  • the growth phase resulted in a tripling of cell volume in approximately 15 hours.
  • Example IV To demonstrate die suitability of the process of the present invention for use with a more cyclie ceU type, as weU as to demonstrate d e abUity of d e process to increase ceUular production of a clinicaUy-significant protein by the raising of dissolved oxygen levels, two separate experiments were run.
  • an apparams was set up which comprised aU die portions of the present invention Ulustrated in Figs. 2 or 3.
  • a system temperature of 30°C was set and a flow rate of 1.0 mL/min was chosen for the hquid media flow input to the pump circuit containing the ceU chamber.
  • a system pressure of 1000 psi was established.
  • the nutrient media was composed of Dulbecco's modified Eagle's medium (DMEM) with 4.5 gm/L glucose and 4.0 mM L-glutamine added. No fetal bovine serum was added to die DMEM.
  • the sterile media was introduced into the ceU chamber circuit at the ceU chamber system priming input
  • ceUs had been grown in spinner flasks in DMEM supplemented with 10% fetal calf serum until "tumor spheroids" composed of self-aggregated AtT-20 ceUs had formed.
  • M aliquot of 0.4 mL of sedimented spheroids in approximately 2.5 ml of DMEM was introduced into the ceU chamber and the chamber pressurized as noted earlier.
  • the high pressure switching valve was turned to aUow the flow of DMEM from the ceU chamber circuit pump to flow through the chamber. After a single-pass sample was taken at 10 minutes for hGH assay, the system output was redirected to recirculate back into the media input reservoir. The total media volume in the system was 75 mL.
  • Experiment B the same apparams and conditions were employed as in Experiment A, except that after the introduction of approximately 100 mL of DMEM into the gas mixing absorption chamber via the nutrient pump input the oxygen tank regulator was set to 150 psi and d e absorption reservoir thus pressurized to 150 psi with oxygen gas.
  • the recirculation pump was set to flow at 15 mL/min and die media recirculated for approximately 30 minutes until the dissolved oxygen content of d e media stabihzed at 11.3 mM.
  • the media reservoir was connected to the input of the nutrient feed pump, the cell chamber priming line valve closed, and die adjacent isolation valve connecting the gas-hquid mixing absorption circuits to the cell chamber circuit opened.
  • the nutrient feed pump and d e ceU confinement chamber feed pump were allowed to flow at 1.0 mL/min, while the recirculation pump continued to flow at 15 mL min.
  • M aliquot of 0.4 mL of sedimented spheroids in approximately 2.5 ml of DMEM was introduced into the cell confinement chamber and the chamber pressurized as noted earlier.
  • the high pressure switching valve was turned to ahow the flow of DMEM from the ceh chamber circuit pump to flow through the chamber. After a single-pass sample was taken at 10 minutes for hGH assay, the system output was redirected to recirculate back into die media input reservoir. The total media volume in the system was 150 mL.

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Abstract

La présente invention se rapporte à un nouveau procédé de culture selon lequel des cellules ou des biocatalyseurs sous-cellulaires sont confinés dans une chambre pouvant être mise sous pression. Les cellules sont immergées dans un milieu nutritif sans qu'il y ait de phase gazeuse en contact avec le milieu. Les cellules ou biocatalyseurs sous-cellulaires développés selon la présente invention présentent des rendements considérablement accrus en produits cellulaires par rapport à des techniques de culture classiques.
PCT/US1992/006640 1991-08-09 1992-08-07 Appareil et procede de culture et de fermentation WO1993003135A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0814900A1 (fr) * 1995-03-07 1998-01-07 Biomolecular Assays, Inc. Reacteur a cycle de pression
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WO2015131046A3 (fr) * 2014-02-28 2015-11-26 Carnegie Institute Of Washington Bioréacteur à haute pression
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Cited By (9)

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Publication number Priority date Publication date Assignee Title
EP0814900A1 (fr) * 1995-03-07 1998-01-07 Biomolecular Assays, Inc. Reacteur a cycle de pression
EP0814900A4 (fr) * 1995-03-07 1999-06-23 Biomolecular Assays Inc Reacteur a cycle de pression
US6036923A (en) * 1995-03-07 2000-03-14 Bioseq, Inc Pressure cycling reactor and methods of controlling reactions using pressure
EP1098005A3 (fr) * 1995-03-07 2002-03-13 B.B.I. Bioseq, Inc. Reacteur à cycle de pression
US6569672B1 (en) 1995-03-07 2003-05-27 Bbi Bioseq, Inc. Pressure cycling reactor
US6107055A (en) * 1997-07-31 2000-08-22 Roche Diagnostics Gmbh Method and device for carrying out biochemical reactions
US10059035B2 (en) 2005-03-24 2018-08-28 Xyleco, Inc. Fibrous materials and composites
WO2015131046A3 (fr) * 2014-02-28 2015-11-26 Carnegie Institute Of Washington Bioréacteur à haute pression
US10280393B2 (en) 2014-02-28 2019-05-07 Carnegie Institution Of Washington High pressure bioreactor

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