EP0207103A1 - Installation et procede pour la culture et le traitement de systemes biocatalytiques - Google Patents

Installation et procede pour la culture et le traitement de systemes biocatalytiques

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Publication number
EP0207103A1
EP0207103A1 EP86900043A EP86900043A EP0207103A1 EP 0207103 A1 EP0207103 A1 EP 0207103A1 EP 86900043 A EP86900043 A EP 86900043A EP 86900043 A EP86900043 A EP 86900043A EP 0207103 A1 EP0207103 A1 EP 0207103A1
Authority
EP
European Patent Office
Prior art keywords
reaction
reaction vessel
liquid
biocatalysts
flat
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP86900043A
Other languages
German (de)
English (en)
Inventor
Hermann Katinger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MBR Bio Reactor AG
Original Assignee
MBR Bio Reactor AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MBR Bio Reactor AG filed Critical MBR Bio Reactor AG
Publication of EP0207103A1 publication Critical patent/EP0207103A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/06—Plates; Walls; Drawers; Multilayer plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/10—Rotating vessel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/18—Flow directing inserts
    • C12M27/22—Perforated plates, discs or walls

Definitions

  • the invention relates to a device and a method for the cultivation and treatment of biocatalysts, such as cells, particles or soluble substances which influence chemical and / or biological reactions, in which a reaction vessel is arranged rotatably about its longitudinal axis and with at least one Reaction space is provided.
  • biocatalysts such as cells, particles or soluble substances which influence chemical and / or biological reactions
  • roller cultures essentially cylindrical bottles are provided which rest on rollers arranged on a frame which can be driven for the rotation of the bottles.
  • a solution is contained, for example, in US Pat. No. 4,238,568.
  • These bottles are partially filled with inoculated reaction liquid, the remaining bottle volume is filled with a gas, for example air, required for the reaction.
  • the rolling culture allows both the cultivation of suspension cells and cells which are anchored to the cylindrical inner surface of the roller bottles, but only in batch-wise cultivation method. It can be regarded as the standard technique for the culture of sensitive cells and is used for the routine cultivation of cells of all kinds in the laboratory. For the technological production or multiplication of animal cells, the technique of rolling culture is used by using up to thousands of roller bottles in order to enlarge the scale, the so-called "scale up" ; to achieve in multiple units.
  • the known rolling culture is disadvantageous in that it can only be operated in batches, i.e. that continuous cultivation or perfusion cultivation are not possible. This is also because a controlled ventilation of the bottle interior is not possible.
  • the enlargement of the operating scale, i.e. the scale up is limited to the use of multiples of the number of roller bottles.
  • immobilization of the cells i.e. an artificial enrichment of the cells beyond the natural equilibrium is not possible in a continuous culture.
  • the scale up of a single roller bottle in the conventional cylindrical form leads to an unfavorable ratio of the wettable surface to the effective volume of the liquid in the roller bottle, as a result of which the interphasial mass transfer is reduced.
  • the object of the invention is to provide a device of the type mentioned at the outset which, in the case of a continuous cultivation and treatment method, enables a higher density of biocatalysts.
  • this object is achieved in that flat parts which extend towards the longitudinal axis thereof are arranged in the reaction vessel, that the supply of the reaction liquid is formed at one end of the reaction vessel and the discharge of the finished reaction product is formed at the same or other end of the reaction vessel and that the inside of the reaction vessel can be charged with gas.
  • Cells such as animal cells, plant cells, parasitic microorganisms, i.e. in vitro systems, which require a high mass transfer under physically mild, i.e. under largely stress-free conditions, are among the most important target groups for their cultivation or Treatment the subject invention is intended. It also enables biochemical conversion reactions that combine high demands on the combination of high interphasial mass transport with physically gentle conditions.
  • the present invention also combines the reaction-kinetic advantages of a tube reactor and the artificial enrichment of the biocatalyst necessary for the reaction, which makes it possible to achieve naturally existing equilibria to positively influence a reaction in favor of a higher production or conversion rate in the direction of an increased reactor space / time yield.
  • the present invention enables, in addition to the advantages of simple procedural procedures, a maximum adaptation to the type of biocatalytic reaction with cells or molecules.
  • the flat parts extending to the longitudinal axis advantageously consist of ring disks, as a result of which a surface enlargement is achieved without the cells or the like containing them being mechanically stressed.
  • the flat, radially inwardly directed parts can be formed by non-perforated annular disks, as a result of which uniform conditions are achieved over the entire circumference of the reaction vessel.
  • the flat, radially inward parts consist of separate segments arranged in a ring.
  • An expedient variant consists in that the flat, radially inward-facing parts are designed as helical conveyor strips. Because this screw conveyor is located inside the reaction vessel, it transports the liquid from one end of the reaction vessel to the other during rotation.
  • the reaction vessel can be divided in the area of the reaction liquid therein by non-perforated ring disks, whereby a division of the reactor interior into individual sections can be achieved.
  • the flat, radially inwardly directed parts can be designed for the surface absorption of the biocatalysts or the reaction liquid.
  • the flat, radially inwardly directed parts can be surface-modified.
  • the biocatalysts immobilizing coatings can be applied to these parts.
  • reaction Liquid can be supplied at several points along the longitudinal extent of the reaction vessel and / or the finished reaction product can be removed at several locations along the longitudinal extent of the reaction vessel, which means that the reactor can also be operated with partial filling.
  • Another form of immobilization of the cells or biocatalysts also lies in the fact that suspended carrier particles for the biocatalysts are arranged between the flat, radially inwardly directed parts in the reaction liquid.
  • biocatalysts mentioned that is to say the biophases, are retained in the reactor by physical forces such as gravity, centrifugal force or the like, while the liquid phases in the reactor are renewed.
  • all essential aspects for a specific process can be optimally designed and implemented in any operating sizes (scale up).
  • the cultivation and treatment of biocatalysts with the device according to the invention is carried out in such a way that the reaction liquid and the biocat introduced, the reaction vessel is set in rotation and the interior of the reaction vessel is continuously charged with reaction gas. After reaching the desired concentration of biocatalysts, further reaction liquid is expediently introduced and the finished reaction product is removed. Sometimes it is advantageous if the reaction liquid is introduced into the reaction vessel at several points and removed from the reaction vessel at several points, for example by suction. For the suction of the reaction product, one or more suction tubes are lowered into the reaction space or spaces between the ring disks. To mix the phases in the reaction vessel, the speed of rotation of the reaction vessel is expediently increased. The centrifugal force is used in this method. If the centrifugal force is sufficient, it is also possible to separate the denser phase containing the biocatalysts from the reaction liquid which has a lower density. For economic reasons, one can also ' connect several reaction vessels in series.
  • FIG. 1a shows the left part of the reaction vessel from FIG. 1 on an enlarged scale
  • FIG. 3 shows the section III-III from FIG. 2a, - 7 -
  • FIG. 11 shows an embodiment in which the sedimented carrier particles are shown schematically
  • FIG. 13 shows a view from the left of the solution according to FIG. 12.
  • the reaction vessel designates the reaction vessel, which in its interior has flat, radially inwardly directed parts 3 which are directed towards its horizontal longitudinal axis 1 'and are in contact with the wall of the vessel.
  • the flat, radially inwardly directed parts 3 are formed by ring disks 3a, b, c which, depending on the area of use of the device according to the invention, are correspondingly surface-modified. For example, they can be roughened or coated for good wettability or have special coverings on which the cells or biocatalysts are immobilized. As such coverings, nonwovens, pile, sponges or the like come into consideration, which are attached to the surface of the flat, radially inwardly directed parts 3.
  • the flat, radially inwardly directed parts 3 are in this case designed as perforated washers 3c, only one non-perforated washer 3a (see FIG. 2) being interposed.
  • the interior of the reaction vessel is subdivided in the area of the reaction liquid, since liquid does not pass through the unperforated annular disk 3a, while the perforated annular disks 3c can be used to exchange liquid between the individual reaction spaces 2.
  • the reaction vessel 1 is on paired Rol ⁇ len 4, by means of which it se about its horizontal Lijnsach ⁇ 1 1 is rotatable.
  • two pairs of rollers 4 are shown as support elements in the upper part of the reaction vessel 1. This construction with four pairs of rollers 4 is intended for the case in which one wants to use high speeds for the rotation of the reaction vessel 1 in order to be able to use the centrifugal force.
  • the reaction vessel is open at its ends, as a result of which it has the character of a so-called tube reactor. Gas and nutrient solution can be introduced via one opening 5 and the finished reaction product can be drawn off and the reaction exhaust gas can be removed via the other opening 6.
  • the gas supply is designated 7 and the gas discharge 8.
  • the reaction liquid is supplied via a through the opening 5 into the nere line 9 of the reaction vessel 1, which can either open directly at the front end of the reaction vessel 1 or extend further into the reaction vessel 1 in order to be able to introduce the reaction liquid into the reactor at various points, as can be seen in FIG 1 is indicated by the arrows 10.
  • the removal of the finished reaction product is denoted by 11, and, as shown by the broken line 12 (FIG. 1), the finished reaction product can be drawn off at the most favorable points in the reaction vessel 1.
  • a suction tube can be provided in a manner not shown in this FIG. 1, which can be introduced into the reaction vessel 1 at different depths and lowered to the desired level of the liquid. Such an embodiment will be explained in more detail later with reference to FIGS. 12 and 13.
  • reaction liquid located in the reaction vessel 1 forms a thin layer 13 on the inner wall of the reaction vessel 1 and on the surfaces of the flat, radially inwardly directed parts 3 in the area of contact with the liquid (see FIGS. 5, 6 and 7).
  • a container 14 or 15 is provided at both ends of the reaction vessel 1, into which the reaction vessel 1 opens via its openings 5, 6.
  • the rotating reaction vessel 1 is sealed with respect to the stationary containers 14, 15 by a labyrinth seal 16, although reaction gas can also escape through this labyrinth seal if there is overpressure in the reaction vessel; this leakage of reaction gas is indicated by the dotted arrows 17.
  • the entire device according to the invention is arranged in a closed room 18 in which the room temperature required for the reaction can be kept constant.
  • the parts shown are also mechanically stored in this room including a drive motor, not shown.
  • FIG. 10 shows a combination of two reaction vessels according to FIG. 1 to form a battery, the reaction product emerging from the first reaction vessel being introduced into the reaction vessel underneath, as a result of which the reaction process continues there, or where the Reaction can be continued under other internal conditions.
  • the rotation of the reaction vessel 1 ensures a very good mass transfer between solid, liquid and gaseous phases.
  • the rotational movement can be carried out continuously with a constant or changing direction of movement or with a changing speed.
  • the type of loading of the reaction vessel with liquid and the removal of the products and the liquids shown in FIG. 1 gives the device according to the invention the character of a tubular reactor, by means of whose reaction-kinetic advantages an improved product implementation is achieved.
  • the interphasial mass transfer takes place on the entire wetted surface of the reaction vessel 1.
  • the gas required for the reaction can flow through the latter in any direction.
  • the flat, radially inwardly directed parts 3 can be designed in the manner of a conveyor screw (FIG.
  • the reaction vessel 1 shown in FIG. 1 is set in a slowly rotating movement about its own horizontal longitudinal axis -1 ', comparable to a rolling culture.
  • the speed of the movement is controlled in such a way that the liquid level in the reaction vessel 1 is predetermined by the level of the outlet opening 6 and the horizontal position of the reaction vessel 1. Due to the larger diameter of the opening 6 with respect to the inlet opening 5, the outlet is deeper than the lower edge of the opening 5, thereby preventing the liquid from escaping through the inlet opening 5.
  • the supply line 9 for the Reaction medium or a pH-regulating medium can be supplied via an additional line (not shown), which, as indicated by the arrows 10, can also be carried out specifically on individual reaction interspaces 2.
  • the majority of the suspending cells or the carriers immobilizing the cells are located in the fluid bulk of the reaction vessel 1.
  • a small proportion of the cell content in the liquid film can also be found on the inner surface of the reaction vessel.
  • the fresh nutrient medium is supplied either when the reaction vessel 1 is rotating, or after a short standstill thereof, preferably into the reaction spaces 2 arranged at the inlet opening 5 of the reaction vessel 1 in order to achieve the reaction characteristics of a tube reactor.
  • the fresh nutrient medium is introduced directly into the rotating reaction vessel 1, since the sedimentation behavior of these particles is usually sufficient to be in the reaction vessel to achieve a cell density which is higher than the equilibrium of cell growth minus wash-out rate in a homogeneously mixed reaction vessel. This is referred to as the immobilization effect, which can be gradually controlled in the device according to the invention.
  • the accumulation of the cell density or the amount of the biocatalyst per unit volume of the reaction vessel 1, which goes beyond the natural cell growth, causes an acceleration of the biocatalytic conversions and thus an increase in the productivity of the system, that is to say the space / time productivity, and leads to higher quantities of the product to be produced per unit volume, that is to say higher titers.
  • a further advantage of the device according to the invention is also given in that the separation of the reaction vessel into reaction spaces, in addition to an artificial enrichment of the biocatalyst, achieves the reaction characteristics of a tube reactor already described, as a result of which the device according to the invention is suitable for all phases of physiological differentiation reproduce growing cells on a continuous basis, which means that regardless of a time factor, the production of the cells, which takes place preferentially in certain phases of the development of cultures, is optimally used for production on a continuous basis.
  • Animal cells that prefer their products as stationary, i.e. as cells which only slowly or not at all release, are particularly advantageous to cultivate in this reactor.
  • the interphasial mass transfer in the reaction vessel 1 and thus the supply of the cells with nutrients and with oxygen or other gases, the immobilization effect on the cells and the characteristics of the tube reactor is determined by the geometry of the reaction vessel 1, specifically by the ratio of the diameter D to the liquid height H ⁇ to the length L R of the reaction space 2 of the individual reaction space 2 shown in FIG. 4, and by the number of combined ⁇ th reaction spaces 2 on the total length of the reaction vessel 1 and the operating conditions.
  • a segmentation of the reaction vessel 1 in favor of a high H ⁇ / LR ratio is preferred.
  • Structural designs of the reaction vessel 1, which aim to enlarge the inner and thus the effective surfaces, can be achieved, for example, by a richer structure such as roughening of the material on the inner surfaces of the reaction vessel 1 or by other measures possible. These measures are suitable for substantially increasing the effect of a high wettable or moldable surface achieved with the device according to the invention in relation to the effective reaction liquid volume.
  • the residence time characteristic of the liquid flowing through, the sedimentation behavior of the cells in the individual reaction spaces 2 of the reaction vessel 1 and the effective ratio of wettable inner surface of the reaction vessel 1 to the liquid volume can be changed by a configuration deviating from the cylindrical shape and thus the physiological requirements of the cells are optimally adapted.
  • the frustoconical reaction vessels shown schematically in FIGS. 5 and 6 are examples of this.
  • the ratio of wettable surface to liquid volume, the sedimentation ratio of the cells and thus the residence time of the cells or biocatalysts can be influenced.
  • FIG. 8 shows an operating state of the device according to the invention, in which the cells are retained in the faster rotating reaction vessel 1 by centrifugal force which is greater than twice the acceleration of gravity, while the reaction liquid in the reaction vessel is continuous is renewed.
  • the cells retained in the reaction vessel by the centrifugal force form a sediment on the inner wall of the reaction vessel 1, while the liquid in the reaction vessel is exchanged by supplying fresh solution through the opening 5.
  • Extensive removal of the liquid from the interior of the reaction vessel 1 is possible by means of additional discharge pipes for drawing off the liquid, which are not shown in this figure.
  • a suction line the opening of which is arranged near the cylindrical jacket of the reaction vessel 1 and which is closed in the normal operating state, is opened, whereby the liquid pressed against the wall of the reaction vessel 1 by the centrifugal forces is drawn off.
  • the flat, radially inwardly directed parts 3 designed as ring disks allow the liquid to flow in from the inlet 5 of the reaction vessel 1 to the suction line and thus largely replace the liquid content of the reaction vessel 1 and also wash away "old" liquid components a reduced liquid volume, which is generally necessary for the renewal of the liquid phase.
  • FIG. 9 shows a schematic operating mode of the device according to the invention, in which the biocatalysts adhering to the inner surfaces of the reaction vessel 1, namely cells, or the functional cell components, such as enzymes, are used for a specific reaction become.
  • the ratio of the settable and wettable surface to the reactive liquid volume in the reaction vessel is important for the following the biological implementation, in particular for the achievable reactor space / time productivity.
  • the loading density of the inner surfaces of the reaction vessel according to the invention with biocatalysts depends on the choice of the material for the coating and, if necessary, on additional measures.
  • Glass, metal alloys, porcelain or plastics, etc. can be used as the material for the execution of the inner surfaces of the device according to the invention, which, because of their charge or because of their chemically reactive groups, enable the binding of a biocatalyst.
  • Inert materials can also preferably be used for the treatment of suspended biocatalysts.
  • Polymerization reactions or reactions which enable the binding of biocatalysts due to a change in temperature can also be used to load the active surface surfaces of the reaction vessel are used.
  • the rotating vessel 1 is rotatably mounted about a horizontal longitudinal axis 1 'and is provided with an end plate 1 "at the left end.
  • Gaps 2 are formed in the interior of the reaction vessel, the upper edge of which determines the liquid level 2' in the lower part.
  • These reaction gaps 2 are delimited by flat, radially inwardly directed parts 3.
  • the flat, radially inwardly directed parts 3 are designed in different ways: 3a is an unperforated washer which can act as a separating washer Provided surface structures, for example with grooves, ridges or other surface deformations.
  • a perforated washer 3c is used to flow through the liquid.
  • the washer 3d consists of segments arranged in a ring. All washers 3a to 3d are provided with central holes 3 '. 3 "are ring-shaped mounts that facilitate the ring slices to keep the reaction vessel first Instead of the ring-shaped holders, it is also possible, for example, to use simple extensions of the ring disks or to insert an O-ring in the circumference of the ring disks.
  • the ring disks are expediently provided with coverings 3 ′′, which can consist of pile, fleece or sponge, so that the active surface is enlarged. Of course, agents can also be used. turn, which increase the wettability of the surface.
  • the rotating reaction vessel is mounted on two pairs of rollers 4, two further pairs of rollers 4 holding the surface of the reaction vessel from the upper part in order to secure the position of the reaction vessel 1 even at high speeds at which the Distribution of the liquid can reach due to the rotational force.
  • the rotational force can be used for mixing as well as for sedimentation.
  • the shafts of the rollers 4 are designated 4 '.
  • the brackets for the shafts 4 ' are not shown, since these are self-evident constructions which are generally known. This also applies to other components, for example the motor drive of the reaction vessel and the fastening means of other containers.
  • the opening 5 serves for the supply of gas and nutrient solution.
  • the opening 6 on the right side of FIG. 1 is intended for the removal or removal of the gas and the nutrient solution.
  • This opening 6 is provided with a flange 6 '.
  • the gas supply is shown with a thick arrow, with a thick arrow in the right part, the gas discharge line 8.
  • a discharge line 19 leads from the left-hand container 14 and two removal lines 20, 21 from the right-hand container 15. The liquid is located in the bottom 22. Sedimented biocatalysts are identified by 23 and sedimented carrier particles by 24. A measuring probe 25 can measure various desired values.
  • FIG. 1a shows in detail and on an enlarged scale the left part of the reaction vessel 1 from FIG. 1.
  • the perforated annular disks 3c allow the flow of the liquid between the reaction interstices 2 and that in contrast the Unperforated washer 3a serves as a separating washer, which separates the left reaction spaces 2 from the right.
  • Rollers 4 also show the shafts 4 'already mentioned.
  • FIG. 2 shows the four examples of the ring disks 3a to 3d already mentioned are shown.
  • the annular disk 2a is also provided with a coating 3 ′′, which is clearly visible in FIG. 3, which shows the section III-III from FIG. 2a.
  • FIG. 2b a structural design is shown where the annular disk 3b has embossed surfaces; in FIG. 2c there is a perforated annular disk 3c and in FIG. 2d the annular disk is divided into four segments 3d, so that there are free spaces between them for the flow of the liquid, which have the same task like the holes 3c 'in the perforated washer 3c according to Fig. 2.
  • Fig. 4 we see two washers 3. In these two washers 3 the outer diameter D, the liquid height HL and the reaction space length LR are shown .
  • the embodiment according to FIG. 5 has a conical reaction vessel 1, the non-perforated annular disks 3a to sufficient for a uniform liquid level 2 1 .
  • the solution according to FIG. 7 shows an embodiment in which cells suspended in the nutrient solution are shown schematically in the reaction spaces 2.
  • the separate segments 3d shown in FIG. 2d are arranged in the form of a screw inside the reaction vessel 1 and can also be designed as at least one continuous screw conveyor.
  • This solution has the advantage that the liquid is transported in the corresponding direction with respect to the rotational movement of the reaction vessel.
  • the segments 2d can also form a screw-like conveyor strip.
  • the reaction vessel 1 is driven at high speed, so that the nutrient solution is distributed over the entire inner circumference of the reaction vessel and the biocatalysts sediment.
  • FIG. 9 shows a state of the device in which 3 cells or biocatalysts are immobilized in layers 13 on the inside of the reaction vessel and the flat, radially inwardly directed parts.
  • Fig. 10 shows a solution in which, for example, two previously described devices are connected in series. Of course, a larger number of these devices can be connected in series.
  • 11 shows an embodiment in which the sedimented carrier particles 24 are shown schematically. The remaining functionality corresponds to that already described.
  • reaction vessel 1 is subdivided with an unperforated washer 3a.
  • This washer 3a is. also provided with the ring-shaped holder 3 ", so that its position in the longitudinal direction in the reaction vessel can be adjusted.
  • the gas supply pipe 7 is mounted in the cover plate 1"
  • the gas discharge pipe 8 12 clearly shows the length of these tubes.
  • a shorter L-shaped tube 26 for the liquid supply line is shown, which is provided with a part 26 ′ bent at right angles.
  • a longer L-shaped pipe 27 for the liquid supply line is arranged, the also has a part 27 'bent at right angles.
  • a shorter L-shaped tube 28 for the removal of liquid with a part 28' bent at right angles is also shown in the same lens 1 ".
  • a longer L-shaped tube 29 for liquid removal is shown, which also has a part 29 * bent at right angles.
  • the latter two pipes 28 and 29 are rotatably arranged in the connecting disk so that their parts 28 1 , 29 'bent at right angles can be moved up and down by rotating the horizontal part of the pipes.
  • FIGS. 12 and 13 show these tubes 28 and 29 in the position in which their ends extend to the bottom of the reaction vessel 1, so that the liquid can be emptied completely.
  • Nutrient medium Dulbecco's DMEM H21 (Gibco) plus 5% fetal calf serum (PAA laboratory, Gallneumaschinen) was used for all experiments without further additives.
  • the cell line was (Denmark disposable goods, the Fa. Nunc AG) preferred initially in Rouxflaschen 4 days at 37 ⁇ C and hier ⁇ of glass on the entire contents of Roux flask to a roller bottle (standard version) transferred with fresh nutrient medium for another 3 days at 37 ⁇ C and a roller speed of approx. 1 revolution per minute. After 3 days, the roller bottle was shaken by hand to suspend cells adhering to the wall and the cell number and the IgG content were analyzed. 100 ml of the culture thus controlled were used for inoculating the device according to the invention.
  • the reactor was spaced apart with 12-hole, lamellar ring disks of about 1 cm packed together. At a distance of 13 cm from the reactor inlet, an annular disc 3a was used, which was only provided with a central opening 3 ', so that the reactor was divided at a distance of 13 cm in the longitudinal direction. A further 30 ring disks at a distance of approximately 1 cm were located in the second segment of the reactor.
  • the total length of the reactor was 50 cm, the diameter was 10 cm and the reactor contained a total of 43 ring disks 3.
  • the material of the reactor was made of glass. Polyester was used for the ring washers, the surface of which was modified in a fibrous manner. The outer diameter of the washers was 10 cm, the inner diameter was about 5.5 cm.
  • the sterile and empty reactor was preheated in an incubation room at 37 C ⁇ and rolled on the roller apparatus (New Brunswick ;.) at about 1 rpm. At the same time, the reactor was gasified with a sterile, moist gas mixture of 95% air and 5% CO2. Hydrophobic filters with an exclusion limit of 0.2 micrometers (Pall) ensured sterility.
  • the empty reactor was inoculated with a 100 ml inoculum from the roller bottle by means of a peristaltic pump through a feed line into the inlet of the reactor. The reactor was rolled at approximately 1 rpm throughout the procedure. The inoculation took approximately 10 minutes, the cell inoculum fed into the first segment being largely absorbed by the 13 lamellae as capillary fluid.
  • the reactor was then further rolled with 1 rpm, and aerated mixed per hour with 95% air, 5% CO2 without further addition of culture medium with about 0.5 liters of gas mixtures and incubated at 37 C ⁇ .
  • Fresh nutrient medium was then metered into the inlet of the reactor until the entire reactor in the first and second segments was filled with a liquid level of approximately 2.5 cm.
  • the metering pump for the nutrient medium was then switched off. During the filling, which lasted approx. 0.5 hour, the rolling speed of the reactor was reset to approx. 1 rpm in individual steps and incubated further at 37.degree.
  • the total volume of nutrient medium in the reactor was then about 950 ml, and the aeration rate with air / CO 2 mixture was arbitrarily increased to about 5 liters per hour.
  • the admixture of CO2 was reduced over the following 3 days from approx. 4% CO 2 content to approx. 1% by hand in order to keep the pH constant.
  • dilution rate 100 ml of fresh nutrient medium per hour, corresponding to a dilution rate (D) of 0.1, was initially metered in over four days. Samples were taken from the outlet of the reactor for the purpose of determining the cell density and the IgG concentration. The dilution rate was then increased arbitrarily to 0.5, corresponding to a flow rate of approximately 500 ml of nutrient medium per hour, and operated for a further four days.
  • the cell number in the samples was determined by counting in the thoma chamber (vital staining with trypan blue).
  • the concentration of IgG in the culture supernatant or the outlet of the reactor was determined using the ELISA technique.
  • Anti-mouse IgG immune serum (from the PAA laboratory, Gallneumaschinen) was precoated in microtiter plates according to standard conditions. The culture supernatants were then incubated on the plates prepared in this way, washed and the IgG content was determined using anti-mouse IgG-goat serum conjugate (from PL Biochemicals, Inc., Milwaukee, WI). Mouse IgG from Sigma (St. Louis, Missouri) was used as the IgG standard. Results
  • the specific IgG production rate (per cell quantity and time) of the hybridoma line used should be a constant for the nutrient medium used, that is to say a variable independent of the cultivation method, then the enrichment factor for the Immobilization of the cells in the film reactor about 6 to 10 times the value compared to other methods.

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Abstract

On aménage dans un récipient à réaction (1) à axe horizontal des parties (3) planes et radiales qui sont dirigées vers l'intérieur et entre lesquelles se trouvent des chambres à réaction (2) où se trouve le liquide de réaction. Ces surfaces radiales (3) dirigées vers l'intérieur peuvent être des disques sans ouvertures (3a) qui constituent des simples séparations. Elles peuvent aussi être percées (3c) de trous (3c') permettant le passage du liquide de réaction. On peut aussi avoir des anneaux constitués de segments circulaires juxtaposés ou ayant une forme à ressort en spirale. Dans la zone de l'axe de rotation (1') du récipient à réaction (1) se situe l'amenée de gaz (7'), l'évacuation de gaz (8) et au moins une conduite (9) ou un endroit d'amenée de liquide. On amène dans le récipient de réaction le liquide de réaction, les biocatalyseurs et le gaz de réaction. Lorsque la concentration des biocatalyseurs est suffisante, on introduit d'autres liquides de réaction et on évacue le produit de réaction.
EP86900043A 1984-12-28 1985-12-23 Installation et procede pour la culture et le traitement de systemes biocatalytiques Withdrawn EP0207103A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AT0412084A AT383142B (de) 1984-12-28 1984-12-28 Vorrichtung zur kultivierung und behandlung von biokatalysatoren
AT4120/84 1984-12-28

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EP0207103A1 true EP0207103A1 (fr) 1987-01-07

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EP86900043A Withdrawn EP0207103A1 (fr) 1984-12-28 1985-12-23 Installation et procede pour la culture et le traitement de systemes biocatalytiques

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EP (1) EP0207103A1 (fr)
JP (1) JPS62501536A (fr)
AT (1) AT383142B (fr)
WO (1) WO1986004085A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4962033A (en) * 1987-12-09 1990-10-09 In Vitro Scientific Products Roller bottle method of culturing cells
US5010013A (en) * 1987-12-09 1991-04-23 In Vitro Scientific Products, Inc. Roller bottle for tissue culture growth
DE4112236C1 (fr) * 1991-04-15 1992-07-09 Forschungszentrum Juelich Gmbh, 5170 Juelich, De
US5151366A (en) * 1991-05-24 1992-09-29 Invitro Scientific Products, Inc. Cell culture flask
US5272084A (en) * 1991-12-18 1993-12-21 Corning Incorporated Cell culture vessels having interior ridges and method for cultivating cells in same
FR2688007A1 (fr) * 1992-02-27 1993-09-03 Bourgogne Universite Bioreacteur a ecoulement piston destine a la culture de cellules et des microorganismes.
EP1304370A1 (fr) * 2001-10-22 2003-04-23 Integra Biosciences Holding AG Insert pour flacon rotatif

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Publication number Priority date Publication date Assignee Title
DE2337056B2 (de) * 1972-07-21 1976-12-02 Worthington Biochemical Corp., Freehold, N.J. (V.StA.) Vorrichtung zur erzeugung eines kontakts zwischen einem immobilisierten biologischen reaktionsteilnehmer und einer substratloesung
US4238568A (en) * 1978-10-10 1980-12-09 Becton, Dickinson And Company Roller bottle
GB2055397B (en) * 1979-06-05 1983-02-02 Univ Strathclyde Rotating biological film contactor
US4317886A (en) * 1980-08-11 1982-03-02 Becton, Dickinson And Company Multiple interior surface roller bottle
GB2097817B (en) * 1981-03-16 1985-01-16 Prendergast Angela Fermentation apparatus
US4377639A (en) * 1982-01-18 1983-03-22 University Of Toledo Tissue culture device for mass cell culture
DE3246590A1 (de) * 1982-12-16 1984-06-20 Josef 8000 München Neubauer Einrichtung zur gewinnung von energie in form von biogas oder rottewaerme

Non-Patent Citations (1)

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Title
See references of WO8604085A1 *

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AT383142B (de) 1987-05-25
JPS62501536A (ja) 1987-06-25
ATA412084A (de) 1986-10-15
WO1986004085A1 (fr) 1986-07-17

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