FLUIDIZED BIOREACTOR AND CELL CULTIVATION PROCESS

Abstract: A fluidized bed reactor (100) and reaction process, particularly well-suited for culturing cells, for example, for tissue culture and fermentation processes, which involve the treatment of at least a portion of the fluid exiting the fluidized bed reactor (100) in a side loop (160) in a manner to alter its temperature or composition, e.g., oxygenation, with recirculation of this treated fluid to the reactor as a portion of the fluid causing bed fluidization.

Description: FLUIDIZED BIOREACTOR AND CELL CULTIVATION PROCESS This application is a continuation-in-part of earlier copending Application Serial No. 527,390 filed on August 29, 1983, in the name of Robert C. Dean, Jr. BACKGROUND OF THE INVENTION 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 < foams, and beds of pebbles. Fluid may be taken from the top of the fluidized bed and can be recirculated through a pump to the distribution resistance element or plate. 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. 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. Many processes for cultivating cells, such as fermentation processes, employ aerobic microorganisms and cells (in general "organ¬ isms''). These organisms demand a continuous supply of oxygen to remain viable. Normally, it is desirable to operate these processes at high solids concentrations, i.e., high cell densities, in order to maximize product yield. Unfortunately, in aerobic processes high cell densities exacerbate oxygen mass transfer demands, which, because of the fragile nature of the biocatalyst beads, cannot be met simply by increasing the level of agitation for increased oxygenation in the bioreactor. Also, in order to operate cell cultivation processes in a continuous manner at optimum conditions, means for controlling the reactor environment, including temperature adjusting means and means for supplying nutrients and other desired reactants to the cell culture and for. removing products and by-products (both desirable and undesir¬ able) from the cell culture, must be provided. Control of reactor con¬ ditions in this way must be accomplished without sacrificing the aseptic integrity of the system. Another problem which can be especially acute in continuous cell culture processes utilizing very small biocatalyst beads containing immobilized microorganisms or enzymes is that conventionally designed perforated distribution plates may become plugged by solids or may per mi t back- flow of biocatalyst beads through the openings, for example, during periods of inactivity. In case of plugging, localized blocking is a typical result. Such blocking causes a change in the hydrodynamic conditions of the fluidized bed upseting bed stabilization and necessitating that the reactor be shut down for the purpose of cleaning. O f course, any solution to these problems must take into account the sensitive nature of the biocatalyst beads to physical impact forces and abrasion that might be encountered during operation as well as the sensitive nature of the immobilized bioactive material, especially mammalian cells. In a continuous process, a single charge of biocatalyst beads is expected to have a useful life on the order of six to eighteen months, so long as excessive attrition can be avoided.. SUMMARY OF THE INVENTION It is therefore an object of one aspect of the present invention to provide a reaction method and a fluidized bed reactor therefor having a stabilized flow, which reactor is not prone to clogging during normal use. It is an object of another aspect of the present invention to provide a reaction method and a fluidized bed reactor therefor, partic¬ ularly suited for carrying out processes for cultivating cells. It is a further object of the invention to provide a reaction method and a fluidized bed reactor therefor, in which mini mal recirculation of solids occurs. It is another object of the present invention to provide a method of continuous cell culture which can accommodate the oxygena- tion demands of an aerobic process without damaging the fragile biocatalyst beads or the cells immobilized in them. 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. It is yet another object of the invention to provide a fluidized bed reactor which achieves the foregoing objects, and is simple in construction and operation, relatively inexpensive to manufacture and relatively easy to maintain in long-term, continuous aseptic use. These and other objects of the invention are accomplished by providing 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. In one embodiment, 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. In this embodiment, 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. The preferred form of 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. In another embodiment, 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. As used herein, the term "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). 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. Also as used herein, 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. BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set out with particu¬ larity in the appended claims, but the invention will be understood more fully and clearly from the following detailed description of the invention as set forth in the accompanying drawings, in which: 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; and F igure 7 is a schematic flow sheet of apparatus useful for practicing continuous aerobic cell cultivation in accordance with the invention. DETAILED DESCRIPTION Although this detailed description is in the context of a fluidized bed reactor used in continuous cell culture processes, including fermentation processes, it is to be understood that the apparatus itself is suitable for use in many different types of processes involving fluid and solid contact. While described primarily in the context of liquid/solid contacting it will be appreciated that aspects of the method and the apparatus are also suited for any fluid, i.e., liquid, gases or mixtures thereof. In such fluidized bed processes it is known that a wide variety of forces can be used to generate and stabilize the counterflow of fluid and solids needed to operate a fluidized bed reactor. While the present invention will be described using an embodiment in which a pressurized liquid and the force of gravity play the primary roles in the operation of the fluidized bed, the present invention is not intended to be so limited. Those skilled in the art will readily appreciate other available embodiments employing other physical phenomena. Referring to Figure 1, 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. During operation, 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. As seen in Figure 1, 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. While this embodiment and others are described in connection with a fluidized bed arrangmeent in which an upwardly flowing stream of pressurized liquid suspends a bed of solids against the downward pull of gravity, as will be apparent to those skilled in this art, the inven¬ tion also is applicable to arrangements employing low specific gravity solids, such as biocatalyst beads having a buoyancy which exceeds the gravitational force, where the fluidizing fluid flow is directed downwardly through the bed of buoyant solids. In a manner known in the art, 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. Moreover, in the pre¬ ferred method for operating the present invention in connection with cell culture processes at high concentrations (densities) of biocatalyst beads, i.e., at a void volume of less than about 75% of the reaction zone volume as will be described in more detail hereafter, 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. To these ends, 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. Moreover, 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. Suitable for this purpose are conventional airfoil types such as those in the NASA 6500 series. The impeller of the present invention should also be adapted to run at a slow speed with low power consumption. Quantitatively, 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. For the biocatalyst system described herein, 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. 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. Thus, 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. Even though the reactor of Figure 1 tends to maintain a stable fluidized bed, a small amount of biocatalyst beads nevertheless may unavoidably escape over the top of reaction vessel 14 and be entrained in the recirculation flow through channels 16 and propeller 24. While the design of propeller 24 minimizes attrition of the biocatalyst beads 36, it may be desirable to further minimize the recirculation of beads 36 by providing, as illustrated in Figure 3, a rotary centrifugal separa¬ tor 40 at the upper end of reaction vessel 14. Separator 40 includes a vaneless rotating diffuser 42 and a bead-separating bladed centrifuge 44, both of which are rotatably driven by shaft 18 which extends upwardly from impeller 24 through reaction vessel 14. 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. As noted, if necessary or desired, other arrangements for separating beads from the fluidizing medium also could be employed including those operating by means of magnetic or electrical forces. Also, a cyclonic separator, e.g., Hydroclone, could readily replace the disclosed rotary centrifugal separator. Various arrangements are readily apparent to those skilled in the art. An especially efficient arrangement for conducting cell culture processes, including fermentation processes, and particularly aerobic processes, is illustrated in Figure 4. 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, such as aeration and/or, CO2 extrac¬ tion e.g., in a membrane gas exchanger 130; heat exchange to heat or cool the culture liquid in exchanger 140; filtration at 150 ; or other treatment such as pH control, sterilization (e.g., by filtration, UV irradiation or ozonation) and altering the composition of the recir¬ culating -fluid such as by adding nutrients or other bioactive materials to the recirculating fluid or by removing desirable and/or undesirable m etabolic products therefrom using any of a wide variety of techniques, for example, 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. Generally, 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. By using a side loop to effect treatment of the culture liquid, and thus treatment of the immobilized bioactive material on or in the biocatalyst beads, it is possible to maintain optimum conditions without jeopardizing aseptic operation or damaging the fragile biocatalyst beads or im mobilized bioactive material, and control of the feed rate of fresh nutrient medium and the flow rate of the recirculating culture liquid. 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. In F igure 5, 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. In the broad practice of the method of the present invention, 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. While 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. As shown, 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. As shown, 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. Preferably, the ports are equally spaced around the circumfer¬ ence of the nozzle. For example, a normal 3/4 inch diameter nozzle may have twelve, approximately 1/8 inch diameter, ports equally spaced about its circumference. In operation, 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. In addition to reducing plugging and back-flow of solids, 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. Generally, 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. In this embodiment, 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. Preferably, the beads comprise a fibrous polymer such as collagen in which the microorganisms are entrapped. Generally, 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. 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. Moreover, certain mediums and products, such as those encountered when culturing mammalian cells, tend to be sensitive to the presence of a liquid-gas interface thereby obviating the selection of any device as the oxygenator which depends upon the generation of a large surface area of small bubbles for gas transfer. 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, based on both design sim¬ plicity and performance characteristics, 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. By properly selecting the tube material, it also is possible to effectively remove carbon dioxide from the recirculating liquid simulta¬ neously with its oxygenation in oxygenators 230. Carbon dioxide typi¬ cally is generated by respring cells in reactors 200. Obviously, in the broad practice of the present invention any of a wide variety of nor¬ mally gaseous constituents may be added to, or removed from the re¬ circulating liquid by properly selecting the membrane material with this arrangement and this aspect of the present invention need not be lim¬ ited solely to aerobic processes. In the arrangement illustrated in Figure 5, 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. If one desires to enhance the rate of mass transfer, generally oxygen is used at an elevated pressure, for example, up to about 10 atmosphere absolute pressure or higher. After exiting the oxygenator, 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. Generally, specially selected recirculation pumps are used (such as blade-less centrifugal blood pumps, e.g., a Biomedicus pump, or flexible rotor vane pumps, e.g., Jabsco pumps) which do not da mage cells entrained in the recirculating liquid. Enzyme released into the liquid as a consequence of cellular damage may degrade the desired product or other components of the culture 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). For example, suitable reagents may be introduced into the reactors through lines 271 and 261. By performing such treatments in a recirculating side-stream, it is possible to maintain optimum conditions in reactors 200 under continuous, aseptic operation without injuring the fragile biocatalyst beads or the im mobilized bioactive material. 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. Typi cally, 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. Preferably, 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. For example, while Escherichia coli typically requires about 0.1 m mol O2 per g-cell per minute, 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, particularly bead size and specific gravity, 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. Referring next to Figure 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. During fluidization, the liquid surrounding the beads typically contains a concentration of organisms of about 1/10th that value. At fluidizing conditions (i.e., average bed void volume of about 60%) the average cell concentration in the bioreactor volume is about 40% of the concentration in the beads themselves. In other words, the actual reactor cell concentration (beads and liquor together) is approximately 4.6 X 10 7 cells per ml. As shown in Figure 7, 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. While only a single recirculating stream and associated equipment is illustrated in Figure 7, it should be emphasized that in actual practice parallel circuits generally will be provided to permit easy isolation of such equipment for maintenance and repair without jeopardizing the aseptic integrity of the system. This ease of maintaining aseptic operation is a particularly preferred feature of the present invention. 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. As noted, 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. In this arrangement, 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. For exam¬ ple, 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. In addition to products comprising primary and secondary metabolites of the cell culture, the product may also comprise the cel¬ lular material or biomass itself. For example, genetically engineered __• col* with an rDNA product that is not expressed, e.g., insulin, could be cultivated in and recovered from reactor 300. With 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. In the practice of the present invention, 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. The following examples are intended to more fully illustrate the invention without acting as a limitation on its scope. Example 1 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. Example 2 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. In order to oxygenate the bioreactor, 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 ( α -HCG). A batch reactor operated under similar condi¬ tions produced product at a rate about 20 times lower. Example 3 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. It will be obvious to one of ordinary skill that numerous modifications may be made without departing from the true spirit and scope of the invention which is to be limited only by the appended claims.

Claims: CLAIMS 1. A method for continuously culturing cells comprising the steps of: (a) fluidizing a bed of biocatalyst beads containing immobilized bioactive material with a liquid nutrient medium in a reaction zone, (b) separating said beads from liquid nutrient medium exiting one end of said reaction zone so that said beads remain in said reaction zone; (c) treating a portion of the separated liquid nutrient medium in a treatment zone, separate from the reaction zone, so as to alter the temperature or composition of the separated liquid; (d) recirculating said treated portion of the separated liquid nutrient medium back to said reaction zone as at least part of said liquid nutrient medium for fluidizing said bed of biocatalyst beads; and (e) recovering another portion of said separated liquid nutrient medium as product. 2. The method of claim 1 wherein said beads are separated from said liquid nutrient medium using a force selected from the group consisting of gravity, centrifugal, electrical and magnetic. 3. The method of claim 2 wherein said beads are separated from said liquid nutrient medium solely by gravity. 4. The method of claim 2 wherein said separating step occurs in a rotary centrifugal separator. 5. The method of claim 3 wherein said beads are porous and are prepared from materials selected from the group consisting of nat¬ ural polymers, synthetic polymers, minerals and metals. 6. The method of claim 5 wherein said beads are prepared from materials selected from the group consisting of natural polymers and synthetic polymers and said beads are weighted with an inert material to increase their specific gravity. 7. The method of claim 6 wherein said biocatalyst beads are polysaccharide gel beads. .8. The method of claim 6 wherein said biocatalyst beads are collagen sponge beads. 9. The method of claim 1 wherein said fluidizing step com¬ prises simultaneously pumping said liquid nutrient medium and stabil¬ izing the flow of said liquid nutrient medium upwardly through a verti¬ cal reaction zone with a pump impeller located at the bottom of said reaction zone to suspend said beads above said impeller, wherein the rotation of said impeller forces said liquid nutrient medium from below said impeller upwardly into said reaction zone, and the blades of said impeller are adapted to stabilize the velocity profile of the liquid nutrient medium above the bottom of the reaction zone without any other stabilizing means above said impeller. 10. The method of claim 1 wherein said fluidizing step com¬ prises flowing said treated and recirculated portion of the liquid nutri¬ ent medium through a distribution plate having at least one nozzle which substantially horizontally directs the flow of said liquid parallel to the surface of said plate at a velocity which stabilizes the velocity profile of the liquid nutrient medium flowing upwardly through a verti¬ cal reaction zone. 11. The method of claim 3 wherein said treatment zone com¬ prises an oxygenation zone wherein the dissolved oxygen content of the separated liquid nutrient medium is increased by contacting said liquid with an oxygen containing gas. 12. The method of claim 11 wherein said oxygenation zone comprises a membrane gas exchanger. 13. The method of claim 12 wherein said bioactive material immobilized on said biocatalyst beads is selected from the group con¬ sisting of aerobic cells and microorganisms. 14. The method of claim 13 wherein said aerobic cells com¬ prise mammalian cells. 15. The method of claim 14 wherein said mammalian cells produce antibodies. 16. A method of continuously contacting a liquid with a bed of particulate solids to effect mass and energy transfer therebetween comprising fluidizing the bed of solids with said liquid in a reaction zone, separating the solids from the fluidizing liquid exiting 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, recirculating 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. 17. The method of claim 16 wherein said fluidizing step com¬ prising simultaneously pumping said liquid and stabilizing the flow of said liquid upwardly through a vertical reaction zone with a pump impeller located at the bottom of said reaction zone to suspend said particles above said impeller, the rotation of said impeller forcing said liquid from below said impeller upwardly into said reaction zone, and the blades of said impeller being adapted to stabilize the velocity pro¬ file of the liquid above the bottom of the reaction zone without any other stabilizing means above said impeller. 18. The method of claim 16 wherein said fluidizing step com¬ prises flowing said treated and recirculated liquid through a distribution plate having at least one nozzle which horizontally directs the flow of liquid parallel to the surface of said plate at a velocity which stabil¬ izes the velocity profile of the liquid flowing upwardly through a verti¬ cal reaction zone. 19. The method of claim 16 wherein said particulate solids are separated from said liquid by gravity forces. 20. The method of claim 16 wherein said separating step occurs in a rotary centrifugal separator. 21. A fluidized bed reactor for fermentation of liquid nutrient medium by biocatalyst beads, comprising: a reaction vessel adapted to contain a suspended bed of said biocatalyst beads containing immobilized bioactive material in a reaction zone; means for pumping said liquid nutrient medium through said reaction zone to suspend said biocatalyst beads in said reaction zone; means for separating said beads from the liquid nutrient medium exiting said reaction zone so that said beads remain in said reaction vesssel; separate means for treating said liquid nutrient medium exiting said reaction zone so as to alter its temperature or composi¬ tion; means for recirculating the treated liquid nutrient medium to said pumping means; means for supplying fresh liquid nutrient medium to said reactor; and means for withdrawing at least a portion of the liquid nutrient medium that has passed through said reaction zone as product. 22. A reactor according to claim 21 wherein said separating means comprises a vertical reaction zone having an upwardly increasing diameter whereby the velocity of the nutrient medium falls below the fluidizing velocity at the top of said vertical reaction zone. 23. A reactor according to claim 21 wherein said separating means comprises a rotary centrifugal separator at the top of a vertical reaction vessel. 24. A reactor according to claim 22 wherein said pumping means comprises a rotary pump impeller at the bottom of said vertical reaction zone which simultaneously pumps said liquid nutrient medium and stabilizes the flow thereof upwardly through said vertical reaction zone without any other stabilizing means above said impeller. 25. A reactor according to claim 23 wherein said impeller and said separator are coupled to and driven by a common drive shaft which extends through said reaction vessel. 26. A reactor according to claim 21 wherein said separate treatment means comprises an oxygenator wherein the dissolved oxygen content of the separated liquid nutrient medium is increased by con¬ tacting said liquid with an oxygen containing gas. 27. A reactor according to claim 26 wherein said oxygenator comprises a membrane gas exchanger. 28. A reactor according to claim 27 wherein said bioactive material immobilized on said biocatalyst beads in selected from the group consisting of aerobic cells and microorganisms. 29. A reactor according to claim 28 wherein said aerobic cells comprise mammalian cells. 30. A reactor according to claim 29 wherein said mammalian cells produce antibodies. 31. A multi-stage fluidized bed reactor comprising at least two of the individual reactors of claim 21 arranged serially with means for passing liquid nutrient medium sequentially through said individual reactors.

Inventors: DEAN ROBERT C JR, GRELA PETER V, KARKARE SUBHASH B, DEAN, ROBERT, C., JR., GRELA, PETER, V., KARKARE, SUBHASH, B.
Applicants: VERAX CORP, VERAX CORPORATION
Application year: 1986
Main classification: C12N5
IPC codes: C07K14, C12M1, C12M3, C12P7