EP2898059A1

EP2898059A1 - Disposable bottle reactor tank

Info

Publication number: EP2898059A1
Application number: EP13760060.7A
Authority: EP
Inventors: Joerg Kauling; Annette WALDHELM; Helmut Brod; Verena HERRMANN; Stefanie JACOB
Original assignee: Bayer Technology Services GmbH
Current assignee: Bayer AG
Priority date: 2012-09-18
Filing date: 2013-09-13
Publication date: 2015-07-29
Also published as: IN2015DN02120A; SG11201501572TA; CN104812888A; US20150218501A1; JP2015531602A; AU2013320374A1; CA2884865A1; BR112015005781A2; WO2014044612A1; KR20150056548A

Abstract

The invention relates to a reactor tank designed as a disposable element, comprising a cover and/or optoelectronically readable sensor patches fastened in the interior, a reactor, comprising the reactor tank and a reactor tank accommodating periphery, which comprises a reactor tank retainer and optionally an optoelectronic measuring system for reading sensor patches, wherein the reactor tank retainer is coupled to a drive unit for producing a rotational oscillating motion of the reactor tank about a center vertical axis of the reactor tank, and the use of said device to cultivate cells and/or microorganisms.

Description

Disposable Fiasefaeiireaktortank

The subject of the application is a reactor tank designed as a disposable element with cover and / or opto-electronically readable sensor patches affixed in the interior, a reactor comprising the reactor tank and a real-estate tank peripheral comprising a catchment heater and possibly an opto-electronic measuring system for reading the Sensorpatche, the Reaktortankhaiterung is coupled to a drive unit for generating a rotationally oscillatory movement of the reactor tank Erend his own central vertical axis, and the use of this device for the cultivation of cells and / or microorganisms.

In highly regulated pharmaceutical production, a great deal of time, technical and personnel costs are incurred in the provision of purified and sterilized bioreactors. In order to reliably avoid cross-contamination in a product change, in a Muiti-Purpose-Aniage or between two product batches, apart from the cleaning, a very elaborate cleaning validation is required, which may need to be repeated during a process adaptation.

This applies both to the upstream processing USP, i. the production of biological products in fermenters as well as for the downstream processing DSP, d. H . the purification of the fermentation products.

Boilers are often used in the USP and DSP as stirring and reaction systems. Especially in fermentation, a germ-free environment is essential for ertolgreiclic cultivation. For the sterilization of batch or fed-batch fermenters, the SIP technology is usually used (SIP = steam-in-place). In order to ensure sufficient long-term sterility in continuous process control, the autoclaving technique is also used, which, however, requires cumbersome transport of the reactors / to autoclaves and is applicable only in comparatively small reactor scales. The risk of contamination during fermentation is particularly critical during sampling and on agitated stirrer shafts. The latter are usually equipped with elaborate sealing systems (e.g., mechanical seals). Technologies that do not require such penetrations of the fermentation casing are preferred because of their greater process robustness.

The failure of the standard reactors due to the provisioning procedures can be of the order of magnitude of the retry effect, especially for short periods of use and frequent product changes. Affected in the USP of biotechnological production, for example, the process steps of media production and fermentation and DS solubilization, freezing, thawing, pH adjustment, precipitation, crystallization, rebuffering and virus inactivation. In order to meet the demand for a fast and flexible resending of the production plant while maintaining maximum cleanliness and sterility, there are concepts of disposable reactors of ever increasing interest on the market. In WO2007 / 121958A1 and WO2010 / 127689 such a disposable reactor for the cultivation of cells and microorganisms is described. It consists in a preferred embodiment of a stable, preferably multi-layer polymer material bag. The deformable disposable reactor is received by a container that supports it. He is preferably introduced from the front into the container. The container is connected to a drive unit. By the drive unit of the container including the disposable reactor is placed in a rotational oscillatory movement about a stationary, preferably vertical axis of the container. By an angular embodiment of the disposable reactor and / or internals in the disposable reactor, a high power input can be achieved in the reactor contents in the oscillatory rotary motion, so that the disposable reactor can be used as a surface-agitated fermenter for the cultivation of cells and microorganisms. The internals for supply and monitoring of the reactor are mounted laterally at the bottom of the reactor via a connection plate. These reactors are predominantly used at reactor volumes of more than 10 liters.

For smaller reactor volumes, the production of a reactor bag together with a suitable container is too time-consuming.

The challenge lies in small disposable reactors in achieving the sensor technology, mixing technology, temperature control and supply of the reactor in a possible compact and cost-effective form.

Small stirred one-way reactors are known in the art.

Sartorius Stedim Biotech offers with its Univers el® SU (http: //www.sart.org/Biotechnology/Fermentation_Technologies/Reusable_Bioreactors/Data Sheets / Data_ UniVessel_SU_SBI2033-e.pdf) a stirred, one-way reactor with a cylindrical reactor tank , The disposable reactor has a stirrer for mixing and, for the gas supply from below, an I. sparger below the stirrer. Above the cover, the drive of the stirrer is ensured via a drive shaft driven from above, the sensors (temperature, pH (former), oxygen (formerly)), the gas and gas disposal for the gas space as well as further supply and sampling via lines , The lid is attached to the reactor tank by means of a clamp connection and sealed in a sterile manner against the reactor tank via an O-ring. The stirrer drive is sealed with 2 lip seals. The sensors for monitoring of H and oxygen content can also be achieved by means of optoelectronic Sensorpatche at the bottom of the reactor tank. For operation, the reactor tank is firmly positioned in a special container, this container having a retaining ring and a foot with an opto-electronic sensor system for reading the sensor patches.

The disadvantage of this or similar reactor systems available on the market is that these agitated systems require moving internals as well as a sophisticated, sterile sealing system in the lid and, due to the high shear forces, for the cultivation of very sensitive cells, e.g. Stem cells are poorly suited.

It is based on the prior art, the task of providing a low-shear system for performing processes with high demands on purity and sterility, which reduces the time, technical and human resources on the provision of purified and sterilized components. The system should be usable for pess measurement volumes of 10 mL to 20 L, in particular 50 mL to 10 L and more preferably 250 mL to 3 L working volume. It is intended to meet the high requirements of the pharmaceutical industry, to be simple and intuitive to handle and to be cost-effective. It is intended to minimize safety risks by discharging substances from the process area to a minimum. It should allow adequate mixing of the reactor contents, be suitable for the cultivation of microorganisms and cell cultures and in this case ensure adequate supply and disposal of the culture medium with liquid nutrient media and in particular gaseous substances. It should be just as suitable for the process development as for the production of cell products, especially for clinical cell products like. For example, human or animal body cells: stem cells, blood lines, leukocytes such. Natural killer cells (NK cells), tissue cells or pharmaceutical agents, e.g. monoclonal antibodies, proteins, enzymes in bioreactors.

According to the invention this object is achieved by the use of a dimensionally stable, square plastic bottle for delimiting the interior of the reactor, wherein the plastic bottle has a bottom, walls, an interior and at least one access to the interior and preferably a pyramidal, inwardly curved bottom, a wide neck and / or. or has one or more laterally in the lower region of the bottle at a position defined with coordinates Sensorpatche. ,

The first object of the present invention is therefore the use of a dimensionally stable square plastic bottle as a bioreactor tank for the cultivation of cells, especially sensitive cells and on (micro) carrier-growing cells such as stem, blood or tissue cells, the plastic bottle a floor, walls, a Interior and at least one lockable Access to the interior in particular has a bottleneck. Usually, in the interior of the plastic glass che on one or more walls in the lower region, attached to a position defined by coordinates, one or more Sensorpatche attached. Another object of the present invention is a reactor tank comprising a dimensionally stable polygonal plastic bottle having a bottom, walls, an interior and at least one closable access to the interior, comprising at least one bottleneck esp. Closable by a lid and / or at least one passage, wherein Interior space, at one or more walls in the lower region of the plastic bottle, at a location defined by coordinates, one or more Sensorpatche are attached. Preferably, bushings are accommodated in the lid.

Support-fixed sensor patches constructed of fluorescent color layers (e.g., Presens, YSI), available e.g. can be stuck on bottle wall. Usually, at least one pH sensor patch and one oxygen sensor trap are used.

Alternatively, the reactor tank or ioreaktor on a bottle wall or in the lid, preferably in the lid, feedthroughs for electrochemical sensors, preferably

Disposable sensors e.g. according to US 20120067724 AI, on.

In order for the reactor to meet the sterility requirements of the pharmaceutical industry, the plastic bottle is usually made from a gamma-sterilizable plastic material. The reactor tank according to the invention is preferably made of a single or multi-layer transparent polymer material that allows insight into the reactor tank during operation.

Plastics or glass are relatively inexpensive materials that can also be processed comparatively inexpensively. The disposal of the used reactor tank and the use of a new disposable reactor tank are thus more economical than the cleaning of used reactor tanks, especially since when using a new disposable reactor tank, a complex cleaning and cleaning validation deleted. The fiction, contemporary reactor tank is manufactured or cleaned in clean room and is preferably sterile packed. The reactor tank according to the invention is dimensionally stable. Suitable materials or combination of materials for the reactor tank according to the invention are all cell biologically compatible, known in the art materials, in particular glass, polyethylene, polypropylene, polyether ketone (peek), PVC, polyethylene terephthalate and polycarbonate. Wall thicknesses of 0, lmm-5mm are preferred and 0.5-2mm is particularly preferred.

The bottle materials are usually brought into the desired shape by means of stretch blown processes known from the prior art. The cross section of the reactor tank or the plastic bottle preferably has the shape of an n-corner with n in the range of 3 to 12, preferably in the range of 3 to 6, most preferably in the range of 3 to 4, most preferably n is equal to 4 ,

Preferably, the side walls of the reactor tank or the plastic bottle according to the invention are at least partially formed as flat surfaces which meet at an angle of 45 ° to 120 °. Preferably, the side walls of the reactor form a polyhedron with the bottleneck attached to one of the surfaces.

Preferably, the reactor tank or plastic bottle is cuboid with edge lengths H, b and c, where H represents the height, b the width and c the depth of the plastic bottle and b <c <H. The broad neck is typically mounted on one of the small surfaces and the opposite surface serves as the bottom of the reactor tank. The reactor tank according to the invention or the plastic bottle have a ratio of bottle height H to maximum width b and depth c in the range of 0.5 to 4, preferably 1 to 3, particularly preferably 1.5 to 2.5. In the preferred embodiment, the reactor has at least one cylinder cross-sectional edge length a = c = D.

For a better mixing of the reactor and a reduced starting volume, the reactor tank or the plastic bottle usually has an inwardly curved bottom. For the design of the floor, the teaching of WO2010 / 127689 is integrated by reference. In particular, the bottom has the shape of an inwardly directed tetrahedron, an inwardly directed pyramid, the shape of a paraboloid or a bell-shaped form. Particularly preferably, the bottom is made pyramidal. The height h _{w of} the curvature is in the range of 0.01 times to 1 times the circle-equivalent diameter Dt of the bottom cross-section. The height h _{w of} the curvature at the circle-equivalent diameter Dt is preferably in the range from 3% to 100%, particularly preferably in the range from 5% to 30% and very particularly preferably in the range from 10% to 20%. The reactor tank according to the invention can be heated and / or cooled via its outer walls. In a preferred embodiment, a disposable heating mat is applied on the outside of the bottom of the plastic bottle or of the reactor tank, with which a very efficient heat transfer can be achieved because of the positive connection of the heating and lateral surface. In this way, the heating surface can be reduced to the floor area. .Dazu this heating mat is usually glued to the outside of the floor. In general, the reactor tank requires no additional cooling, since switching off the heating mat in reactors with a small volume and thus large specific heat exchange surface causes a sufficiently rapid cooling. An additional cooling would be applicable, for example, in microbial applications at lower fermentation temperature and higher heat of respiration by attaching Peltier elements on the side surfaces of the reactor tank or the tank holder if necessary.

The reactor tank according to the invention preferably constitutes an externally sealable space for carrying out chemical, biological, biochemical and / or physical processes. In particular, the reactor tank serves to provide a sterile space for culturing cells and / or microorganisms. In a preferred embodiment of the reactor tank according to the invention for this purpose, the bottleneck of the reactor tank is sealed by means of a lid, wherein the lid has at least passages and / or connections for the gas and liquid supply and removal of the reactor tank. According to the invention, the cover has no passage for a drive axle [Fig. 2-5]. The lid is another element of the reactor tank according to the invention. Preferably, the connected gas lines are equipped with sterile filters, wherein the sterile filter of the exhaust pipe is preferably equipped with a heating mat to keep condensate from Fiiterflächen. Alternatively, the exhaust gas for condensate avoidance at the filter with egg nen exhaust gas cooler, the e. is cooled down to a lower dew point (condensation temperature <ambient temperature) via an electronic cooling element (e.g., a Peltier element) mounted on a heat transfer surface made of sheet materials

Furthermore, the lid can, if necessary, further bushings and / or connections for elements from the group comprising:

one or more electronic, optoelectronic or electrochemical sensors, in particular one-way electrochemical sensors from US 2012/0067724 A1 or PT100

Resistive sensor for temperature control and / or capacitive sensors for level control or cell density measurement,

an internal cell separator and / or

a trial system

exhibit. Depending on the application, the reactor tank is equipped with one or more of these elements.

In a preferred embodiment of the invention, the lid is composed of a plug and a cap sleeve. The plug is usually made of plastic selected from the group of polyetheretherketones, thermoplastic or silicone. Usually, the plug is designed as a disposable plug, in a special imple mentation form reusable.

Preferably, the plug is inserted into the neck of the reactor for closure, sealed by means of a circumferentially mounted O-ring seal against the inner side of the bottle neck and with a separate locking means such. B. screwed over a nut üb screwed to the thread of the bottle neck or jammed with a clamping ring. Alternatively, the introduced into the bottleneck can be sealed by means of a sealing lip placed on the bottle opening and clamped with a separate screw-cap sleeve and screwed to the plastic bottle. Another alternative is a lid containing the identical feedthroughs as the plug, which is screwed to the plastic bottle and sealed with an O-ring against the bottle neck and / or the bottle opening. Preferably, the stopper inserted into the neck of the bottle is used, which is sealed with an O-ring seal on the neck of the bottle and screwed to the plastic bottle with a separate screw-on union nut [Fig. 2]. This embodiment has the advantage that the O-ring is subjected to little mechanical stress and no twisting of the hose occurs, as would be the case when turning a blanket.

The reactor tank with lid is preferably designed as a disposable element, i. It is preferably provided not to clean the entire reactor tank after use, but to dispose of it. Therefore, the reactor tank preferably comprises only the essential elements necessary to provide a sterile reaction space:

The plastic bottle is usually produced and used as a disposable item. For the cultivation of sensitive cells or production of clinical cell products, fumigation preferably takes place exclusively via the surface. In this case, the lid does not have a passage for a bubble gasification element and the reactor tank according to the invention has no devices for the bubble gassing. For applications in the context of process development with the focus on scale transfer to large fermenters, for perfusion processes with high cell densities or for microbial processes, installation for additional micro- or Macrobegasung (eg with hose lines from above the lid supplied and adhered to a container wall sintered body) be provided. Preferably, the reactor according to the invention can be made completely from inexpensive elements and thus makes possible the use of the reactor as a one-way system. Alternatively, all high quality elements are integrated into a reusable lid and only the reactor tank is used as a disposable element.

In a particular embodiment of the reactor, a cell separator in the reactor tank is used for cell retention. According to the invention, the internal cell separator is formed by a central vertical separator tube and a separator head with a collector for extracting cells liberated culture solution, the lid has a passage for the collector and the Zellabscheider on the lid is either rotatably mounted or statically attached. The tube and the separator head can have different lengths, conical and straight geometries and diameters, as well as various tube installations (conical and ring installations, rectifiers.) Special embodiments are shown in FIGS. 6 to 8. The cell separator can be made of steel, glass or metal Preferably it is made of plastic such as polyethylene, polypropylene, polyethylene terephthalate, polyether ketone and / or polycarbonate and used as a disposable element.

Another object of the present invention is therefore an internal vertical cell separator for bioreactors, formed by a central vertical separator tube and a separator head with a collector for aspirating cell-depleted medium, the cell separator is attached to a cover for a reactor tank or rotatably mounted and the lid a Performing for the collector.

When a cell separator is used, the reactor tank usually has a wide neck so that the prefabricated cell separator attached to the lid can be inserted through the neck of the bottle. If the cell separator is movably attached to the cover (= rotatably mounted), it is only minimally influenced by the rotational movement of the reactor tank due to its inertia. As a result, the circulating flows transmitted from the separator into the sedimentation space and interfering with the sedimentation process can be avoided, and the retention can be remarkably improved, as shown in FIG.

The cell separator is preferably designed so that the inner and outer regions of the cell separator are largely separated from one another by corresponding constrictions. In this way, a transfer of the sedimentation disturbing flows from the well-mixed outdoor space in the S edimentations zone can be reduced. In other words, the inner volume of the cell separator should be as small as possible of flows in the outer volume (= culture volume) However, a return transport of the retained lines must remain ensured in the mixed, supplied reactor area

Preferably, the internal cell separator for a reactor tank with the dimensions of cross-sectional edge length D = 120 mm, H = 235 mm, a separator tube (310) with a Abs length 1 (370) of 40 mm to 200 mm, in particular from 90 to 190 mm, preferably 190 mm (Figures 6 and 7). the case is a ratio l H of 0.2 to 0.9, preferably 0.5 to 0.9, in particular 0.8 used.

The separator tube has a round cross-section with a tube diameter d (350), wherein the ratio of tube diameter d to Flasch chenquers chnittkantenlänge D usually from 0.25 to 0.90, in particular from 0.5 to 0.85, preferably 0.83 , The tube diameter d is essential for the cell retention for the realization of the separator surface.

It is preferred to select the bottleneck and cell separator sections so that the cell separator can be easily inserted into the bottle. This is particularly necessary if a reusable, autoclavable lid is to be used which is to be connected under the clean bench to the γ-sterilized reactor tank.

In a first embodiment of the bioreactor, the gassing takes place exclusively via the surface (FIGS. 6 and 7). For this purpose, the diameter of the pipe d (350) of the precipitator is selected so that a ratio of over the surfaces fumigated Kuiturvoiumen VK defined by formula (I) and trap volume VA defined by formula (II) of 0.01 to 1 0 preferably from 0.2 to 2 is present ,

V _K = D ² * L - d ² {L - S) (I)

V _A = ~ d ² l (II)

Further parameters of the cell separator are the separation surface (= clarifying surface) A defined as A = -d (III) and the clarifying surface loading v = q / A, where q is the harvesting current. At the separator head is the collector (320) for aspirating cell-free culture solution. Usually, the ratio dv / d of the collector diameter dv (360) to the pipe diameter d 0, 1 to 0.7 is preferably 0.3-0.5

Preferably, the collector (320) has a conical shape. This form has the advantage that there is room for the introduction of further elements (sensors, sample collection line, etc.) over the lid. Also, the fumigation * is reduced slightly. Preferably, the separator is used in the reactor tank with a ratio 1 / s from the separator length 1 to the bottom clearance s of the separator tube of 0.75 to 9.

On internals in the separator tube and collector (320) is preferably omitted in Zellabscheider.

In hydrodynamic investigations with the model particle PAN-X, a tube with different tube lengths, geometry (conical / straight head) and diameters as well as various tube internals (conical and ring inserts, rectifier, etc.) was statically installed on the lid. According to the experiments available so far, the static built-in (= co-rotating) internal cell separator for solids in the area of the clarifying surface loading v of 0.025 <v [m / h] <0.2 shows a comparable P / VK of 3 W / m3 Retention level as static external systems on (eg Plattenabs checker, Dortmund fountain) (Fig. 13). In particular, the cell separator according to the invention for the culture of well sedimentable particles, such as. B. carrier-fixed ized cells applicable depending on the rate of descent of the

Trägerermateriaiien in significantly higher power input ranges P / VK of>> 3W / m3 can be used.

In a particular embodiment of the invention, the reactor according to the invention also has an automatic sampling element.

In a first embodiment, the Probennahmeeiement consists of a receiving line, which is performed by the lid (= Deckelprobennaiimeeiement, see Figure 10). Outside the reactor, the line can be shut off by means of clamps and pinch valves. Via an adjoining Y-piece, on the one hand the vertical suction of the sample from the fermenter takes place by means of negative pressure and the subsequent transport to the sample preparation station by means of overpressure. This Y-shaped sampling element is for the realization of an automatic sampling module consisting of hoses, pinch valves, sterile filters, a positive and negative pressure supply particularly advantageous. Usually commercially available lines, valves and plastic Y-pieces are used so that the sample taking element which can be integrated in the cover is provided and used as a disposable element. The basic principle of the Y-shaped sampling element is shown in WO 2007/121887, and is integrated by reference, in which two burettes are driven in order to ensure the transport and the aliquoting of a sample. After sampling, the Probenahmeeiement is sterilized with EtOH and dried. Preferably, filter elements for air and EtOH are incorporated to prevent contamination of the sampling element. Preferably, the sampling element is coupled with the biological chromatograph for automated analysis by Bayer Technology Services GmbH. In a further embodiment, the reactor tank on a bottle wall, in particular on the wall opposite the sensors (sensor patch or electrochemical sensors), has a passage and / or a connection in the area near the ground for the attachment of a sampling system. Examples of feedthroughs and / or inserts include standardized Ingold nozzle or PG 13.5 threaded nozzles. A suitable sampling system is z. B. described in DE102008033286 AI.

The mixing within the reactor tank according to the invention is carried out by a periodically reversing rotation of the reactor tank, which causes in combination with the polygonal shape of the plastic bottle inwardly directed wavy currents to the surface of the reactor contents. For the design of the reactor movement, the teaching of WO2010 / 127689 is integrated by reference.

All other elements which are required for operating a reactor, in particular for cultivating cells and / or microorganisms, in particular a drive unit for generating the periodically direction-changing rotation of the reactor tank and optoelctronic sensor system for reading the Sensorpatche be provided by a periphery and are reusable. Of the

Reactor, which usually represents a coherent unit in the prior art, is thus preferably divided in the present case into separate parts, which are designed according to their functions.

A further element of the reactor according to the invention is therefore the periphery. In particular, a reactor tank storage periphery is used as the periphery, which has one or more reactor deposits, wherein the reactor tank and the reactor tank aging are adapted to one another as separate parts of an overall system in such a way that the reactor tank into the reactor tank Reactor tank holder introduced or in particular can be trapped there and is supported by this in the liquid-filled state.

The reactor tank receiving periphery for accommodating a reactor tank according to the invention is a further element of the reactor according to the invention and comprises at least:

One or more Reaktortankhaiterungen for receiving in each case a reactor tank comprising a matched on the Reaktorlank footprint and one or more lateral fastening elements. For example, the eaves on k ha 11 eru n g an adjusting plate and lateral lemmarme or surfaces on. - Associated with the Reaktortankhaiterungen, especially with the footprint of

Reactor Tank Holdings, is a drive unit for performing a periodically reversing rotation, such as Z. B is a stepper motor. Preferably a stepper motor without gears with direct coupling of the motor and the drive is used. By means of the drive unit, the reactor tank can be displaced about its stationary, vertical axis in a periodically inclined rotating rotation, so that a direct coupling of the drive unit to the reactor tank itself is not required. Preferably, a stepper motor without gear is used for the realization of the reactor movement. Such an arrangement has the advantage of being very quiet Preferably, the drive unit is controllable by means of a control unit. Usually, the controller is part of the drive unit.

- One or more installed at the Reaktortanichalterung, in particular on one of the lateral fasteners opto - electronic Chen sensor systems for reading the laterally attached to a coordinate point of the reactor tank Sensorpatche, in particular a pH Trans mitler and / or a Sauerstoffgehalttrans mitter. The direct coupling of the light excitation and detection units to the sensors can be the

Reduce light intensities to produce evaluable measurement signals and thus the generation of Radikaien significantly, which benefits a prolonged life of the sensor patches. According to the invention, the data transmission is conducted via differential, serial

Interfaces and / or non-powered via radio such as Bluetooth or WLAN. Preferably, the optoelectronic sensor system has the differential, serial interface for symmetrical signal transmission type EIA485 / RS485 due to the robust data transmission and a high Toieran / against electromagnetic interference. For improved data transmission, a stepper motor without a gearbox with direct coupling of the motor and the drive has been identified as particularly advantageous, because this allows a particularly trouble-free data transmission.

FIG. 11 shows a particular embodiment of the reactor including the reactor tank receiving periphery.

Preferably, the footprint adapted to the reactor is replaceable or adaptable, so that the reactor frame can be used with reactors of different sizes.

The present invention also provides for the use of the inventive reactor and reactor tanks and a method for cultivating cells and / or microorganisms. During operation, a ratio of liquid level to reactor tank width of preferably 0.05 to 2 and more preferably 0.1 to 1 is present in the reactor tank, wherein the liquid level may change as a result of replenishment with the growth of the cells. In addition, the reactor tank is operated while maintaining its preferred hydrodynamic and process properties with sufficient headspace between reactor tank head and liquid level (= level 180, Hi) of at least 5% to 50% liquid height, preferably at least 25% liquid level, to provide adequate foam spacing to ensure a gas discharge line equipped with a sterile filter. To control the level usually a capacitive sensor for level control is used by the lid or on one of the container walls.

It has surprisingly been found that a comparatively small angle amplitude is sufficient for the rotationally oscillating movement of the reactor in order to achieve a thorough mixing and / or a sufficient intensification of transport processes. In particular, it is hardly necessary to realize 3600 ° revolutions (corresponding to 10 revolutions) of the reactor, so that no structurally complex solutions for the connection of the oscillatory rotating reactor to the static environment (eg for the supply and removal of media and gases, of electrical energy and electrical signals) are required. In accordance with the invention, the reactor tank is moved at an angular amplitude α in the range of 2 ° <jaj <3600 °, preferably 20 ° <jaj <180 °, particularly preferably 45 ° <jaj <90 ° in a rotationally oscillating manner Deviation of ± 5 ° may be present. In particular, jaj = 60 ° is considered to be very particularly preferred when using particularly low-shear surface-treated bioreactors. In total, the oscillating motion thus covers an angle of 2 jaj.

Experiments have shown that when the power input is increased, states of motion can occur in the reactor in which gas bubbles are introduced into the reactor medium. Gas bubbles are introduced from a power input of P / VK> 10 W / m ³ , For cells and / or microorganisms that are not damaged by a bubble fumigation, can be realized in this way a very simple increase in gas supply. By an additional bubble gas via a preferably laterally installed in the bottom of the sintering tube mass transport can be significantly improved. The flow generated by the rotational oscillation ensures a gentle demolition of the microbubbles from the sparger and thus for a large phase boundary a or a large mass transfer coefficient km. The invention will be explained in more detail with reference to figures, but without limiting it to the embodiments shown from management forms.

Description of the figures:

Figure 1 shows schematically the lateral longitudinal section of a preferred embodiment of the reactor tank according to the invention and reactor tank holder in side view. FIG. 2 shows a schematic view of a plug-type thermoplastic construction from above.

Figure 3 shows a schematic section of a Stop fen- Silikonaus guide with hose nozzles (135) secured with cap sleeve (120) and O-ring seal (140).

Figure 4 shows schematically in front view a section through a plug Siiikonausführung attached with cap sleeve (120) and sealed by means of sealing lip (140b). FIG. 5 shows a schematic front view of a section through a screwable plastic cover with hose nozzles (135).

Fig. 6 shows schematically in front view a straight Rohrabscheiders with straight Kop (= sudden cross-sectional constriction) in longitudinal section.

Fig. 7 shows schematically in plan view a straight tube separator with a straight head.

Fig. 8 shows schematically in the front view of a straight pipe separator with straight head (= sudden cross-sectional constriction) and flow inverters.

Fig. 9 shows schematically the experimental set-up together with lines, wherein a reactor is shown by way of example with a statically fixed in the lid straight tube separator with conical head. 10 shows the schematic structure of an automated lid sampling element with a sample suction line (1110) passing through the lid, which via Y-pieces (1170) with a sample line (1120) to an automation platform (1190) for fully automated

Removal and plug-flow transport of liquids is connected as well as to other lines for air supply and ETOH cleaning or sterilization (1210).

Fig. 11 shows in particular the guidance of the rector tank of the reactor receiving tank periphery. FIG. 12 shows test results R = f (P / V) when comparing the static and co-rotating separator according to FIGS. 6 and 7 and proves the highly surprising necessity of a rotatably mounted sedimentation tube which, in contrast to the co-moving variant, also has higher power inputs P / VK> 3 W / m3 ensures good particle retention.

Fig. 13 shows comparative experiments to other sedimentation. Reference / calibrate:

8, 9 port (ID 2mm, OD 3mm, port length: 15mm)

10 to 15 ports (I D 3mm, OD 4mm, Pon length: 15mm, port 10 with double-sided hose nozzle)

16, 17 PG13.5 port

18 identical port to PC i 1 3.5 with I D4mm

100 containers

101 rotation axes

110 bottleneck

120 Outer nut / 'Cap sleeve

120b he screwed lid

130 stopper

1 35 hose nozzle

136 silicone hose

140 O-ring

140b sealing lip

140c seal

150 nozzles

160 floor

170 gas distributor

180 level H _L

185 bottle height H

186 bottle cross-section D

190 bottle wall

198 culture volume V _B

200 gas supply

210 sterile filters

220 gas discharge

230 sterile filters

240 medium 250 adjusting agents

260 Harvest

261 PG13,5 connection (cover introduction) 270 Filter heating (19Janl2)

280 lateral sampling (19Janl 2) 290 sensor port (chem.) (19Janl2)

300 settlers / cell separator

310 separator tube / cylinder

320 collectors / cone

330 collector installation / flow inverter

331 opening

332 gap

333 sediment discharge

340 drain / suction tube

350 pipe diameter d

360 collector diameter dv

370 separator length 1

380 ground clearance s

390 level

395 separator volume V _A

400 holders

410 heat-conducting element

420 oscillation

430 heating mat

610 pH measurement

61 1 pH spot

620 p02 measurement

621 p02 spot

630 temperature measurement

640 level measurement

700 drive unit with motor

710 opto-electronic measuring system 20 control cabinet

30 Temperature measurement40 Knob

50 display

60 reactor rack 00 sample valve

10 union nut

1 1 seal

12 pipe

21 check valve

22 check valve

23 membrane

30 sample line

3 1 sample

50 flushing line

51 Sterile filing

52 gas

53 water vapor

60 sample distribution

70 pressure line

71 negative pressure

72 overpressure

80 pH measurement

81 pH sensor

982 buffers

983 waste

990 Coupler (Luer-Lock)

1 100 lid sampling element

1 1 10 Sample suction line

1 120 trial line

1130 clamp

1 140 fluid filter

1 150 air filters

1 160 pressure reducer

1 170 Y-piece

1 180 pinch valves 1190 automation platform for the fully automatic collection of liquids, transport, sample preparation and subsequent analysis, eg from EP-1439472 AI and EP-2013328A2 known (BaychroMAT®)

1200 feed cleaning solution

1210 air supply

B spit

bioreactor bottle

As a container 100 was a disposable plastic bottle with square cross-section and cross-sectional edge length D = 120 mm, a height H = 235 mm and a round neck 110 with a neck diameter of 105 mm. The container had rounded edges (Figures 6 and 7); however, this hardly affected the characteristics of the system. The drive was carried out with a stepper motor, which acted directly on the bottle holder (Fig. 11).

A cell separator 300 was installed in the container 100 to operate the bioreactor as a perfusion system (Figure 9). The cell separator 300 comprised a suction pipe 340 passed through the lid 120b and perpendicularly connected to a cylindrical separator pipe 310 having a pipe diameter d (= 350) of 70 mm, wherein the transition region between the suction pipe 340 and the upper portion of the separator pipe 310 formed the crop stream collector 320 and the downwardly open lower part of the separator tube 310 formed the trap volume VA (Fig. 7).

In order to dispense with moving seals, the suction tube 340 has been firmly integrated into the cover 120b and therefore followed the periodically direction-alternating rotary motion (also called oscillation movement) about the fixed axis 101 of the bioreactor (co-rotating embodiment). For comparative experiments, the suction tube 340 was alternatively mounted on a tripod; in these experiments, the cell separator 300 was then used statically.

In the perfusion mode, the separator tube 310 protruded into the suspension in the container (fill level 390> ground clearance s, 380).

The suspension was sucked from below into the separator volume V _{A of} the separator tube 310 by means of a peristaltic pump (peristaltic pump from Watsen & Marlow) connected to the harvesting current collector 320. Within the separator tube 310, the suspension rose and was clarified by sedimentation of the lines / particles (vertical precipitation). The particles fell back against the flow direction out of the separator volume into the culture volume VK (FIG. 7). The clarified solution was collected from the crop stream collector 320 of the separator tube 310 and discharged through the suction tube 340.

The surface A of the separator tube corresponds to its Kreisförmi en cross section and is according to Eq. III calculated. PAN-X examinations:

The particle ytem PAN-X (polyacrylonitrile, spherical particles from Dralon GmbH) was used as the model particle for the investigation of the separation efficiency of the reactor bottle according to the invention with integrated cell enabels in cell culture.

To verify the identity of the physical properties, the particle size distribution and the particle sinking rate were compared since these are the determining factors for the sedimentation. The particle size distribution was determined by the laser diffraction method (Mastersizer 2000, measured according to the instruction manual). The application of the results is carried out as a particle volume in%, based on the total volume, as a function of the particle size in μιη. The modal value XM _OÜ indicates which particle size is most frequently represented by volume and was about 21 μπι.

The rate of descent was analyzed by means of a sedimentation balance. For this purpose, a suspension was prepared which has the same concentration as that used in the experiment. The PAN-X was suspended in deionized water (= deionised water) and had a mass concentration of about 3 g 6 or a volume concentration of 0.88 vol%. For the analysis, a temperature of 20 ° C was chosen. , Sinking rates v _s measured from these experimental conditions, from 0.129 m / h to 0.137 mh, were determined on different PAN-X batches and correspond to the conditions of non-obstructed sedimentation.

For example, C HO cells have a sedimentation rate of 0.0145 m / h [Searles J A, Todd P, Kompala D S, Biotechnol Prog (1994) 10: 198-206] and are relatively slow-sedimenting cells. The hybridoma cell line AB2-143.2 has a sedimentation rate of 0.029 m / h [Wang Z, Belovich J M (2010) Biotechnol Prog 26 (5): 1361-1366]. For the preparation of the model suspension, 3 g of PAN-X were weighed and suspended in 1000 ml of deionized water using a magnetic stirrer. For sampling, the crop stream was collected in a measuring cylinder, while the volume removed was replaced by DI water up to a level H D = 1 by means of a second peristaltic pump.

Unless otherwise specified, all experiments were performed using the following standard parameters:

Acceleration a = 1000 s ² (P / V = 1 1, 12 W / m ³ )

Co-rotating separator Kiärflächenbelastung v = 0.1 mh

Ground clearance s = 70 mm

Gravimetric Determination of the Particle Concentration: The determination of the particle concentration in the harvest stream was carried out by filtering off (suction filtration) a defined volume of crop stream and then drying and weighing the filter by means of a drying weigher.

The influence of the acceleration or the power input on the retention level at different separator lengths 1 (= 370) was determined. For this purpose, tests are compared with static separator and co-rotating separator (results see Fig. 12). To a separator tube with a diameter d = 70 mm, the harvest current collector with sudden cross-sectional constriction of Fig. 6 and 7 was grown. Length L of the separator of 90 mm and 170 mm at accelerations of 600 to 2000 ° / s ² were investigated.

The performance comparison in the retention of the PAN model particulates! Enter at different line P / V of up to P / V = 50 W / m ³ and Kiärflächenbelastung v = 0.1 m / h showed that the retention degree R with increasing power input into the bioreactor in a co-rotating installation of the sideriderrohr it drops off, the degree of retention being significantly influenced positively by the increased length L of the separator. In further experiments the influence of different harvest current collectors at a separator length 1 = 108 mm and a power input of P / V = 11.12 W / m ³ (a = 1000 ° / s ² ) and v = 0.1 mh was investigated. The harvest current collector with sudden cross-sectional constriction according to FIGS. 6 and 7 with the diameter ratio d _v / d = 5/70 (also called simple collector) or a harvest current collector with conical cross section s constriction according to FIG. 8 with an opening angle of 54 ° and a separator length 1 = 58 mm were co-rotatingly installed in the lid 120 b of the bioreactor. The results showed the clear advantage of the conical harvest current collector. The retention performance of this separator was surprisingly almost constant at separator lengths 1 of 90 to 143 mm in the entire examination area. In harvest crop collectors with sudden expansion, the separation efficiency increased with increasing separator lengths 1 from 90 to 170 mm, but without fully achieving the performance of the conical cross-sectional constriction.

Influence of ground clearance:

To investigate the ground clearance, a separator tube with a simple harvest current collector according to FIGS. 6 and 7 and from 1 to 90 mm long was used. It was set with floor spacings s from 10 to 90 mm. Under standard experimental conditions of PIV = 1 1 W / m ³ and v = 0, 1 m / h could with the separator tube d = 70 mm and / = 90 mm im

Range of ground clearances of 10 <s [mm] <70 no influence of ground clearance can be determined. Only above s> 70 mm is an influence of the ground clearance visible. Influence of the clarification:

The Klärflächenbelastung v of Ab s cheiderrohre s corresponds to the speed of the vertically rising medium and has according to Eq. IV direct influence on the Partikeirückhaltung. The influence of the clarification area load was investigated on the separator tube according to FIGS. 6 and 7 (d = 70 mm, I = 170 mm). The separator tube was fixed in the bottle cap with a bottom clearance s = 70 mm. The reactor was operated at a power input of 3.43 W / m ³ (a = 600 ° / s ² ) and a Klarsflächenbelastungen of v = 0.025 mh to v = 0.2 m / h. In the tested load range of 0.025 <v [m / h] <0.2, the backhaul degree R decreases almost linearly with increasing surface loading or rate of climb v of the separator. This result is due both to the characteristic of the descender and to the sedimentation characteristics of the model suspension used. PA-X particles are present as a polydisperse suspension with widely distributed particle size and sink rate. It can therefore be assumed that the retention characteristic in the almost monodisperse cell suspensions has a steeper course which has been shifted towards smaller ascent rates.

A comparison of the performance of various separation systems is shown in FIG. 13 in the form of retention levels R over the treatment surface load. Compared with 2 power packers of P / V ~ 3 W / m ³ , the evaporator with built-in evaporator with separator surfaces of 8 cur and 39 cm ² (inner tubes 1 and 2) with the stationary external gravity separator variants, such as the classic Dortmund well , a Schägkanalabscheider according to EP 1451290 and a cube-shaped separator according to EP 1 2001 1 21 .8. The test results prove a nearly equivalent retention performance of all separators over the entire areas of the examined Kiärflächenbelastung of 0.025 to 0.2 m / h with slight Vortei len for the cube.

T he e rf o rm e n ts o u r th e s e rf o rt o f th e s, o f the e ective grant agreement "Bio. RW: ProCell - Innovative Platform Technologies for Integrated Process Development with Cell Cultures "funded under the European Regional Development Fund (ERDF)

Claims

claims

1. Use of a dimensionally stable square plastic bottle having a bottom, walls, an interior and at least one access to the interior as a bioreactor tank for the culture of cells.

2. Reactor tank comprising a dimensionally stable square plastic bottle having a bottom, walls, an interior and at least one closable access / to interior, said closable access consists of at least one bottleneck and / or at least one passage, and wherein in the interior, on a or more walls in the lower one

Area of the plastic bottle, at a location defined by coordinates, one or more Sensorpatche are attached.

A reactor tank comprising a dimensionally stable square plastic bottle having a bottom, walls, an interior and at least one closable access to the interior, the closable access consisting of at least one bottleneck, and wherein the bottleneck is closed with a lid, the feedthroughs and / or or connections for the gas and liquid supply and discharge into and out of the reactor bank and has no implementation for a drive axle.

4. Reactor tank according to claim 3, wherein for the gas discharge equipped with a sterile filter with a heating mat Gasaustragsleitung is performed by the lid.

5. Reactor tank according to one of claims 3 to 4, wherein one or more electronic, optoelectronic or electrochemical sensors are performed by the lid.

6. A reactor tank according to any one of claims 3 to 5, further comprising an internal vertical

Cell separator, itself comprising:

a. a central vertical separator tube with a round cross-section with a tube diameter d, where w is the ratio of tube diameter to diameter

Bottle cross-sectional edge length D is 0.25 to 0.90, and

b. a separator head with a collector for aspirating cell-freed Kuiturlösung, wherein the Zellabscheider is attached statically or rotatably mounted on the lid of the reactor tank and the collector is performed by the lid.

7. A reactor tank according to any one of claims 3 to 6 further comprising a disposable sample taking a t, wherein the disposable sampling element on the wall or on Cover of the reactor tank installed Probeneinsaugleitung (11 10), which is connected by means of a Y-piece (1170) to a sample transport line (1 120), which connects the sampling line to an automation platform for fully automated sterile removal of liquids (1 190) wherein the sample reagent port is assisted by means of an air supply (1210) connected to the Y-piece.

Reactor comprising:

a reactor tank formed by a dimensionally stable square plastic bottle having a bottom, walls, an interior and at least one closable access to the interior, the closable access consisting of at least one bottleneck and / or at least one passage, and

a reactor tank receiving periphery for receiving the reactor tank, the reactor tank receiving periphery comprising:

At least one reactor tank mount for receiving in each case one reactor tank with a footprint adapted to the reactor tank and one or more lateral fastening elements,

- A connected to the Reaktortanichalterung drive unit for carrying out a periodically direction-changing rotational movement. 9. Reactor according to claim 8, wherein in the interior of the reactor tank, on one or more walls in the lower region of the Kunststo ffflas che, at a location defined by coordinates, one or more Sensorpatche are attached and wherein at the reactor tank mount directly or by means of optical waveguide or several opto-electronic sensor systems for excitation and readout of the Sensorpatche are attached.

10. Reakt r according to claim 9, wherein the opto-electronic sensor system transmits collected data via the cable via differential, serial interfaces and / or unbundled via radio. 11. Reactor according to claim 10, wherein the drive unit is a stepper motor without a gearbox with direct coupling of the motor and the drive.

12. Reactor according to one of claims 8 to 11, wherein the lockable access of the

Reactor tank is a bottleneck and is closed with a lid, the feedthroughs and / or connections for the gas and liquid supply to the reactor tank and no

Has implementation for a drive axle. Realdortankaumahmeperipherie for receiving a s reactor tank, which ee the reactor tank receiving periphery comprises:

At least one reactor tank mount for accommodating each of a reactor tank with a footprint adapted to the reactor tank and one or more lateral fasteners;

one or more optoelectronic sensor systems attached to the reactor tank mount for reading sensor patches attached laterally to a location of the reactor tank defined with coordinates,

- Connected to the reactor tank mount, a drive unit for performing a periodic direction-changing rotational movement, wherein the drive unit is a stepper motor without a gearbox with direct coupling of the motor and the drive.

A method of culturing cells in a reactor according to any one of claims 8 to 12, wherein the reactor tank with an angular amplitude α in the range of 2 ° <jcxj <3600 ° with a power input> 10 W / m3 about its stationary, vertical axis in one Periodically reversing the direction of rotation is offset.

The method of claim 14 for culturing cells characterized by a rate of descent of 0.01 to 100 cm / h, the reactor tank having an internal vertical cell separator formed by a central vertical separator tube and a separator head with a collector for aspirating cell-depleted medium the Zellabscheider is fixed statically or rotatably mounted on a lid for a reactor tank and the collector is performed by the lid, wherein a Kuituriösung separated from cells with Hil e of Zellabscheiders is sucked from the reactor tank.