CA1305681C

CA1305681C - Fluidized bioreactor and cell cultivation process

Info

Publication number: CA1305681C
Application number: CA000502887A
Authority: CA
Inventors: Peter V. Grela; Robert C. Dean, Jr.; Subhash B. Karkare
Original assignee: Verax Corp
Current assignee: Verax Corp
Priority date: 1985-02-28
Filing date: 1986-02-27
Publication date: 1992-07-28
Anticipated expiration: 2009-07-28
Also published as: AU5452986A; EP0215820A1; WO1986005202A1

Abstract

FLUIDIZED BIOREACTOR AND
CELL CULTIVATION PROCESS
ABSTRACT
A fluidized bed reactor and reaction process, particularly well-suited for culturing cells, for example, for tissue culture and fermentation processes, are described which involve the treatment of at least a portion of the fluid exiting the fluidized bed reactor in a side loop in a manner to alter its temperature or composition, e.g., oxygen-ation, with recirculation of this treated fluid to the reactor as a por-tion of the fluid causing bed fluidization.

Description

FLUIDIZED BIOREACTOR AND
CELL CULTIYATION PROCESS

BACKGROUND OF THE INVENTION
This invention relates to fluidized bed reactors for contacting iluids and solids, such ~s for carrying out ~hemical reactions, snd par-ticularly rel~tes to pro~esses for cultiv~ting cells, e.g., tissue cultures ~nd fermentations9 using such reactors.
Fluidized bed reactors ~re known in which the nuid is delivered upwardly from the bottom of the re~ctor through fl distribution plate or other r esistance which stabilizes the fluidized bed. Stabili2ation is achieved by virtue of the positive resistance to flow offered by the distribution pl~te. The distribution plate tends to prevent gross distortion of tbe flow in the fluidized bed by offering lower resistance in regions naving lower fluid ~relocity, and high resistawe In regions of high velocity. Thus, t}~e tluid flow tends to redistri~ute itself toward uniformity across the cros~section ~ail~ble for flow. A uniîorm fluid velocity profile Is import~nt to ~vold channeling and other aberrant flow phenomenon whieh prevent good solids suspension and good fluid/solid contact, Typical ex~mples of distribution plates known in the ~rt ~re perforated metal plates, sintered m~terials, open-cell fo~ms, ~nd beds of pebbles. Fluid may be taken from tlle top of the fluidized bed ~nd can be recirculsted through A pump to the distribution resistance element or plate.

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_ ~ _ Fluidized bed reactors provide a convenient way for conducting chemical processes which require m~ss nnd energy transport between a solid flnd fl liquid or gas. Such reactors potentially offer the advantages of high mass and energy transfer rstes over a wide rMnge 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 be~ds. Although often quite fragile, these beads generally are sui table 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 Q 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 exflcerbate 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 ~nd other desired reactants to the cell culture and for removing products and by-products (both desirable ~nd undesir-able) from the cell eulture, 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 ~ntinuous cell culture processes utilizing very small biocatalyst beads contAining immobilized microorganisms or enzymes is th~t conventionally designed periorated distribution plates mdy become plugged by solids or may ~3~

permit back-flow of biocAthlyst beads through the openings, ~or example, during periods of inactivity. In CIISC Or plugging, localized blocking is a typical result. Such blocking c~uses ~ change in the hydrodynamic conditions of the fluidized bed upseting bed stabilizstion and necessitating thut the reactor be shut down for the purpose of cleaning~
Of eourse, any solution to these problems must take into sceount the sensitive nature of the bioc~talyst beads to physical impact for~es ~nd abresion that might be encountered during operation as well ~s the sensitive nature of the immobilized bioactive material, especially mammQlian cells. In a continuous process, a single charge of biocatalyst beads is expected to h&ve a useful life on the order of six to eighteen months, so long ~s excessi~e sttrition c~n be ~voided.
SUMMARY OF THE INVENTlON
It is therefore an object of one aspect of the present invention to provide a reQctlon method and a itluidized bed reactor therefor having a stflbilized flow, which reactor is not prone to clogging during normsl use.
It is an object of an aspect of the present invention to provide ~ reflction method ~nd a flu~dized bed reactQr therefor, partic-ulnrly suited for c~rrying out proce~ses for cultivating cells.
It is an object of an aspect of the invention to provide a reaction method ~nd a nuidized bed reactor therefor, in which minim~l recircul~tion Or solid~ occurs.
It is an object of an aspect of the present invention to provide a method.of continuous cell culture which can accommodate the oxygena-tion demands of ~n aerobi~ process without damaging the fragile biocatalyst beQds or the cells immobilized in them.
An object of an aspect of this invention is to provide a fluidized bed reactor and method for continuously cultivating cells At high cell densities under optimum conditions while m~lntaining aseptic operation It is an object ~ an aspect of the invention to provide a fluidized bed reactor which achieves the foregoing objects, and is simple in ~3~5~

construction ~nd operation, relatively inexpensive to manufacture and relatively easy to maintain in long-term, continuous ~septic use.
These and other objects of the invention ~re accomplished by providing a method of continuously contflcting a liquid wi th 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 ~t one end of the reaction zone; treating a portion of the separated liquid in a treatment zone, separ~te 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 o~
said liquid for fluidizing the bed of particul~te solids; and recovering another portion of said separsted liquid as product.
The method of the present invention has specific application in continuous aerobic cell culture processes wherein a bed of rel~tively fra~ile bioc~talyst beads containing immobilized bioactive material is fluidized with a liquid nutrient medium in a reaction zoneO A liquid stream containing unconsumed nutrients and biochemical (metabolic) products is separated frorn 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 th0 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 reflction zone to sus-pend the solids above the impeller, and the blades of the impeller are sdapted to stabilize the velocity profile of the liquid above the bottom of the reaction zone without the need for any other stabllizing means abbve the impeller. ln this embodiment9 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 nuidizing velocity range for the solids in the central portion of the reaction zone, the liquid in the bottom portion OI the reaction zone is at a velocity above the fluidi~ing velocity range and liquid in the top portion of the reaction zone is at a velocity below the fluidizing velocity renge.
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 reflction vessel for simultaneously pumping the liquid nutrient medium and stabilizing the flow thereo~ 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 re~ction 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 fluidiæed bed at typical fluidization velocities of from about 0.01 to about 0.5 meters per second and which, through its dynamic action, stebilizes the bed in order to counter velocity distribution distortion that leads to nonuniform ~luidized bed operation. A study of the veloci ty di~grams of the propeller shows that, where the axi81 (through flow) liquid velocity is low, the propeller adds more work because the ef fectiYe angle of attack on the blflde is increased. Conversely, where the axial velocity is high, the effective nngle of atta~k decreases and so does the work input. Where the work input is high3 there tends to be an increase of axial velocity countering the defect and vice-versa.
The propeller has an l'open" design, i.e., with large spaces through which the biocatalyst beads can pass. The probability of collision wi th the blades is therefore low and, because the ~lades move rel~tiYely slowly and with low power consumption, the incidence of dflmege to the beads and the bioreactive material~ e~g., microorganisms, within the m is low.

In another embodiment, the bed o~ solids is ~luidi2ed in a st~ble f~shion by pumping the liquid into the bed o~ solids in ~ YertiC~I
reaction zone through ~ distribution plate h~ving one or more nozzles which horizontally direct the flow of liquid subst~ntially p~rQllel to the surface of the plnte comprisir!g the bottom of the nuidized bed.
As used herein, lthe term "bioc~talyst be~d" Is used to gen-eri~lly cst~gorize supports contsining immobilized bioactive materi~ls such ~s enzymes, microorg~nisms ~nd the cells o~ higher organisms, particul~rly microorganisms ~nd cells requiring a constant supply oî
oxygen for proper development, including withvut limit~tion bacteris, fungi, plAnt cells and m~mmslifln cells (8.g., hybridomas). Such beads can be used in connection with 5 wide varlety of processes Qnd the present Invention is directed to a nuidized bed method and ~pparatus ~or c~rrying out such processes. Norm~lly, suit~ble besds will have porous structure and m~y be fibrous or sponge-like in ~ppe~r~nce. The beads can be prepared using ~ wide variety o~ m~teri~ls including, i_ alia, natur~l polymers such ~s polysaccarides ~nd fibrous proteins, synthetic polymers, such a9 poly~lmides (nylon), polyesters ~nd polyureth~nes, miner~ls including cerQm~cs and metalsO
Also as used hereln, phr~se3 such ~s "process for cultiv~ting cells," ncell culturs process~ and the llke are intended to embr~ce ~
wide variety o~ biochemic~l processes involving bio~cffve materials.
These phrases embrace processes in which microorganisms or the cells of higher organisms ~re eultured, using an appropriate nutrient medium, to enh~nce the production of desired metabolic products. For inst~nce~
monoclonAl sntibody ~nd other metabollte production by continuous culture of mammali~n cells (e.g., hybridom~s~ is specific~lly ineluded within the intended mean~ng Or these phra~esO

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6a Other aspects of the invention are as follows:
A method for continuously culturing cells comprising the steps of:
(a) providing a reaction zone containing a bed of porous biocatalyst beads, said beads having immobilized therein microorganisms or cells;
(b) fluidizing said bed of biocatalyst beads with a liquid nutrient medium;
~ c) separating said beads from liquid nutrient medium exiting one end of said reaction zone so that said beads remain in said reaction zone;
(d) 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;
(e) recirculating said t:reated portion of the separated liquid nutrient medium back to said reaction zone as at least part of said liquid nutrient medium ~or fluidizing said bed of biocatalyst beads;
(f) recovering another portion of said separated liquid nutrient medium as product; and (g) feeding fresh nutrient medium into said reaction zone at a rate equal to the recovery of separating liquid nutrient medium as product, said rate yielding a feed dilution rate above the maximum specific growth rate of said microorganisms or cells.
A fluidized bed reactor system for fermentation of liquid nutrient medium by biocatalyst beads, comprising in combination:
a reaction vessel containing in a reaction therein a suspended bed of porous biocatalyst beads having immobilized therein microorganisms or cells;
means for pumping said liquid nutrient medium through said reaction zone to suspend said biocatalyst beads in said reaction zone;

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6b means for separating said beads from the liquid nutrient medium exiting said reaction zone so that said beads remain in said reaction vessel;
separate means for treating said liquid nutrient medium exiting said reaction zone in a treatment 20ne separate from said reaction zone so as to alter its temperature or oxygen concentration;
means for recirculating the treated liquid nutrient medium to sai.d pumping means;
means for supplying fresh liquid nutrient medium to said reactor at a rate equal to the recovery of separated liquid nutrient medium as product; and means for withdrawing at least a portion of the liquid nutrient medium that has passed through said reaction zone as product.

BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set out with particularity in the appended claims, but the invention will be understood more fully and clearly from the following detailed cdescription of the invention as set forth in the accompanying clrawings, in which:

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Figure 1 is ~ sche mstic vertical section~l view of a fluidized bed reactor in ~ccordance with one embodiment of the invention;
Figure 2 is a propeller ~har~cteristic plot of fluid pressure rise as a function of fluid flow;
Figure 3 is a schematic view of a i~luidized bed reactor in accordance with a another embodiment of the invention;
Figure 4 is 8 schematic view of a rnulti-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 pl~te in accord-ance with another embodiment s)f this invention;
Figure 6 is a schematic illustration of a nozzle which when used on the distribution plate of a vertical reRCtor substantially horizontally direets the flow of liquid parallel to the distribution plate at the bottom of a fluidized bed reactor; snd Figure 7 is 8 schematic flow sheet o~ app~ratus useful for practicing continuous aerobic cell cultivation in accordance with the invention.
DETAILED DESCRIPTION
Although this detailed description is in the context of ~ fluidized bed reactor used in continuous cell culture processes, including fermentation processes, it is to be understood that the appar~tus itself is suitable for use in many different types of processes involving fluid and solid cont~ct. While described primarily in the context of liquid/solid contacting it will be apprecisted that aspects of the method ~nd the appRratus are ~lso suited ~or any fluid, i.e., liquid, gases or mixtures thereof. In such fluidized bed processes it is known that a wide variety of forces can ~e used to generate and st~bilize the counterflow of fluid and solids needed to operate ~ fluidized bed reactor. While the present Invention will be described using an embodiment in which a pressurized liquid and the force of gravity pl~y 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 C~

will readily appreciate other available embodiments employing other physical phenomenfl.
Referring to Figure 1, ~ nuidized bed reactor 10 in accordance with one embodiment of the invention comprises a containment vessel 12 having within ~ stationary, tapered reaction vessel 14. An ~nnular recircul~tion channel 16 piovides fluid communication between the top and bottom portions of reaction vessel 14. A rotatable shaft 18 is journ~lled in bearings 20, 22 in the upper and lower portions of containment vessel 12. A propeller 24 is ~ixed to ~nd rotated by shaft 18, ~long 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 ss described below. An inlet 30 is provided for supplying fresh liquid nutrient medium to the reactor. Outlet 32 permits the withdrawal of Qt 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 ~nd the recircul~tion channel 16 are substantially filled with liquid nutrient medium, while a fluidized bed of biocstalyst beads, for example polysaccharide gel beads containing entrapped microorganisms, is m~intained suspended in the central portion of reaction vessel 14 ~bove the propeller.
As seen in Figure 1, the inner di~meter o~ 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 beflds 36 in the central portion of the re~ction vessel, the liquid nutrient medium in the bottom portion of the reaction vessel, just above propeller 24, is et a velocity above the fluidizing velocity r~nge, and the liquid nutrient medium in the top portion of the reaction vessel is at a veloci ty below the fluidizing velocity rangeO The fluidizing velocity range is, of course, that range of upward fluid velocity OI liquid nutri-ent medium 34 which overcomes the gravitation~l force on the 6~.~

biocatalyst beads 36 ~nd mflintains them in suspension with substanti~lly little or no net movernent of the beads either upward or downward.
Typical ~luidization velocities may be on the order of about 0.01 to about 0.5 meters per second for biocat~lyst beads having a size of about 0.1 mm up to about 0.5 mm or rnore.
While this embodiment nnd others are described in connection with a fluidized bed arrangmeent in which an upwardly flowing streflm of pressurized l;quid suspends ~ bed of solids against the downward pull of gl'RVity, IIS will be apparent to those skilled in this art, the inven-tion ~lso 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 solid~s.
In a IT anner known in the ~rt, the t~pered bed can be replaced with other forms so long AS meflns 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 (electric~l) 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 bioc~talyst 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 rom the fluidizing medium is very distinct ~nd only a minimal amount of freeboard, without necessity for precautionary designs such as tapered or stepped expansion zones, is needed to ensure s~tisfactory removal of the be~d solids from the fluidizing liquid.
Propeller a4 is designed to effect suspension of the particulate bed and stabilizRtion of the fluid velocity above the propeller without ~ny other stabilization means and to avoid damage to any recirculating solids. To these ends, the propeller should have an open design hflving lflrge spaces through which biocatalyst beads 36 c~n pflSS undamaged.

More specifically the preferred propeller design hfls ~ solidity value of less than 1Ø Solidity is the r~tio of the propeller blade chord to the blade spacing. Moreover, the blade angle should be set very n~t, i.e., typically not more than ~bout 15~ off a tflngent to the axis of rotation. The propeller M~de profile is designed to move high volumes of fluid with ~ small rise in pressure, flS in the case of cooling tower fflns 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 la to 2~ 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 speci~ic gravity and fluidizing medium viscocity. For the biocfltslyst 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 funetion of now Across the propeller. The characteristic graphic~lly illustrates the stabiliz~tion effect that the propeller has on the fluidized bed. When the local flow up into the fluidized bed tends to decrease (represented by R 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 ~rom 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 ~ction. The "steeper" the propeller's characteristic (i.e. the flatter the blade setting), the stronger the stabilizing action.
Even though the reactor o~ Figure 1 tends to maintain a stable fluidized bad, a small amount of biocatalyst beads nevertheless may unflvoidably escape over the top o~ reaction vessel 14 and be entrained in the recirculation flow through channels 16 ~nd propeller 24. While the design of propeller 24 minimizes attrition of the blocatalyst beads 369 it may be desirable to further minimize the recirculation of beads 3 6 by providing, as illustrated in Figure 3, a rotary centrifugal separa-tor 40 Qt the upper end of reaction Yessel 1~. Separator 40 includes a vaneless rotating diffuser 42 and M bea~separstirlg bladed centrifuge 44~ both of which are rotatably driven by shaft 18 which e~tends upwardly from impeller 24 through reaction vessel 14. Bea~separating centrifuge 44 slings any beads 36 whieh rise within it ~long with upwardly flowing liquid nutrient medium 3d~ 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 desired9 other arrangements for separating beads from the fluidizing medium also could be employed inclùding 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 arrangennent for conducting cell culture processes, including fermentation proce~ses, 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 illustr~ted in Fi~lre 1. All of the propeller~ 124 of the se~eral individual reactors 100 are driven by a common shaft 118. Treatment of the recirculating culture liquid to maintain opti-mum conditions in reactors 100, such as aeration and/or, C2 extrac-tion e.g., in a membrane gas exchanger 130; heat exchange to heat or cool the culture liguid in exchanger 140; filtration at lS0; or other treatment such as pH control, sterilization (e.g., by filtration, UV
irradiation or ozonatiorl) and altering the composition of the recir-culating fluid such as by adding nutrients or other bioactive materials to the recircul~ting nuid or by removing desirable and/or undesirsble metabolic products therefrom using any o~ a wide vAriety of :~3~

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. ~uch 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 strPams 170 can he taken off at any desirable stage in the process. The reactor may be operated hyperbarically to ten or more atmospheres in order to proportionally enhance the oxygen carrying capacity of the recirculating liquor. By using a side loop to effect treatment of the culture li~uid, 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 immobilized bioactive material. This arrangement also permits separate control of the feed rate of fresh nutrient medium and the flow rate of the recirculating culture liquid.
Figure 5 shows an alternative arrangement of 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 Figure 5, elements corresponding to elements in the Figure 4 arrangement are identified by reference numerals having the same last two digits. Figure 5 differs from Figure 4 in that Bl l;~OS~

12a 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 vari.ety of available distribution designs including, inter alia, a perforated plate or sieve tray, a slotted tr~y, a pebble bed, e.g.9 glass beads, a porous ceramic, an open cell foam and a sintered metfll.
While Figure 5 illustrates a conventional perforated distribution plate, in order to avoid plugging flnd back flovv of solids through the distribution means, for example, during inoperatiYe periods~ particularly in the case where fragile biocatslyst beads are being fluidized by and reacted with a liquid nutrient medium, the normal perforations in the conventional distribution plate csn be replaced with one or more hori-zontal flow~directing nozzles in a suitable arr~y~ 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 wi th a perforstion 403 In the distribution plate 425. The stem has a cen-trslly locRted bore 404 which extends into head member 401 (indicated by dotted outline). The side wall of the head member is provided with substsntially horizontal ports 407 which communicate with bore 404. PreferQbly, the ports are equal]ly spaced around the circumfer-ence of the nozzle. For example, a normal 3/4 inch diameter nozzle msy have twelve, approximately 1/8 inch diameter, ports equnlly spaced about its circumference. In oper~tion, liquid (and biocatalyst beads when occasionally recircul~ted) passes through the distr;bution plate by flowing through bore 40~ 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 ~nd arrangement of such nozzels in any application is, among other things, a function of the size of the ~eactor and the characteristics of the biocatalyst beads. For ex~mple, in fl 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 ~3~

diameter of ~bout 8 inches) about 16 nozzles positioned in Q symmetric pattern on the distribution plate should be employed. If advantageous, a bed of pebbles9 e.g., glQss beads, OI an appropriate diameter, also can be supported by the distribution plate fitted with said no~zles to further stabilize performance. The number and arr~ngement of such noz~les for any particular ~pplication is within the skill of the art.
Generally, the nozzles are designed so that fluidization velocities in the range of ~bout 0.001 to 0~01 m/sec are achieYed at a pressure drop through the nozzle on the order of about 0.1 to flbout 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., 25%-60% solids), however, which is preferred, it is suf-ficient to rely on the force of gravity simply by providing ~ small dis-engagement zone (fre~board) above the expected (design) level of the expanded bed. When operating ~t such high concentrations of the biocatalyst beads, the separQtion of solid particles from the ~luidizing liquid is very distinct, eliminating the need for any prec~utionary 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 al 0 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 9S feeds for these st~ges. Product is removed from the re~ctor ~rrangement prim~rily throug31 line 27û of stage 3.
Lines 271 can ~e 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 embodiment9 e~ch bed of biocatalyst besds 238 contains an immobilized bioactive material that requires oxy~en -to remain active. Exemplary bioactive msterial includes aerobic microorgenisms and cells such as aerobic bacteria, fungi and mammalian cells. The beads may consist of a polysaccharide gel such as carrageenan or agQrose gels entrapping the microorganisms and cells. Other bead supports include natural ~nd synthetic polymers, ceramics and metals.
The beads gener~lly are porous and may be fibrous or sponge-like in appearance. PreferMbly, the beads comprise a fibrous polymer such as collagen in which the microorgsnisms are entrapped. Generally, the beads will be treated to alter thek specific grRvity. 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 m~y 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 mRjor portion of the unconsumed nutrient medium discharged from each reactor stage in lines 2~0 through a side loop.
The portion o~ the unconsumed nutrient medium discharged in line 260 ~nd circulated for fluidizing the bed of biocatalyst beads is first passed through the oxygenators 230 where the dissvlved 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 reeirculating fluid, including porous fine-bubble diffusers, mechanical aerators, or membrane oxygenators, membrane devices generally will be preferred for aerobic cell culture applicationsO ~ine 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 Q liqui~gas interface thereby obviating the selection of any device as the oxygenator which depends upon the ~ 16 -~eneration of a large surface area of small bubbles for gas transfer.
M em brane oxygenators trsnsfer the oxygen directly into the liquid on a molecular level without any gas-liquid interface. Membrane oxygenfltors suitable ~or fermentation ~pplic~tions include commercially flveilable blood oxygenators such as available from Cobe, Denver, Colorado;
American Bentley Corp., Irvine, California, Qnd SciMed Co., Minneapolis, Minnesota. Microporous filters such as the ~elman A croflux cartridge Gelman Sciences, l[nc., Ann Arbor, Michigan, and the Millipore Millidisk, Millipore Co., Milford, Massachusetts, may nlso be used in appropriate circumstances.
A particularly preferred oxygenator, b~sed on both design sim-plicity and performance characteristics, is a shell and tube oxygenator employing tube material ha~ring a suitable oxygen permeability. Any of the well-known shell ~nd tube-type designs used for example in the heat transfer art c~n be employed. See, for example, Perry, R.H. and Chilton, C.H., Chemical Engineers' ~andbook. A particularly useful design simply comprises a single helical strand of suitable tubing in a pressure vessei. Silicone tubing having aln oxygen permeability on the order of about 1.3-1.4 X 10-6 mmols 2 - mm per cma-cm Hg per minute has proven to be particularly e~fe~ctive as tube materi~l. Of course, other materials having different oxygen perme~tion characteris-tics can be employed. The selection of suitable materi~ls and gas exchanger designs is wi thin the skill of the art.
~ y properly selectin~ 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-c~lly is gener~ted by respring cells in reactors aoo. Obviously, in the broad practice of the present invention ~ny of a wide v~riety of nor-mally gaseous constituents may be edded to, or removed from the re-circulating liquid by properly selecting the membrane materi~l with this arrangement and this ~spect of the present invention need not be lim-ited solely to ~erobi c processes.

~l3~

In the arrangement illustrated in Figure 5, the liquid to be oxy-gensted is flowed through the tubes of the gas membrane exchænger and the oxygen-containing gas~ normally comprising dil', enriched air or oxygen, is flowed through the exchanger on the shell side. If one desires to enh~nce the rate of mass transfer, generally oxygen is used st an elevated pressure, for example, up to ~bout 10 atmosphere absolute pressure or higher. After exiffng the oxygenator, the liquid is recirculated using pumps 235 to the fluidi7ed bed through he~t exchangers 240, which adjust the temperature of the liquid to optirnize conditions in reactors 200. Generslly, specially selected recirculation pumps are used (such as bl~de-less centrifug~l blood pumps, e.g., a Biomedicus pump, or flexible rotor vane pumps, e.g., Ja~sco pumps~
which do not damage 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.9 by adding reagents to control pEI;
by adding nutrients, drugs or other materials to influence the metabolism and/or growth of the cells or microorganisms in reflctors 200; and by removing metabolic products and by-products, both desirable ~nd undesirable, ~rom the recir~ulating liquid by fl variety of techniques including ultra~iltration, affinity absorption, ~dsorption and many others~. For example, suitable re~gents m~y 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 re~ctors aoo under continuous~ aseptic operation without injuring the ~ragile biocatalyst beads or the immobilized bioactive materialO
The recirculation rate needed in any cell culture flpplication is a function of many variables including, inter alia, the targeted solids (biocatalyst bead) concentration during ~luidization; the density of the bioactive materi~l, i.e., the concentrfltion of organisMs, e.g., mæmmfllian cells such as hybridomas, immobiliz~d on or inside o~ the biocatalyst beads; the nature of the bioflctire material (e.g., its oxygen demand), the morphology o~ immobilizfltion (i.e.~ whether the bio~ctive material is contained on or in the bead matrix~, the nature of the bioc~talyst beads, e.g., the size of the beads and their specific gravity, the nature of the culture medium ~e.g., its chemical eharacteristics and oxygen c~rrying capacity), and the nature o~ the treatment to be per-formed on the recirculating liquid.
Typically, in order to maximize efficiency the cell culture process is operflted under fluidization conditions that yield a fluidized bed void volume within the range of about 80% to ~bout 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 ~nd culture medium resulting in poor mass trAnsfer, while operation at a nuidized bed void volume of above about 75~6 constitutes an inef-ficient usage of the reactor volume ~nd impedes the natur~l 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 trflnsfer needed, the bead attri tion r~te, etc.
The recirculation rate also is influenced by the concentration and nature of the cells or mlcroorganism~ in the biocatQlyst beads.
For example, for any specific oxygen-consuming microorganism, as the concentration tdensity) of the microorgflnisms inside the biocatalyst beQds increases, higher recirculfltion rates are required in order to maintain ~dequate oxygen and/or nutrient transfer in the fluidized bed.
Additionally, certain microorganisms, such ~s bacteria, generally have very high speci~ic oxygen demands, flnd there~ore normally will require higher recircul~tion rates and smaller, denser beflds to maintain proper operating conditions than certain other organisms such as eukaryotes and mammalian cells which have much lower specific oxygen demands.
For exflmple9 while Escherichia coli typic~lly requires about 0.1 mmol ~ 3g~

-- lg --2 per g-cell per minute, Penicilliurn chrysogenum gen~rally requires only about 0.02 mmol 2 per g-cell per minute ~nd a typical hybridoma mflm mali~n cell mfly require only about S x 10-7 mmol 2 per 106 cells per minute.
The properties of the biocatalyst beads, particularly bead size and specific gravity, also in~luence 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 um generally have proven to be suitable~ Bead specific gravities between about 1.05 and 2.0 ~lso have been investigated and found to be suitable depending upon the circumstances. Generally, beads having higher specific gravities within this range rnQy be preferred since a higher specifie gravity permits a higher recircul&tion rate and a higher reactor ~spect ratio. At higher reacter aspect r~tios ~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 beflds containing immobilized hybridom&s 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 example7 of the type illustrated above in Figure 5. The cell concentration inside su~h biocatalyst beads typically is about 108 cells per ml of beads itor hybridomns of a 14 um diameter. During fluidization, the liquid surrounding the beads typically contQins a concentr&tion of organisms of about 1/lOth that value. At fluidizing conditions (i.e., ~verage bed void volume of about 60%) the average cell concentration in the bioreactor volume is about 40% of the concentr~tion in the beads themselves, In other words, the actual reactor cell concentration (beads and liquor together) is approximately 4.6 ~ 107 cells per ml~

~a~3~

As shown in Figure 79 fresh nutrient medium ~s fed to Q
fluidized bed bioreactor 300 from a medium storage vessel 302 through pump 303 and vAlves 30~ and 305. A liquid stre~m 360 is separated from the top of the bioreactor 300 to ~orm e recircul~ting culture liquor. The culture liquor is circuIAted outside the fluidlzed bed through line 360 and ~alves 306 and 307 and pump 335 in order to appropriately treat the culture liquor in separste trefltrnent means, which liquid then is returned to the bottom of the biore~ctor 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 pHr~llel circuits generally will be provided to permit easy isolation of such equipment for maintenance and repair without jeopardizing the aseptic lntegrity of the system. This ease of maintaining aseptic operation is a particularly preferred ~eature 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 snd 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 rnembrane gas exchanger 330 in counter-current flow with ~ stream of air or enriched oxygen, preferably pressurized, delivered from oxygen storage tank 310 through control valve 311 and îilter 312. Waste gas ~ontaining CO2 is discharged through filter 313, ~,alve 314 ~nd flow meter 315.
As noted, ~n 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 di~
solved oxygen probe 342, temperature sensors 343 and 3~4, and a turbidity probe 345, are installed in the external recycle loop for easy access and calibration.

1~0~i6~

-- 2~ --In this arrangement, the recycle dilution rate can be adjl}sted independently of the feed dilution rate in order to control ench separately to achieve optimum performance in reactor 300. For exam-ple, the recycle dilution rate, which normally is the m~jor fluidizing flow, can be very high, e~g., up to about 1000 hr~1, while the fresh nutrient medium throughflow dilution rate can, at the sarne time, be very low, e.g., down to about 0.006 hr 1 Separation of the reci~
culating and throughflow dilution is a very convenient and Qdvantageous feature of the present invention.
A product stream of harvest 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 ;s collected in storage vessel 318. Thereafter, the liquid is removed from vessel 318 flnd the antibodies can be separated and recovered by first remoYing additional eells and cell debris ~rom the harvest liquor, wi th a microporous filter, filter 319, followed by removal of about 95% of the water by tangential-flow ultrafiltration. An ion-exchange chromotography 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 ~or final purification before filter steri-lization and Iyophilization, or the bottling of a steril~liquid product containing the recovered antibocly.
In addition to products comprising primary and secondary met~bolites of the cell culture, the product may also comprise the cel-lular materi~l or biomass itself. For example, genetically engineered E. coli with an rDNA product that is not expressed9 e.g., insulin, could be cultivated in and recovered from reactor 300. With certain mate-rials îor the biocatalyst beads9 e~g., porous or fibrous structures as described in the above-noted copending ~pplication, 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 ~3L3~
~ ~2 -entrained component of the culture medium. In the practice of the present invention, other methods for recovering the biomass directly from the biocat~lyst beeds themselves also can be employed and the present invention is not intended to be limited to any particular embodi ment.
The following examples are intended to more fully illustrate the invention without acting as a limitation on its scope.
Example A nuidizing bed reactor of the configuratiorl 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 lûO0 liters.
The propeller should have a tip diameter of ~bout 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 m~trix weighted with a mineral (i.e., kappR-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 predominflntly glucose with trace amounts of other nutrients normally employed in this type of fermentation reaction (flnaerobic). The medium from the top of the reaction zone would be partially recovered continuously to ~ive an average yie~d of about 50 kg/hr of ethanol.
Exa m ple 2 A fluidized bed bioreactor was constructed having A diameter of 2 inches and fl height of about 8 inches. The capacity of this reactor is about ~00 ml. Bio~atalyst 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 Framingham, . .
Massashusetts, immobilized therein. A nutrient medium, also obtained from Integrated Genetics~ containing glucose ~nd other nutrients nor-mally employed in this type of aerobic fermentation process, was 5~

pumped to the bioreactor at a r~te sufficient to yield a residence time (based on the feed rate) of ~bout 2 hours. The bed of bioc~talyst be~ds was nuidized by recirculating the fermentor (culturej liquol, using a magnetically coupled genr pump, at a rate sufficient to yield a residence time of less than about one minute in the re~ctor.
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 ~s 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 biore~ctor was operated continuously for more th~n 1000 hours producing a product stream containing approxi-mately 100 nanograms per milliliter o~ alpha human chorionic gonadatropin ( -HCG). A batch reactor oper~ted under similar condi-tions produced produ~t at a rate about 20 times lower.
Example 3 A ten liter capacity fluidized bed bioreflctor 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 ~welve approximately one-eighth inch diameter holes equnlly spaced about its circumference loc~ted 9.5 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 bioc~talyst beads formed from a weighted fibrous polymer (i.e., collAgen with silic~
powder) containing immobilized mammalian cells (hybridom& VX-7). The beads will have an aver~ge diameter of about lOOI)u9 a specific gravity of about 1.15 nnd the cell density within the beads can be about 7 x 107 cells/ml.

~ 3~:~S~8~

The biocatalyst beads will be fluidized by recirculating the culture medium in the reactor at ~ flow rate of ~bout 2 liters per minute. Fresh nutrient medium also will be introduced into the bioreactor at a r~te of about 1 liter per hour and a product stream having An equivalent flow r~te will be removed.
The bioreactor will be aerated by passing the recirculating culture medium through a gas membrane oxygenhtor. The oxygenfltor 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 ~n oxygen permeability of about 1.34 x 106 mmols 02-mm per cm2Hg per minute. Air can be fed to the oxygen~tor Qt a rate of about 1 liter per minute and a carbon dioxide-containing ~as will be dischflrged ~t 8 substantially equivalent rate.
It will be obvious to one of ordinnry skill that numerous modifications mQy be made without departing from the true spirit ~nd scope of the invention which is to be limited only by the appended clai ms.

Claims

1. A method for continuously culturing cells comprising the steps of:
(a) providing a reaction zone containing a bed of porous biocatalyst beads, said beads having immobilized therein microorganisms or cells;
(b) fluidizing said bed of biocatalyst beads with a liquid nutrient medium;
(c) separating said beads from liquid nutrient medium exiting one end of said reaction zone so that said beads remain in said reaction zone;
(d) 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;
(e) 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;
(f) recovering another portion of said separated liquid nutrient medium as product; and (g) feeding fresh nutrient medium into said reaction zone at a rate equal to the recovery of separating liquid nutrient medium as product, said rate yielding a feed dilution rate above the maximum specific growth rate of said microorganisms or cells.

2. The method of Claim 1 wherein said fluidizing step comprises simultaneously pumping said liquid nutrient medium and stabilizing the velocity profile of the flow of said liquid nutrient medium upwardly through said reaction zone with a stabilizing means, wherein said stabilizing means is 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 flow of said liquid nutrient medium above the bottom of the reaction zone without any other stabilizing means above said impeller.

3. The method of Claim 1 wherein said fluidizing step comprises flowing said treated and recirculated portion of the liquid nutrient 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 flow of said liquid nutrient medium flowing upwardly through said reaction zone.

4. 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.

5. The method of Claim 4 wherein said beads are separated from said liquid nutrient medium solely by gravity.

6. The method of Claim 4 wherein said separating step occurs in a rotary centrifugal separator.

7. The method of Claim 1 wherein said beads are prepared from materials selected from the group consisting of natural polymers, synthetic polymers, minerals and metals.

8. The method of Claim 7 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.

9. The method of Claim 8 wherein said natural polymer is a polysaccharide gel.

10. The method of Claim 8 wherein said natural polymer is collagen in the form of a sponge.

11. The method of Claim 1 wherein said immobilized microorganisms or cells are from the group consisting of aerobic microorganisms and aerobic cells.

12. The method of Claim 11 wherein said treatment zone comprises 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.

13. The method of Claim 12 wherein said oxygenation zone comprises a membrane gas exchanger.

14. The method of Claim 13 wherein said aerobic cells comprise mammalian cells.

15. The method of Claim 14 wherein said mammalian cells produce antibodies.

16. A fluidized bed reactor system for fermentation of liquid nutrient medium by biocatalyst beads, comprising in combination:

a reaction vessel containing in a reaction therein a suspended bed of porous biocatalyst beads having immobilized therein microorganisms or cells;
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 vessel;
separate means for treating said liquid nutrient medium exiting said reaction zone in a treatment zone separate from said reaction zone so as to alter its temperature or oxygen concentration;
means for recirculating the treated liquid nutrient medium to said pumping means;
means for supplying fresh liquid nutrient medium to said reactor at a rate equal to the recovery of separated liquid nutrient medium as product; and means for withdrawing at least a portion of the liquid nutrient medium that has passed through said reaction zone as product.

17. A reactor system according to Claim 16 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.

18. A reactor system according to Claim 16 wherein said separating means comprises a rotary centrifugal separator at the top of a vertical reaction vessel.

19. A reactor system according to Claim 17 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.

20. A reactor system according to Claim 18 wherein said impeller and said separator are coupled to and driven by a common drive shaft which extends through said reaction vessel.

21. A reactor system according to Claim 16 wherein said separate treatment means comprises an oxygenator wherein the dissolved oxygen content of the separated liquid nutrient medium is increased by contacting said liquid with an oxygen containing gas.

22. A reactor system according to Claim 21 wherein said oxygenator comprises a membrane gas exchanger.

23. A reactor system according to Claim 22 wherein aerobic cells are immobilized on said biocatalyst beads.

24. A reactor system according to Claim 23 wherein said aerobic cells comprise mammalian cells.

25. A reactor system according to Claim 24 wherein said mammalian cells produce antibodies.