Patent Application
20060141614
The present invention relates to a device for cultivating cells, an arrangement of devices of this type, a agitation system suitable for this purpose and also a culture method for cells. Devices and methods of this type are required for cultivating cells on a millilitre scale. These are used in particular for parallel batches during strain or bioprocess development in the chemical industry, e.g. for reaction optimisation or catalyst optimisation, in the field of environmental protection, for the optimisation of sewage treatments or chemical or biological treatment of solid materials or exhaust air, or in the field of food technology.
Agitated flasks or mixing vessels have been used to date as reactors for cultivating cells in liquid columns on a millilitre scale, as is required in particular for parallel reactions for testing specific biotechnological techniques.
The standard parallel reactor in biotechnology is the agitated flask, with which simple batch experiments in a parallel batch have been implemented manually for the last century. Agitated flasks are mounted on agitated tables, set in a rotational movement with given eccentricity in incubators at a prescribed temperature at a specific agitation frequency. Due to the movement of the reaction vessel, mixing of the liquid which is contained in the reaction vessel and in which the biochemical reaction takes place, is effected.
Via surface aeration, the oxygen required for many biochemical reactions is fed into the liquid phase from the gas phase. High oxygen transfer rates are consequently only possible if a very large surface/volume ratio is set. This means that very large agitated flasks (1-21 volume) with very little reaction medium (10-20 ml) must be operated with the greatest possible eccentricity and agitation frequency (400 rpm). Under these conditions, oxygen transfer coefficients kLa of up to 0.07 s−1 are achieved.
The power input in an agitated flask is effected by the friction of the liquid on the inner wall of the rotating reaction vessel. Hence, a relatively uniform energy dissipation is effected.
The advantages of the agitated flask cultivation are simple handling and relatively low technical outlay.
Alternatively hereto, also stirred tank reactors can be used. Substantial reaction engineering differences between the reaction vessel, agitated flask, and the reaction vessel, stirred tank reactor,—the standard production reactor of biotechnology—are the lower oxygen transfer, the far smaller ratio of the maximum local energy dissipation to the average power input and the inadequate control of important process variables (such as for example pH or PO2). This leads to the fact that reaction courses in most cases cannot be directly transferred from the reaction system, agitated flask, to the reaction system, stirred tank reactor, and hence, in bioprocess development, additional personnel and time-intensive sequential experiments in laboratory bioreactors are required.
Technical attempts to resolve this problem are the provision of parallel mixing vessel units with completely individual measuring and control technology. Parallel reactor systems with 4 or 6 stirred tank reactors with a volume of up to 0.5 l are commercially available. The obtained process data can generally be transferred readily to larger stirred tank reactors. The capital, personnel and time expenditure is however exceptionally high if a plurality of these parallel reactor units need to be used for bioprocess development.
A newer development is the operation of parallel small-scale reactors in an incubator with intermittent substrate dosage and parallel pH control (DE 197 09 603 A1). Either bubble columns or agitated columns with an operating volume of 200 ml are used as parallel small-scale reactors (DE 195 29 099 A1). Hence, oxygen transfer coefficients and volume-specific power input, as in the standard mixing vessel reactor, can be achieved. The number of parallel bioreactors is however restricted (≦16). A further parallelisation is practically impossible on the basis of this technology. For simultaneous concentration measurement of key components of the reaction medium (substrate or product concentration), parallel sampling and analysis systems are used (EP 0995 098 A1).
A further simple possibility of operating far more reactors in parallel is the use of microtitre plates in incubation agitators for batch cultivation of cells. Microtitre plates with 24, 48, 96 or more wells for cultivating cells have however, to an even greater extent, the same technical reaction restrictions as agitated flasks. In addition, the evaporation on this scale has proved to be problematic, since the relative evaporation volume flow relative to the initial volume, due to the very large surface/volume ratio and the small reaction volume (≦1.5 ml) is very much larger than for example in the agitated flask or stirred tank reactor.
In order to be able to rapidly implement new biological knowledge into technically achievable and economical methods, the sequential procedure to date with simple parallel batches in the agitated flask and subsequent optimisation of the reaction conditions in the controlled laboratory stirred tank reactor must be overcome. This is only possible if as large a number as possible of mixing vessel reactors can be parallel-operated in an automated fashion under technical, controlled reaction conditions.
In order to make parallelisation of mixing vessel reactors possible, these must be constructed as simply as possible and must be operated as far as possible without baffles. Ideal reaction vessels are for example sterilisable reagent glasses or microtitre plates with correspondingly large wells.
The power input can be effected simply by magnetic agitator drives on this scale. In technical agitation reactors, the oxygen transfer into the reaction medium with volume aeration is determined primarily by the power input of the impeller and secondarily by the superficial gas velocity. Primary dispersion of the gas phase, as is effected in the standard stirred tank reactor via a gas distributor on the reactor base, can however only be achieved in a very complex manner in millilitre parallel-operated stirred tank reactors. The reaction vessels would have to be provided with an individual gas supply and a gas sparger. The gas sparger would have to ensure that the desired superficial gas velocity was achieved exactly in each of the parallel reaction vessels.
It has only been possible to date to implement either a large number of simple, uncontrolled parallel reactions under non-technical conditions in microtitre plates or in agitated flasks or to operate a relatively small number of bioreactors under controlled, technical conditions.
Since it is necessary to achieve a high gas transfer into the reaction vessels, one option is to implement the sterile gas supply into the vessels from above. For this purpose, the simplest sterile boundary would be use of a sterile filter as a cover of the individual reactors or of an entire arrangement of reactors. Such a sterile filter, in addition to the mechanical barrier for contaminants, would however require to have good gas transfer properties in order to avoid oxygen limitation in the reaction vessels.
Furthermore, the cover of the reaction vessel represents the only possibility for intervening in the reaction course, for example for adding substrate, titration media or inductors during the reaction, for sample removal for process control or for introducing measurement probes. In order to be able to implement these interventions perfectly in a technically sterile manner, the sterile filter is usually configured as a septum. The gas permeability of septums, which are generally based on silicon, is however inadequate so that both functions cannot be fulfilled by one material. It is therefore problematic to have a simple and guaranteed sterile access to the reaction vessel or vessels, this access however being intended to be sterile in all circumstances.
It is therefore the object of the present invention to produce a device for cultivating cells in liquid columns on a millilitre scale, with which, on the one hand, a high gas and power input is achieved and, on the other hand, individual or also a large number of devices of this type can be operated effectively in a parallel manner. Furthermore, it is the object of the present invention to make available an arrangement of this type of parallel devices and also an agitation system with which the desired high power input and gas transfer can be achieved. The object of the present invention is in addition to make available corresponding culture methods of cells in liquid columns on a millilitre scale.
This object is achieved by the device according to claim 1, the agitation system according to claim 21, the arrangement according to claim 31 and also the method according to claim 49. Advantageous developments of the respective device, arrangement, agitation system or of the method according to the invention are given in the respective dependent claims.
With the solution according to the invention, it is possible to operate individual or also a multiplicity of automated stirred tank reactors, for example 24, 48 or 96 or more stirred tank reactors, both for strain and also for bioprocess development in a time-effective manner under technical reaction conditions. Hence, the parallel, automated cultivation of cells on a millilitre scale under individually controlled reaction conditions, such as temperature, hydrogen ion activity, oxygen feed, power input and also media supply is possible, so that reaction courses, as are achieved in the standard stirred tank bioreactor, can be implemented in a parallel batch. There is thereby understood by millilitre scale advantageously a range for the mixed liquid volume of 0.5 to 50 ml, preferably from 1 to 30 ml, preferably from 5 to 20 ml. By means of the device according to the invention and the method according to the invention, direct transfer of the thus obtained (fed batch) process courses from the millilitre scale according to the invention to the litre scale (and vice versa) is possible. The millilitre agitation reactors according to the invention permit an equally efficient oxygen supply of organisms in liquid culture as stirred tank reactors of a larger scale with volume gassing.
When using the stirred tank reactors according to the invention, parallelisation of a large number of bioreactors in one bioreactor block is possible, operation thereof being able to be automated for the first time by using laboratory robots, for example pipetting robots and the like and thus an efficient individually controlled parallel operation is made possible. Only the device according to the invention and also the cover according to the invention make possible the use of a laboratory robot and hence a quantum leap in obtaining relevant process data.
If the laboratory robots are operated with suitable screening and optimising routines, then process optimisation, for example with respect to media composition, induction methods and dosage profiles, can be achieved systematically and with high time efficiency. The complete digitalisation of the parallel process development makes possible furthermore a novel data transparency and data availability.
Since the volumes of the mixing vessel reactors, with which nevertheless meaningful information about the respective process course can be obtained, can now be greatly minimised, for example instead of 500 ml now merely 5 ml, with the same total volume by using 100× more reaction vessels, a multiple of information can be obtained or with the same quantity of information the time expenditure can be minimised by the achieved automation. When using suitable experimental planning algorithms, a quantum leap in the effectiveness of the bioprocess development can be made possible.
The present invention is based crucially on the fact that it was detected that the gas transfer from the surface of the liquid column into the liquid column in a mixing vessel reactor is improved as a result of the fact that either the container and/or the agitation system are configured in such a manner that the flow velocity is modified locally and/or temporally along a streamline or flow line which, in the case of a stirred tank reactor, extends in a circle. This leads to a spatially and/or temporally pulsating Bernoulli effect. This can lead for example to a flow field of the culture suspension which is directed towards the base of the mixing vessel reactor (container), which leads to intensive entry of gas bubbles. There are described as streamlines thereby lines in the flow, the direction of which is identical to the direction of the velocity sector at each ramming point. There are described as flow line lines through which liquid particles flow.
On the basis of this knowledge, it is now possible to configure either the container (stirred tank reactor) and/or the agitation system in a suitable manner.
It is possible, on the one hand, to configure the container itself such that its inner volume has no rotationally symmetrical shape. Where the liquid flow then widens, there is a region of low flow velocity and hence high pressure, whilst where the liquid flows through between the agitation system and the container wall, a region of high flow velocity and hence low pressure is present there. Hence, a flow velocity which varies spatially along the circumference of the container or a liquid pressure which varies is given.
The same effect is achieved if the agitation system is disposed off-centre or eccentrically within the container which has any shape or is shaped rotationally symmetrically. In this case, spacings between the agitation system and the wall of the container are again produced, which spacings vary along the circumference of the container and consequently induce different flow velocities and pressure ratios.
A further possibility for achieving this pulsating Bernoulli effect is to dispose baffles along the circumference of the container. There are thereby understood by baffles elements which are situated in the flow of the mixed liquid and thus represent a flow resistance for this. Baffles lead to a narrowing of the flow cross-section. Since the spacing between impeller and wall of the vessel is greater than between impeller and baffle, again regions of high flow velocity are formed between impeller and baffle and regions of low flow velocity between impeller and free wall regions. The baffle must thereby be disposed not in the rotational plane of the agitation system but can be disposed below, in the rotational plane or also or in addition also above the rotational plane of the agitation system. In all these cases a pulsating Bernoulli effect is produced. The baffles can advantageously be configured in one piece with the mounting of the agitation system or in one piece with the container, for example in the injection moulding method.
The gap spacings should thereby be chosen such that an adequate pulsating Bernoulli effect is produced, but the shear forces should not become so great that the cells contained in the suspension are destroyed. Gap spacings >0.05 mm, preferably >0.1 mm and/or <20 mm, preferably <3 mm, are particularly suitable for this purpose. There are suitable as containers standard mixing flasks, reagent glasses or also the wells of a microtitre plate or a specially prepared plate with the same cavity arrangement with adequate diameters.
A further possibility of producing a pulsating Bernoulli effect resides in configuring the agitation system in a suitable manner. For this purpose, a boring is introduced into the agitation system which boring extends from the underside and/or side wall of the agitation system to a side wall or to the upper side of the agitation system. Advantageously the boring extends at an angle α with 0°≦α<90° relative to the rotational or central axis of the agitation system, this angle opening upwards.
Advantageously also further through-channels with a corresponding opening can extend downwards from the upper side and/or side wall of the agitation system to the side wall or underside thereof.
These channels can also extend only partially through the agitation system at an angle α and then meet a further channel which, with respect to the plane perpendicular to the rotational axis, extends in this plane or linked at an angle <90° upwards or downwards to this plane and discharge in said further channel which for its part ends at the lateral outer wall of the agitation system with an opening.
Through borings of this type, a flow is likewise induced from the base of the container to the side wall of the agitation system, which leads to an altered pressure at the opening of the borings or channels which is orientated away from the rotational axis and thus induces a pulsating Bernoulli effect.
Advantageously, the vessels have a closure or cover which covers the vessel or an arrangement of vessels in a sterile manner. On the one hand, gas distributor structures can be introduced into these closures for the supply of sterile gas. For this purpose, the closure can comprise for example a double base plate, the gas distributor structure being disposed in the intermediate space between the two plates of the double base plate. The closure can also comprise one or more plates, the gas distributor structures being disposed on the underside of t he lowermost plate.
Advantageously, the gas distributor structures are set up in such a manner that, starting from a central gas supply, individual channels lead to the respective containers as branches. Advantageously the channels are thereby guided such that they both have the same cross-sections, the same length and the same number of bends or kinks. Consequently, a uniform gas pressure is effected on all the containers. Merely the branches can either be set up thereby in the same manner or else the entire system can be.
Furthermore, the closure advantageously has an opening for each individual container, through which sterile gas supplied externally of the container flows. This opening can for example be a tube through which a cannula or any other elongated sample removal or sensor unit can be introduced. Since this is then effected in counter-flow, it is merely required to sterilise the respectively introduced unit in advance and then to introduce the tube into the suspension in order to avoid contamination of the reaction vessel.
Furthermore, the closure can have webs which, in an arrangement of containers, isolate the individual containers from each other in a sterile manner. A further web, which is likewise assigned to the respective container, can be configured such that it is immersed into the suspension and thereby separates the inlet for the sterile gas from the above-mentioned outlet. This leads to the fact that sterile gas is forced into a route through the culture suspension and consequently gassing of the culture suspension is further improved.
Examples of the device, arrangement of device, agitation systems and associated methods according to the invention are described subsequently.
There are shown
FIGS. 4 to 6 further devices according to the invention;
FIGS. 8 to 13 further agitation systems according to the invention;
FIGS. 20 to 22 the results of the cultivation of Escherichia coli in agitation systems according to the invention of a different type;
FIGS. 24 to 26 further agitation systems according to the invention;
FIGS. 27 to 28 further devices according to the invention;
In the following, the same or similar reference numbers are used in all Figures for the same or similar elements.
This bioreactor block 1 contains up to 96 cavities or borings 8a, 8b, these being able to be disposed in different formats, for example 4×3, 4×5, 8×3, 8×6 or 8×12 borings. The diameters of the borings 8a, 8b are advantageously between 10 and 35 mm. They are disposed in the bioreactor block 1 such that correspondingly dimensioned micro-reaction vessels 9a, 9b can be introduced into the latter in a form-fit. The bioreactor block 1 is thereby constructed from a multiplicity of horizontal layers 3, 4, 5, the lowermost layer 3 forming a baseplate, the layer 4 thereabove a central part and the layer 5 thereabove an upper part. Borings 6a, 6b, 6c are disposed between the baseplate 3 and the central part 4 through which, as heat exchanger, a fluid at a suitable temperature flows and which thus moderates the temperature of the entire bioreactor block 1. Furthermore, the borings 8a, 8b surrounding magnetically inductive magnetic drives 7a, 7b are disposed in the central part 4, as are known for example from U.S. Pat. No. 4,568,195. The upper part 5 contains a laterally projecting edge 12 along the outside of the entire bioreactor block 1, into which a sterile gas supply 13 is introduced for supplying sterile gas from the outside into the bioreactor block 1.
In the inner region of the bioreactor block 1, a distance disc 11 can be placed on the upper part 5. Since the individual mixing vessel 9a, 9b has an annular flange 10a, 10b on its upper side or upper edge, this flange 10a, 10b is supported on the distance disc 11. By choice of a suitably thick distance disc 11, the height of the reactor vessel 9a, 9b can be adjusted. Hence, the mixing height of a magnetic mixer 21a, 21b disposed in the vessel 9a, 9b above the base of the reaction vessel 9a, 9b is then defined. A one-piece baffle 20a, 20b is disposed in the respective reaction vessel 9a, 9b lying on its base, said baffle narrowing the cross-section of the reactor vessel at two positions on the circumferential line of the reactor vessel 9a, 9b. The baffle 20a, 20b ends with its upper edge in the present example below the mixing plane of the agitation system 21a, 21b. In other examples, the baffle can also however extend laterally beside the agitation system or even protrude upwardly beyond the latter. It is also possible that the baffles are disposed only above and/or in the mixing plane of the agitation system.
The lid or cover 15 applied on the reactor block 1 has central webs 14a, 14b which extend up to the distance disc 11 and thus isolate the individual reaction vessels 9a, 9b from each other in a sterile manner as separating walls. Furthermore, webs 18a, 18b are provided which extend into the reaction chamber 9a, 9b and separate this likewise as separating walls into two compartments. Finally, the cover 15 also has another boring 16a, 16b respectively, through which respectively one tube 17a, 17b extends. This tube 17a, 17b represents a constantly open connection between the outside of the bioreactor block 1 and respectively one of the reactor vessels 9a, 9b.
If now a culture suspension 30a, 30b is introduced into the respective vessels 9a, 9b, then the web 18a, 18b separates the surface 19a, 19b of the liquid 30a, 30b into two regions 19a, 19a′ or 19b, 19b′ which are separated from each other. If now the mixer 21a or 21b is set in rotation, then a convex liquid surface 19a or 19b is formed because of co-rotation of the liquid. This effect is not so pronounced for the liquid 30a or 30b at the surface 19a′ or 19b′ and is not represented here further.
If now a gas under excess pressure is guided via the sterile gas supply 13 to the surface 19a′ then, in order to again leave the container 9a, said gas must flow through the left member 31a of the liquid column below the web 18a into the right member 32a of the liquid column and from there again leave the vessel via the tube 17a. Since the flow though the tube 17a is constantly in this way from inside to outside, the reaction vessel 9a is sterile although the tube 17a is open and forms a constantly open access to the vessel 9a. The same applies correspondingly for the reaction vessel 9b.
Advantageously, interventions into the reaction course can now be implemented readily via the tube 17a or 17b. This means for example that substrate or titration media or inductors can be added via the tube 17a, 17b, that samples can be removed or measurement probes, for example pH electrodes, can be introduced into the liquid 30a or 30b. The introduction of corresponding probes is thereby effected in counter-flow to the outflowing sterile gas, so that contamination of the vessel 9a or 9b is avoided.
The cover 15 therefore produces a sterile cover for the bioreactor 1 with a central gas feed 13 via a sterile filter. An individual distribution of the sterile gas via gas distributor structures leading to the individual millilitre stirred tank reactors can likewise be effected.
The convective air flow through the tube 17a, 17b therefore prevents, in operation, the introduction of extraneous germs via the surrounding air. The open conducting pipe 17a, 17b is manufactured here for example from aluminium and is consequently also suitable as access with sterile pipette tips or piercing cannulae.
If gas distributor structures are inserted in the sterile cover, then these should be configured such that cross-contamination by aerosol entrainment or foam formation is precluded.
The bioreactor block 1 and the cover 15 are configured in such a precisely adapted manner that they can be assembled inside a sterile workbench, if necessary after sterile filling of the individual reactors 9a, 9b with reaction medium 30a, 30b, to form a functional unit.
The sterilisation of the bioreactor block 1 and of the cover 15 can either be effected together in an autoclave or also as individual components. In order to sterilise the bioreactor block 1 in the autoclave, a cost-intensive encapsulation of the inductive drives 7a, 7b is necessary in order to make direct autoclaving possible.
Alternatively, as also represented here, bioreactor inserts 9a, 9b can be used (corresponding to microtitre plates) which can be sterilised separately from the bioreactor block 1. These bioreactor inserts 9a, 9b can also be configured as sterile single-use or disposable articles as long as the material and production costs thereof are low.
In
In
In
In
In
In the individual cases of
In all the examples 2b to 2d, it is possible, as long as the shaft 23 does not serve for actuation of the magnetic agitation system 21, to manufacture the shaft 23 and the baffle 20 in one piece and to insert it as a unit into the reaction vessel 9 in a precise fit.
Examples of agitation systems according to the invention and reaction vessels according to the invention are now illustrated in the following. By using a suitable agitation system, the possibility exists in principle of dispensing with a primary dispersion of the gas phase via a gas distributor, as in the state of the art, since small reaction vessels, in comparison to laboratory stirred tank reactors, have a far higher surface/volume ratio.
In the present invention, suitable advantageously steam-sterilisable magnetic agitation systems have been developed which effect axial conveyance from the liquid surface to the base of the reaction vessel (absorption of the gas phase) and effective dispersion of the gas phase into as small as possible gas bubbles with a high oxygen transfer area (high local energy dissipation) in the reaction medium and also release of the spent gas bubbles at the liquid surface. These magnetic agitation systems have a basic body which can be manufactured advantageously from Teflon and contain one to four magnetic cores (ferrite or rare earth magnets, such as e.g. SmCo (samarium cobalt) or NdFeB (neodymium iron boron)) as actuation means. The subsequently represented magnetic agitation systems advantageously have the following dimensions and shapes:
These basic bodies are advantageously provided with borings or channels. These are between 3 and 20 mm long and have diameters which should be adapted to the size of the agitation system, advantageously from 0.5 to 5 mm, advantageously from 0.5 to 3 mm. Different arrangements of the borings can be hereby produced:
These magnetic agitation systems are thereby accelerated to speeds up to 4000 rpm, for example by a suitable magnetic rotary field or by a shaft.
Absorption of the gas phase into the reaction vessels with these agitation systems begins at a minimum rotational speed of the magnetic agitation system and becomes stronger by increasing the rotational speed. This minimum rotational speed is dependent upon the magnetic agitation system which is used, upon the position of the magnetic agitation system below the stationary liquid surface and upon the material properties of the liquid.
A particularly effective absorption of the gas phase and dispersion in gas bubbles in the reaction medium can be effected in reaction vessels with baffles which are disposed along the vessel wall in the circulating liquid flow. These advantageously one to four baffles can be disposed either below and/or above or over the entire vessel height on the vessel wall.
The magnetic agitation system is preferably operated in a self-centring manner, in a suitable rotating magnetic field. However, as shown in
FIGS. 3 to 13 show different embodiments of mixing vessels or agitation systems according to the invention.
If the agitation system 21 rotates about its central axis 22 in a liquid 30, the channel 33 induces an annular flow in the reaction vessel together with the horizontally extending channel 35a. The channels 34a and 34b for their part absorb gas from above and likewise lead to improved gassing of the liquid 30 situated in the vessel 9.
a shows a cross-section along the line A-A in
Via the borings 33, 34a, 34b, 35a and 35b, temporally pulsating variations in the flow velocity which run along the openings 45a and 45b in the vessel are furthermore induced and lead likewise to the aeration of the reaction volume.
The actuation of the agitation system 21 is effected magnetically inductively via magnets 25a to 25d which are incorporated in the agitation system 21.
In
The borings thereby have a longitudinal cross-section as can be detected in
In
The side face 26 of this agitation system extends from its surface 28 perpendicularly downwards to the plane of the horizontal borings 35a to 35d and merges then conically parallel to the borings 33a to 33d downwardly.
The borings 33a to 33d in turn convey liquid from the base of a vessel upwardly whilst the borings 34a to 34d draw in gas and liquid or a mixture thereof from the surface 28 of the agitation system 21 and produce turbulences at the interface of gas and liquid. As a result, a pulsating Bernoulli effect is also generated or possibly increased.
A further possibility for generating a pulsating Bernoulli effect resides in using a non-rotationally symmetrical agitation system.
In the case of the agitation system 21 illustrated in
An agitation system 21 of this type can be used for example in a non-rotationally symmetrical mixing vessel or in a mixing vessel with baffles.
In
In
b now shows a cross-section along the line A-A in
Starting from a gas supply boring 50, this boring branches via individual further borings 51, 52, 53, 54 to 55 continuously and finally discharges in respectively one gas outlet above the individual containers 9a to 9e. In
Now individual measurements with reaction vessels according to the invention or with a bioreactor block according to the invention are represented subsequently.
If the reactor block 1 is configured correspondingly geometrically (spacing and arrangement of the individual millilitre stirred tank reactors, e.g. respectively 8 millilitre stirred tank reactors in parallel—since pipetting robots are generally equipped with 8 parallel dosage stretches—etc.), simple automation can be effected. By means of a pipetting robot as actuator,
By means of suitable process control systems, an automated process control is hence possible in the parallel batch; pH adjustment, individual substrate dosage, automated off-line sampling and analysis and the like.
In principle, the following operational steps are necessary for automated implementation of parallel cell cultivations. Time planning is designed thereby such that the operational steps can be implemented in the course of the entire process in the same time cycle, i.e. maintenance of a constant timespan ΔT between the operational steps is made possible. Such a timeplan is represented in
In a first step, a microtitre plate 60a with samples from the reactor block 64 is now filled via the pipette tips 65. The microtitre plate 60a is then transported by the bearing arm 61 to the photometer 62 where the individual cups of the microtitre plates 60b are measured photometrically. The thus measured microtitre plate 60b is transported by the bearing arm to the washing device 63 where the microtitre plate (now microtitre plate 60c here) is washed and cleaned. Hence, the microtitre plate 60c is again available for samples and the measurement cycle and is conveyed by the bearing arm 61 back to the table 58.
According to the process requirements, microtitre plates filled with samples can be withdrawn from the process cycle at specific times and be cooled and stored in the interim. A new microtitre plate can be supplied automatically to the process in order to be able to maintain the analysis cycle.
Determination of materials dissolved in the aqueous reaction medium, such as the hydrogen ion activity (pH), must be effected individually in each reaction vessel. Use of 48 or 96 individual pH sensors, for example sterilisable pH glass electrodes are not economical. Also the use of economical pH field-effect transistors (“disposable sensors”) is in practice not possible due to the additionally required standard reference electrodes and the lack of thermal stability (ability to be sterilised).
The number of necessary pH sensors for parallel reactors can in principle be reduced if a sensor can be used for a plurality of reaction vessels. One possibility for technical production is the integration of commercially available miniature pH electrodes in piercing cannulae, which are immersed intermittently into the individual millilitre agitation reactors by means of a pipetting robot. pH single-rod measurement sequences with an external diameter of 1 mm and a response time of ˜6 s are suitable for this purpose.
A further possibility is sterile removal of samples from the individual millilitre stirred tank reactors with cannulae and parallel measurement of the pH value in the samples with pH-sensitive microtitre plates. On the base of the cavities of these commercially available microtitre plates, a sensor spot is integrated in which two fluorophores are immobilised. These fluorophores can be read in a correspondingly equipped photometer-fluorimeter. The fluorescence properties of the indicator-fluorophore vary with pH value of the solution whilst the reference fluorophore produces a fluorescence signal which is independent of the pH value. The ratio of indicator signal to reference signal can be correlated to the pH value of the solution via a sigmoid function. Use of a reference fluorophore increases the measuring precision and the lifespan of the sensors since a decrease in signal intensity by “bleeding out” of the sensor (diffusion of the fluorophores in the measuring solutions) results in a smaller effect on the measuring signal.
The produced pH measurement data are read by the process control system and made available to control algorithms. These calculate the necessary volume of titration means per reactor vessel in order to maintain a desired pH reference value in the reactor vessel. The process control system calculates the dispensing steps taking into account the necessary dosage for pH control.
The hence possible high oxygen and energy supply into a culture medium is essential in the present invention. Hence measurements of the oxygen transfer coefficients kLa of different magnetic agitation systems according to the invention are described subsequently.
Agitation systems of the Types I to V corresponding to
In order to determine the kLa values, 0.5 M Na2SO4 solution is used, which guarantees non-coalescing conditions. In addition, a concentration of 10−3 M CoSO4 is present as catalyst for the chemical oxidation of sulphite into sulphate. Implementation of the dynamic sulphite method begins with aeration of the liquid phase with air until the latter achieves saturation. After addition of a sufficiently large material quantity of sulphite in order to consume all the oxygen dissolved in the liquid phase, the dissolved oxygen concentration of the liquid phase drops abruptly to zero. After stoichiometric sulphite conversion, the dissolved oxygen concentration in the liquid phase increases again. From this so-called reconcentration curve, the kLa value is determined by assuming ideally mixed conditions in liquid and gas phase. The response time of the used oxygen probe in the model is thereby taken into account.
Conventional technical stirred tank reactors are operated with oxygen transfer coefficients of kLa <0.25 s−1. Hence, in millilitre stirred tank reactors according to the invention or with the agitation system according to the invention, the same or similar oxygen transfer rates can be achieved (by way of comparison: in agitated flasks or microtitre plates, oxygen transfer coefficients of at most kLa=0.07 s−1 can be achieved under optimal conditions).
Oxygen transfer coefficients of the agitation systems of Type III and V were furthermore implemented with 8 ml 0.5 M Na2SO4 solution likewise in a millilitre agitation reactor with 20 mm diameter (
In order to verify the oxygen transfer properties of the agitation systems, parallel cultivations with Escherichia coli (wt) were implemented as example system.
100 ml of a sterile, defined medium were inoculated with 0.25% inoculum from the feedstock in a 500 m agitated flask sealed with Alucap and incubated for 14 h at 37° C. and 200 rpm in an agitated incubator with an eccentricity of 5 cm. On the following day, 4-6 ml of this preculture were transferred into millilitre agitation reactors. In the millilitre agitation reactors, the cells were incubated for 3.5 h at 2000 rpm of an agitation system according to the invention and for a further 2.5 h at 2200 rpm at 37° C. The pH value was controlled to 6.8.
The agitated flask (AF) was incubated further with the approx. 40 ml residual volumes of preculture in the agitated incubator under the same conditions.
The measurement results in
FIGS. 24 to 26 show further embodiments of agitation systems according to the invention. These agitation systems are all prepared for mounting on a shaft 23, in that borings 50 and 51 are introduced into the agitation system 21. In the upper region of the agitation system, the boring 51 has a clearance width so that it makes possible passage and mounting of the shaft 23.
The borings 33a and 33b are now continuous from a lower edge of the agitation system to the oppositely situated upper edge of the agitation system. The borings 33a and 33b are thereby situated in a section plane so that they intersect in the region of the boring 50 and form a common cavity. The openings of the borings 33a and 33b now extend both over a part of the underside 29 or of the upper side 28 of the agitation system 21 and over its side wall. As a result, four partial borings, in a star-shape, thus extend towards each other in the centre from opposite sides on the upper and the lower edge of the agitation system.
This agitation system can be self-centring or also, as illustrated in
The cultivation was effected here in a mineral medium with 15 gL−1 glucose at the beginning of the batch phase as starter concentration. After consumption of this glucose after 4.15 h, a glucose solution with 250 gL−1 glucose content was fed intermittently every 4 minutes. The pH value was set to 6.8 by means of 2.5% NH4OH.
In summary, it can be consequently established that parallel millilitre stirred tank reactors have been made possible by the present invention, in which comparable growth as in controlled processes in bioreactors of a laboratory scale is made possible.
Surface-aerated millilitre agitation reactors equipped if necessary with special magnetic agitation systems permit an equally efficient oxygen supply for organisms in liquid culture as mixing vessel reactors of a greater scale with volume aeration.
The use of up to 96 and more millilitre stirred tank reactors in one bioreactor block which can be automated by means of pipetting robots makes possible for the first time an efficient, individually controlled parallel operation. The use of a laboratory robot for automation of the parallel reactions hence makes possible a quantum leap in obtaining relevant process data.
By implementing suitable screening and optimising routines, screening methods and process optimisations (media composition, induction methods, dosage profiles) can be automated systematically and with high time efficiency in the parallel batch. Complete digitalisation of the parallel process development makes further possible a novel data transparency and availability.
By means of this new tool for high-throughput bioprocess development, new bioprocesses can be developed in a time-efficient manner under technical reaction conditions since, for example instead of an experiment in the controlled 0.5 l mixing vessel reactor with the same reaction volume, 100 experiments can be implemented at the same time in 100 parallel 5 ml mixing vessel reactors in an automated manner—i.e. an information yield per unit of time, at a multiple of 100, becomes possible. When using suitable test planning algorithms, the effectiveness of the bioprocess development is further increased.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10260691.9 | Dec 2002 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/14752 | 12/22/2003 | WO | 12/8/2005 |
