EP0207103A1

EP0207103A1 - Installation and method for the culture and treatment of biocatalysts

Info

Publication number: EP0207103A1
Application number: EP86900043A
Authority: EP
Inventors: Hermann Katinger
Original assignee: MBR Bio Reactor AG
Current assignee: MBR Bio Reactor AG
Priority date: 1984-12-28
Filing date: 1985-12-23
Publication date: 1987-01-07
Also published as: JPS62501536A; ATA412084A; WO1986004085A1; AT383142B

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.In a reaction vessel (1) having a horizontal axis, planar and radial portions (3) which face inwards and between which there are reaction chambers (2) in which the reaction liquid is located are arranged. These radial surfaces (3) facing inwards can be disks without openings (3a) which constitute simple separations. They can also be pierced (3c) with holes (3c') allowing the passage of the reaction liquid. It is also possible to have rings made up of juxtaposed circular segments or having a spiral spring shape. In the region of the axis of rotation (1') of the reaction vessel (1) there is located the gas supply (7'), the gas outlet (8) and at least one line (9) or a liquid supply point. The reaction liquid, biocatalysts and reaction gas are supplied to the reaction vessel. When the concentration of the biocatalysts is sufficient, other reaction liquids are introduced and the reaction product is discharged.

Description

Device and method for cultivating and treating biocatalysts

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.

In the known devices of this type, the so-called 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. Such 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 advantages of the rolling culture and its routine use for cultures of all kinds can clearly be attributed to this. - 2 -

that the rotation, that is to say the rolling, of cylindrical bottles in slow motion represents the gentlest method of aerating and / or keeping a cell system in suspension.

However, 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. Furthermore, the enlargement of the operating scale, i.e. the scale up, is limited to the use of multiples of the number of roller bottles. In addition, immobilization of the cells, i.e. an artificial enrichment of the cells beyond the natural equilibrium is not possible in a continuous culture. Finally, 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.

According to the invention, 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. This makes it possible to use the gentle physical principles of roller cultivation and, moreover, to operate with all known methods of cultivation continuous cultivation of cells or biocatalysts, the immobilization of cells or other biocatalysts with practically unlimited interphasial mass transfer. Thus, a high interphasical mass transfer is ensured under physically gentle conditions between the solid biophase, the liquid phase and the gas phase.

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.

In addition to the conditions mentioned for maximum interphasial mass transfer under gentle physical 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. Finally, 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 general problem in the cultivation of sensitive cells is described, for example, in Acta Biotechnologica 2 (1982) 1, 3-41. 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. According to an expedient solution, the flat, radially inward parts consist of separate segments arranged in a ring. One advantage of this solution can be seen in the fact that there are free passage gaps between the segments through which the liquid can flow. Another type kon¬ structurally simple since s ^* is rich communicating the gaps to ER that at least some of the flat radially perforated inwardly directed parts. 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. For a particularly good interphasial mass transfer, the flat, radially inwardly directed parts can be designed for the surface absorption of the biocatalysts or the reaction liquid. For this purpose, the flat, radially inwardly directed parts can be surface-modified. In order to be able to fix the biocatalysts in a particularly favorable production ratio on the flat, radially inwardly directed parts, the biocatalysts immobilizing coatings can be applied to these parts.

In order to achieve a slight scale up, the 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.

Further advantages of the device according to the invention can also be seen in the fact that an artificial enrichment of cells, an immobilization of soluble or suspended biocatalysts or the achievement of the reaction kinetics of a tube reactor, a so-called plug flow reactor, to increase the space / time yields and Amount of product formed per unit volume is enabled, and that the _# artificial enrichment of cells or biocatalysts can be achieved through chemical bonds. Furthermore, an artificial enrichment of single cells in the free suspension of cell aggregates or pellets, of cells on microcarriers or in microcapsules is possible beyond that which is predetermined by the principles of continuous culture (flow cultures). The 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. In addition, due to the design of the geometric configuration of the apparatus and its operating conditions, 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.

The invention is explained in more detail with reference to some drawings:

Show it:

1 shows a schematic section through an overall system according to the invention, the guidance of the phases through the reaction vessel being indicated,

1a shows the left part of the reaction vessel from FIG. 1 on an enlarged scale,

2 four embodiments of the flat, radially inwardly directed parts,

3 shows the section III-III from FIG. 2a, - 7 -

4 two ring disks in perspective view,

5 shows a solution with a conical reaction vessel and with the same liquid level,

6 shows a variant with a conical reaction vessel and with annular disks arranged in stages,

7 schematically shows the culture of suspended cells with a screw-like conveyor strip,

8 shows the distribution of the liquids in the reaction vessel in the case of cells retained in the reaction vessel by the centrifugal force of the rapidly rotating reaction vessel,

9 shows the mode of operation of the device according to the invention with cells or biocatalysts immobilized on the inside of the reaction vessel,

10 shows a combination of individual reaction vessels to form a reactor assembly, any combinations being possible depending on the use of the device,

11 shows an embodiment in which the sedimented carrier particles are shown schematically,

12 shows a schematic arrangement of the supply and discharge pipes and

13 shows a view from the left of the solution according to FIG. 12.

1 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. In the execution Examples, with the exception of FIGS. 2d and 7, 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. These parts 3 lie with their outer edges directly against the inner wall of the reactor vessel 1. As shown schematically in FIG. 1, 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. As a result, 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 Längsach¬ 1 ¹ is rotatable. In Fig. 1, 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. For this purpose, 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.

During operation, the 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).

In the overall arrangement shown in FIG. 1, 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. However, it is also possible to carry out the liquid supply or removal specifically for each individual intermediate space 2, the intermediate spaces 2 being divided by interposed non-perforated annular disks 3a. As a result of the geometric configuration, a ratio of wettable surfaces with respect to a certain filling volume, which is ideally adapted to the process, can be achieved, the number and structure of the flat, radially inwardly directed parts 3 and also the jacket shape of the reaction vessel 1 are decisive. 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. 7), which, in addition to the surface enlargement, also has a promoting effect on the reaction fluid, for example in the case of sedimented cells If the biocatalysts are immobilized or immobilized on microcarriers, the cells are moved against the flow of liquid through the reaction vessel by a counter-rotating screw, which correspondingly extends the dwell time of the cells in the reaction liquid.

For the cultivation of cells, 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. If it _* voltage by improper handling or a possible malfunction to a liquid outlet through the Öff¬ 5 come, then the liquid in the tank 14 is moved up caught and discharged via line nineteenth The finished reaction product exits according to the arrows 11 along the flange 6 'into the container 15, from which it is removed via the line 20 or 21. Part of the liquid, that is to say the nutrient medium, forms a liquid film over the entire area of the inner surface of the reaction vessel 1 lying above the liquid bulk and the flat, radially inwardly directed parts 3. Through the large interface between the liquid and the liquid film On the one hand and the introduced gas on the other hand - usually air and / or CO2 - a high interphasial mass transport is guaranteed, which means that both the supply of cells with oxygen and / or other gases and the setting of a desired pH Value is made.

In the event that the control of the pH value via the gas phase is insufficient, 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.

In the case of flat, radially inwardly directed parts 3 which are not provided with a coating that immobilizes the cells, the majority of the suspending cells or the carriers immobilizing the cells are located in the fluid bulk of the reaction vessel 1. Depending on the sediment behavior of the cells, 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.

In the case of the culture of cell pellets, cell aggregates, microcarriers or of cells or biocatalysts in microcapsules, 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.

If non-aggregating individual cells with slow sedimentation are cultivated in the device according to the invention and the flushing out of the cells from the reaction vessel or from the individual reaction spaces is to be kept lower than the natural cell growth rate. speaks, then a brief interruption of the rotational movement of the reaction vessel 1 must be inserted before the nutrient solution is metered in. The duration of the standstill of the reaction vessel depends on the one hand on the sedimentation behavior of the cells and on the other hand on the desired degree of enrichment of the cells. The operating state brought about by the standstill of the reaction vessel is illustrated in FIG. 9.

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. For biological reactions with high demands on the interphasial material transport, 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. Depending on the opening angle of the jacket, the distance and the height of the individual ring disks 3a to one another, 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. 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. In this variant of the operation, 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. As already mentioned above in connection with suspended particles, in this case 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.

For the use of the device according to the invention using biocatalysts which are specifically bound to the effective surfaces, regardless of whether they are cells or their functional components, analogous principles apply as compared to particles suspended in the liquid with regard to the interphasial mass transfer in relation to the surface / volume ratio and the residence time behavior of liquid particles and the biocatalyst. The effective surface, which is available for binding the cells and / or other biocatalysts through electrostatic conditions or other binding mechanisms, is important for the result of the reaction carried out in the reaction vessel. 9 shows a schematic representation of the biocatalyst layers 13 immobilized on the inner surfaces of the reaction vessel for converting the reaction liquid in a continuous process.

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 previous description based on a few drawings had served to explain the subject of the invention in a simple manner. It goes without saying that all drawings represent the subject of the invention or its components and its mode of operation. In all drawings, the same parts are provided with the same reference numbers. All drawings are clearly described below, the reference numbers being described numerically one after the other.

1 shows a schematic section through an overall insert, the guidance of the individual phases through the reaction vessel 1 being indicated. 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. 1, 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 '. In the left part of the drawing, the gas supply is shown with a thick arrow, with a thick arrow in the right part, the gas discharge line 8. In the left part, a line extending into the interior for the nutrient solution is shown in broken lines, with the arrows 10 indicating the Show directions in which the nutrient solution can flow. In the right part you can see the discharge 11 of the finished reaction product, which is shown with a dashed arrow. Tapping points of the reaction product are designated by reference number 12. A thin layer 13 of reaction liquid forms on the parts which are effective for the reaction, which promotes the exchange between the reaction liquid and the reaction gas. In order to create the connection between two lateral containers 14 and 15 with the rotating reaction vessel 1, labyrinth seals 16 are implemented on both sides. The walls of the containers 14, 15 serve as part of these labyrinth seals, with annular disks 16 ′ of the labyrinth seal 16 opposite them - 1 9 -

are attached. Dotted arrows 17 show escaping reaction gas. The entire device is stored and fastened in a connected room 18. 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. In this drawing it is clearly visible that 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. Both

Rollers 4 also show the shafts 4 'already mentioned.

2 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. In 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. In 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 .

6 again uses a conical reaction vessel 1, but the annular disks 3a are arranged in stages, so that a gradual gradation of the liquid level 2 'is also achieved. With the reference number 13, thin layers of reaction liquid which form during operation are provided on the annular disks 3a and inner surfaces of the reaction vessel 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. In this example, 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.

8 shows a use of the device according to the invention as a centrifuge. 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.

12 and 13 show the left part of a reaction vessel, this part being used both for supplying and for removing liquids and gases. In this example, the 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. As can be seen particularly well in FIG. 13, the gas supply pipe 7 is mounted in the cover plate 1", likewise the gas discharge pipe 8 12 clearly shows the length of these tubes. In the lower part of the cover plate 1 ″, a shorter L-shaped tube 26 for the liquid supply line is shown, which is provided with a part 26 ′ bent at right angles. Similarly, 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 ". In addition, 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. 13 shows that the tubes 28 and 29 can be rotated in the directions of the arrows 30 in such a way that the ends of the said tubes come above the liquid level 2 'and therefore no suction of the liquid can take place. The exact function of the device according to the invention is described below with reference to a method example carried out with this device.

EXAMPLE OF ILLUSTRATING THE FUNCTION OF THE REACTOR

Materials and methods used

Cell line: Mouse / mouse hybridoma line 3RFUD6C3 from the collection of the Institute for Applied Microbiology (IAM) produces a monoclonal antibody of the IgG class with unspecific specificity and was used for all experiments.

Nutrient medium: Dulbecco's DMEM H21 (Gibco) plus 5% fetal calf serum (PAA laboratory, Gallneukirchen) was used for all experiments without further additives.

Preparation of the inoculum:

The cell line was (Denmark disposable goods, the Fa. Nunc AG) preferred initially in Rouxflaschen 4 days at 37 ^β 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 ^β 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.

Experimental arrangement with the device according to the invention (reactor for short)

For carrying out the experiment, 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 reactor constructed in this way was thoroughly cleaned in succession with 0.1 N HC1, with tap water, with 0.1 N NaOH and finally with distilled water and then sterilized in an autoclave at 120 ° C. for 1 hour (n = normal).

The sterile and empty reactor was preheated in an incubation room at 37 C ^β 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.

After the inoculum was in the reactor, about 150 ml of fresh nutrient medium were metered in with the same peristaltic pump via a T-piece in the suction tube. The first The segment of the reactor was then filled to a liquid level of approximately 2.5 cm.

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 ^β.

After approx. 24 hours the CO 2 content of the gas mixture rose slightly to approx. 3-4% CO2. 96-97% air reduced in order to keep the pH to approx. PH 7.0, visually recognizable by the indicator color of the nutrient medium. A visual inspection through the glass container could not detect any visible clouding of the nutrient medium. Approximately 60 hours after the start of the culture, the rolling speed of the cultivation was increased to approximately 30 rpm. Just a few minutes after the rolling speed increased, a clear turbidity of the nutrient medium indicated that hybridoma cells suspended from the surface of the lamellar ring disks in the nutrient medium.

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.

Approximately 72 hours after the reactor was refilled, i.e. approximately 100 hours after the first inoculation, - 25 -

allow the reactor to be fed continuously with fresh nutrient medium in order to initiate a continuous perfusion culture. 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 processes from the reactor were analyzed with regard to cell number and IgG concentration. The results are shown in Table 1.

Analytics

Cell count

The cell number in the samples was determined by counting in the thoma chamber (vital staining with trypan blue).

IgG concentration

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, Gallneukirchen) 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

Table 1: Results of the culture of 3RFUD6C3 during the culture in the film reactor with DMEM + 5% FCS

Culture method reactor type cell number IgG

Time (10 ^β Z / ml) (µg / ml)

(Hours)

Batch approx. 96 Roux bottle 1.2 260

Batch approx. 72 roller bottle 1.4 240

Batch approx. 72 film reactor - -

Perfusion 1 / D ca. 1 / 0.1 film reactor approx.0.08 * 190 *

Perfusion 1 / D approx. 1 / 0.5 film reactor approx. 0.06 * 85 *

* in the process

The results from Table 1, in particular the comparison of the individual cultivation times with the different cultivation methods, allow the following conclusions:

1) Assuming that 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.

2) If the immobilization factor stated under point 1) should not apply to the cells, this would mean that an increased specific production rate of the formation of the IgG can be ascertained in the film reactor.

3) Whatever the cause, whether point 1) or Point 2) is more valid, the volumetric productivity of the monoclonal antibody formation (= IgG / reactor volume x time) is increased by 6 to 10 times in the film reactor according to the invention and in the concrete experiment compared to a stationary suspension culture.

It goes without saying that the subject matter of the invention is not limited to what is shown. The number of annular disks 3 can thus be adapted to the specific one, which of course also applies to the supply and discharge lines. The drive of the device according to the invention can also be designed in a manner known per se differently than shown.

Claims

Patent claims

1. A device for culturing and treatment of Biokata¬ catalysts, such as cells, particles or soluble chemical and / or biological reactions influencing ^'Substan¬ zen, in which a horizontally disposed reaction vessel (1) about its longitudinal axis (1' rotatably) arranged and provided with at least one reaction chamber (2), characterized in that in the reaction vessel (1) to its longitudinal axis (1 ¹ ) extending flat parts are arranged that the supply of the reaction liquid at one end of the reaction vessel ( 1) and the discharge of the finished reaction product are formed at the same or different end of the reaction vessel (1) and that the inside of the reaction vessel (1) can be charged with gas. (Figs. 1, 4 to 12)

2. Device according to claim 1, characterized in that the flat parts (3) extending to the longitudinal axis (1 ') consist of ring disks (3a to 3c). (Fig. 2)

3. Device according to claim 2, characterized in that at least some of the flat, radially inwardly directed parts (3) - are designed as perforated washers (3c). (Fig. 2c)

4. The device according to claim 2, characterized in that at least some of the flat, radially inwardly directed parts (3) are designed as annular segments (3d). (Fig. 2d)

5. The device according to claim 1, characterized. that at least some of the flat, radially inwardly directed parts (3) are designed as screw-like conveyor strips. (Fig. 7)

6. The device according to claim 1, characterized in that the flat parts extending to the axis

(3) have formed their surface for receiving biocatalysts or the reaction liquid. (Fig. 2b)

7. The device according to claim 6, characterized in that the flat parts extending to the axis

(3) are surface modified, e.g. with the help of the surface structure or with the help of increased wettability. (Fig. 2a, b)

8. The device according to claim 7, characterized in that on the flat parts extending to the axis

(3) immobilizing documents are applied. For example, Pile, fleece. (Fig. 8)

9. The device according to claim 1, characterized in that in the reaction vessel (1) at several points (9, 10) the reaction liquid can be supplied and / or the finished reaction product at several points (12) of the longitudinal extension of the reaction vessel (1) can be removed . (Fig. 1, 12)

10. The device according to claim 1, characterized in that between the flat, radially inwardly extending parts (3) reaction intermediate spaces (2) for the suspended in the reaction liquid carrier particles for the biocatalysts are arranged. (Fig. 11)

11. Process for the cultivation and treatment of biocatalysts with the device according to claim 1, thereby indicates that the reaction liquid and the biocatalysts are introduced into the reaction vessel (1), the reaction vessel is set in rotation and the interior of the reaction vessel is continuously charged with reaction gas.

12. The method according to claim 11, characterized in that after reaching the desired concentration of Biokata¬ analyzers further reaction liquid is introduced and the finished reaction product is removed.

13. The method according to claim 11, characterized in that the reaction liquid is introduced into the reaction vessel (1) at a plurality of points (9, 19) and discharged from the reaction vessel (1) at a plurality of points (12), e.g. is suctioned off.

14. The method according to claim 13, characterized in that one or more suction pipes (28, 29) are lowered into the reaction space or spaces (2) between the ring disks (3a) for suctioning off the reaction product.

15. The method according to any one of claims 11 to 14, characterized in that the speed of rotation of the reaction vessel (1) is increased in order to mix the phases in the reaction vessel (1).

16. The method according to any one of claims 11 to 14, characterized in that for separating the denser phase containing the biocatalysts, from the reaction liquid having a lower density, the reaction vessel (1) is set in rapid rotation, by sufficient To generate centrifugal force.

17. The method according to any one of claims 11 to 15, characterized in that a plurality of reaction vessels (1) are switched in series.