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Conical or pyramidal, tapered solids collection area At the top they lead to a common Emtestromsammelbereich 56, which has at least one through 80, through which the crop from the Vessel can be transported. The separation of cells and cell culture solution takes place in the channels.
In the lower segment of the middle region of the plastic bag or plastic bottle, one or more feedthroughs or internals 84 are introduced for uniform horizontal flow distribution of the reactor mixture Usually, depending on the width of the disk pack one to four Feedthroughs with straight ports at the same height in the wall plastic bag or the plastic bottle introduced.
Depending on the height and distance between such bushings, horizontal manifolds 85 may be advantageous as internals. The cells sediment within the slanted channels, slip back out of the channels as in conventional slant channel separators in countercurrent crop flow, and accumulate in the conically tapered solids collection area The channels of the disk set I can have a polygonal, elliptical, round or semicircular cross section FIG.
The dimensioning of the channels number, diameter, length depends on the type of cells to be retained, the size of the bioreactor and the throughput. The required separation area A er f results from the sedimentation rate ws, the perfusion rate qlV medium throughput q per bioreactor volume V and the bioreactor volume according to Eq. Binder, Sedimentation of single- and multi-grain suspensions in inclined, laminar flow-through circular and rectangular tubes, Dissertation Berlin, the gin.
I Ii erb ei sind 7. The length LK of the bag to be realized depends on the fill levels to be realized in the plastic bag and on the hydrostatic pressures to be realized in the plastic bag. Excessively large hydrostatic pressures may be transmitted to suitably dimensioned, non-product-contact and therefore reusable housings.
Sufficient pressure losses can be regarded as technically feasible for channel lengths from 0. Short channel lengths L can lead to distribution problems due to the reduced pressure losses, which may necessitate withdrawal speeds, in particular when the crop stream is removed from the harvested crop collecting area 56 e i n e distribution. Bevorzugt umfasst das Lamellenpaket 1 von 1 bis Stegplatten, besonders bevorzugt 1 bis 50 Stegplatten.
The separator according to the invention may usually comprise 1 to 10 6 channels, preferably 10 to ,, particularly preferably 10 to 10, The channels are possibly distributed over one or more web plates in a disk set 1 to optimize space requirements. Preferably comprises the disk set 1 from 1 to web plates, more preferably 1 to 50 web plates. In one embodiment of the invention, the web plates can be bonded to a support plate 30, which provides support and can be connected to the plastic bag by gluing or welding for exact positioning.
The width to height ratio of the disk set I consisting of single or multi-layer web plates including the support plate can be adapted to the geometry of the plastic bag. Das Lamellenpaket 1 kann aus einer profilierten Platte oder gebildet werden siehe Figur 4.
Dabei werden die Rinnen zur offenen Seite hin jeweils durch die glatte Seite einer benachbarten Lage bzw. For the welding of a 2D bag with the disk pack inserted, it is advantageous to maintain a minimum distance ratio 0. The disk pack 1 may be formed of a profiled plate or see FIG. A profiled plate preferably has a smooth side and a side with a series of webs and grooves at equal intervals.
Channels form in the stacking of the plate in one or more layers, e. The gutters are closed to the open side in each case by the smooth side of an adjacent layer or through the wall of the stator. It is also possible to extrude a disk pack or - part package one or more layers and connect to a disk set 1.
The geometry of the channels is determined by the ratio of the web height hs to the channel width d Figure 4. Elasticity, deep drawability in the range 0. Usually, hs should be greater than or equal to 1 mm, or preferably greater than or equal to 3 mm. The web widths bs are determined by the mechanical stability of the film material. The web widths bs should be as small as possible in order to enable high separation surfaces per metered volume.
At the same time they should not be too low to allow a positive connection with the lower layer without changing the shape can. Das aus Stegplatten aufgebaute Lamellenpaket ist entweder als gerader Quader Fig. The lamellae set up of web plates is designed either as a straight parallelepiped FIG. The latter solution is preferred in order to prevent a concentra tion caused by sedimentation to the lower channel openings. The channels are preferably uniformly flowed through by the reactor mixture by means of the horizontal distributor A preferred connection of the multi-wall sheets is via gluing or welding.
The multi-plate package should primarily be spatially fixed by the connection. In addition, it is desirable to keep the so-called dead zones spaces not used for deposition around the outer surfaces of the web plates as small as possible.
However, a complete avoidance of dead zones is not necessarily required. Suitable adhesives are the adhesive components known to the person skilled in the art and adapted to the material and surface properties of the channels. In particular, preference is given to using adhesive which is available on the market in the required FDA quality grades.
For welding, thermal bonding techniques such as heat, laser, ultrasound can be used. Welding technology has the advantage that the number of plastics introduced in the pharmaceutical process is not increased by this connection technology.
Die profilierte Platte kann durch Formgebung unmittelbar bei der Plattenherstellung oder durch z. The profiled plate can be made by molding directly in plate making or by e. The material properties of the embossed and smooth plates can be optimally adapted to their different functionality good sliding properties and dimensional stability of the embossed plates, good sealing properties of the smooth plates , i. Kunststoffstegplatten z. Usually commercially available, inexpensive, and suitable for pharmaceutical processes web plates esp.
Plastic sheeting, e. To Hersteilung of the separator, the bushings and other fittings are also prepared in the appropriate places and possibly installed in a plastic film. Subsequently, a plastic bag 50 of the plastic film, which includes the disk pack 1, in a Kunststoffb bag 50 with weld 55 welded together Fig. Als Befestigungsband eignet sich auch eine Kunststofffolie, die um Beutel und Lammeilenpaket straff herumgewickelt wird.
The disk set 1 including the support plate is then usually pressed against the inner surfaces of the plastic bag 50 to prevent the ingress of cells between the plastic bag 50 and disk set 1 and thus fouling. In a first embodiment of the manufacturing process, the plastic bag 50 is tightened to the lamella pack I Figure 5 and the formed pleat 52 is flattened and secured by one or more straps 60 Figure 6. As a fastening tape is also a plastic film, which is wrapped tightly around bags and Lammeilpaket.
Favorable tightening properties are e. Household films upper flexible, thin films made of silicone. A welding of the disk pack 1 with the bag wall may be suitable for producing a tight connection between the bag and disk pack. The processes described allow the simple and inexpensive production of the solids separator according to the invention for the retention and recycling of cells.
Due to the variably variable configuration of the disk packs, the geometry of the later device can be easily and precisely defined and, in contrast to systems made of stainless steel, also be made available for very large bioreactors. The described methods allow, in particular, the cost-effective production of disposable elements, through the use of which the outlay for providing a retention system cleaned according to the basic principles can be reduced to a minimum.
Auf der Auflage wird das Lamellenpaket 1 inkl. To the footprint. On the support , the disk set 1 incl. This reduces dead spaces and corresponding fouling. Deckel zur Aufnahme des Lamellenpakets 1. In this case, the tightening process can also take place when mounting the solids separator according to the invention on the frame and in particular in the housing and cover FIGS.
Preferably, the lid is attached to the housing at one side, e. With the aid of the frame , it is simpler to operate the start-up of the solids separator according to the invention. Such a shape-fitting container is u. Systems with larger hydrostatic forces, as they are to be expected when connecting to large bioreactors. To improve the slipping behavior of the cells in the channels of the disk pack and on the inner walls of the downwardly, esp. Conical or pyramidal, rejuvenated solids collection area di e device can be made by suitable means, such as pneumatic or electric vibrators to vibrate.
Alternativ zu dem Kunststoffbeutel kann ein Lamellenpaket 1 in einer Kunststoffflasche 50 mit eckigem Querschnitt eingebaut sein Fig. Preferred perfusion rates are in the range of 0. Preferred separation surfaces per bioreactor volume, depending on the sedimentation properties of the line depending upon the concentration, size and agglomeration tendency of the cells is in the range of 0.
As an alternative to the plastic bag, a disc pack 1 may be incorporated in a plastic bottle 50 of angular cross-section Figure 21 , the plastic bottle having a downwardly tapered region terminating in a bottle neck and the downwardly tapered region forming the solids collection region 57 Collect the solids with the help of Gravity forms.
For operation, the plastic bottle neck is closed by means of a cover , which has the passages for the passage of the connecting lines. In an alternative embodiment, the plate pack 1 is replaced by one or more attached to the walls of the plastic bottle 50 floors as a deposition area. As a material for the plastic bottle z. The solids separator according to the invention is preferably designed as a disposable article to avoid the problem of cleaning.
Usually, the invention e cell separator to a bioreactor z. The supply of the separator according to the invention is ensured by at least two pumps, preferably low-shear peristaltic pumps FIG. Pall, Fa. Sartorius, Fa. Der Anschluss, der Betrieb und die Wartung sind problemlos. The storage of the present invention solids separator is space-saving, since they can be easily stacked on each other and are placed only at startup with the appropriate angle.
They can then simply be connected and operated outside a bioreactor. The connection to the fermenter he carried by means attached to the end of the tubing sterile coupler various manufacturers Pall, Fa. Sartorius, Coulder within or outside of safety workbenches, but preferably by tube welding.
The hose lines fastened to the solids separators according to the invention are therefore preferably-at least partially-equipped with a hose element which is suitable for the hosech. In addition, the hose lines for conveying the suspension usually contain at least two special hose elements which are mechanically highly loadable for example made of elastomers and which can be loaded non-invasively in peristaltic pumps without endangering the sterility of the separators.
Connection, operation and maintenance are easy. The embodiment of the device according to the invention or parts of the device according to the invention as a disposable element eliminates cleaning problems. Further objects of the present invention are therefore a method for producing a Festsstoffabscheidevorrichtung invention with lamellar package in a plastic bag and A direct use of the disk set 1 in aerobic bioreactors is in principle conceivable if the gas bubbles required for the gassing can be kept away from the access openings.
In this case, the conical collection part of the plastic bag and the back! Bioreaktors vorgesehen. Bioreactor provided. Ecke des Polyeders oder der Kegels gebildet wird. In a wide Ausfii form of the cell separator according to the invention, the plastic bag is poiyeder- or cone-shaped, the plastic bag is set during operation so that the bottom conically tapered solids collection area 57 formed by the walls of the plastic bag and a tip or corner of the polyhedron or cone becomes. The volume of the separator may for example assume values of 0.
For a simple production from a plastic film Disphenoid esp. Tetrahedron, cones and cubes are preferred. The cell inventions according to his invention usually has a ratio of height to the maximum width in the range of 0. The connection of the Zellabscheiders invention with a bioreactor or other external equipment is preferably via connections and connecting lines at the top of the separator. This has the advantage that the container for receiving the separator without performing, d.
In this way, the emergence of genetically modified production cells can be prevented without additional safety equipment. The inlet of the bioreactor mixture usually takes place via an installation 84 for introducing the bioreactor mixture along the vertical axis of the plastic bag set up for operation. In the cell separator according to the invention at the Inlet opening of the installation!
The inlet is thereby by means of annular nozzles or by means of two or more outlet openings horizontally directed internals such. As tees or downwardly directed fittings such. Y-pieces on introduction surface preferably in the direction of the corners of the inlet distributed.
In the case of the Y distributors, the inlet flows are directed downwards. Better retention was achieved with a simple T or Y manifold than with dual T or Y manifolds. Vorzugsweise wird der Einbau 88 zum zentralen, vertikalen Absaugen der Feststoffe verwendet Fig. The removal of the solids from the solids collection region 57 is usually via one or more passages 88 near the bottom tip of the precipitator. The assembly 88 is connected to the bioreactor into which the collected cells are returned through the pressure drop or by pumping.
Preferably, the insert 88 is used for central vertical suction of the solids Figures 14, 15, 16, This simplifies the manufacture and placement of the separator in its container for operation. In a further preferred embodiment, the trigger can also be withdrawn via a welded into the Spit e of the separator implementation. In this case, for a leak-free execution of the housing, the connection hose is to be laid inside the housing for accommodating the separator i.
This fitting 80 usually has the shape of an inverted screen with the outer contour of the harvesting current collecting area 56 in the case of a tetrahedron, a triangular installation is used with an edge length or diameter a, where 0. In a preferred embodiment of the inventive he inventive separator is located in the upper region on the wall of the Kunststoffb eutels tetrahedron or at a corner cube one or more connection plates 90, preferably one, which may also be a lid that the bushings for contains the passage of the connecting lines and in the region of the terminal allows a holder of the separator.
The maximum separation surface is located above the inlet surface The separation of the cells takes place in the vertical separators according to FIGS. The dimensioning of the crop sediment depends on the type of cells to be retained, the size of the bioreactor and the throughput.
The required separation area A er results, as in the first embodiment of the invention, from the sedimentation rate ws, the perfusion rate qlV medium throughput q per bioreactor volume V and the reactor volume according to Eq. A, A,. Rondendurchmesser D ergibt: Diameter D gives:. By dimensioning the plastic bag 50 , the edge length D to be realized or the length LK of the booty depends!
Usually, the inlet surface is at a height h with respect to the height of the plastic bag HK of 0. For proper use, the separator is placed in a container which supports the flexible walls of the plastic bag when the separator is filled. Therefore, the shapes of container and separator are preferably matched. Another object of the present invention is therefore a Feststoffabscheidevorrichtung comprising a Festsstoffabscheider invention and a container for receiving the Feststoffabscheiders, the container at least: an interior space for receiving the solids separator, wherein the interior is adapted to the dimensions of the solids separator by means of walls adapted to the shape of the solids separator and enclosing the interior space and the outside world, an opening for introduction of the solids separator from above the container.
The opening for introducing the Zellabscheidevorrichtung is preferably closable. The container according to the invention is preferably made liquid-tight in the closed state, i. Diese Kunststoffb eutel bzw. Kunststoffflasche aus Fig. Also possible is a plastic bag or bottle with a quadrangular cross section and at the bottom of a downwardly tapered solids collection area 57 for collecting the solids by gravity Figure Baffles and bushings 80, 88 and 84 may be welded into the plastic bag or plastic bottle.
Alternatively, the downwardly tapered solids collection area 57 terminates in a throat. Both the plastic bag neck and the plastic bottle neck are then closed with a lid or stopper in which all necessary feedthroughs are installed. In both embodiments, the shape of the crop stream collecting area 56 may be arbitrary. These plastic bags or bags are particularly advantageous for small fermenter volumes of 0. For operation, one or more plastic bags or puffs are hung on a rack.
For a space-saving application of several separators in parallel di e hanging plastic bag or plastic bottle of Fig. Die Bioreaktoranlage ist beispielsweise ein Perfusionsreaktor, der in bekannter Weise betrieben werden kann. Another object of the present invention is a bioreactor system consisting of a bioreactor and a Zellabscheidevorrichtung invention described. The bioreactor is preferably a one-way reactor, in particular a reactor as described in US The bioreactor plant is for example a perfusion reactor, which can be operated in a known manner.
Nutrient medium is fed continuously into the bioreactor, and cell-poor cell culture supernatant is continuously removed. In this case, the flow through the separator takes place continuously. Likewise, the perfusion reactor can be operated in such a way that a culture is initially allowed to grow up batchwise. If the medium is used up so far that no appreciable build-up of biomass is possible anymore, the culture supernatant, which is almost free of biomass, is withdrawn via the external cell separator.
The space obtained in the bioreactor can then be used to supply fresh nutrient medium, thereby allowing further growth and thus a higher total biomass productivity R epeat cd-Bat cli-M odus. In this case, the flow through the line separator takes place intermittently. This method can be used as an example for precultures that are intended to be used to inoculate very large bioreactors because it can increase the productivity of existing preculture reactors.
For operation on bacteria, a continuous flow of the cell separator according to the invention is preferred. The bioreactor or perfusion reactor can be used to grow cells that grow in vitro and in free suspension or on microcarriers.
Among the preferred cells include protozoa and adhesive and non-adhesive eukaryotic cells human nerve, blood or tissue cells, as well as stem cells of embryonic or adult origin , animal or vegetable origin, the z. Produce enzymes, antibodies, neurons, tissue cells or diagnostic structures. Particularly suitable for high-performance pharmaceutical production cells are used, for example, ciliates, insect cells, baby hamster kidney BUK cells, Chinese 1 lambda ovary CHO cells, 1 IKB cells by the fusion of human HEK cell line with the human bacterial line 2B8 , hybridoma cells and stem cells.
The present invention further provides a process for the retention and recirculation of solids, in particular cells, in a vessel through which the vessel is fed continuously or batchwise solids-containing medium and solids-free medium is discharged, characterized in that the vessel is a flow-through gamma sterilizable plastic bag or bottle is inclined at the bottom Areas, conveniently one down, esp.
Conical or pyramidal, rejuvenated. Feststoffsammeibereich 57 zum Sammeln der Feststoffe mit Hilfe der Schwerkraft aufweist. Solid collection area 57 for collecting the solids by gravity. Die Reynolds-Zahl Re kann nach Gl.
In inclined channels, there is a lower flow velocity at the channel inner walls than in the channel centers. The cells sediment in the channels and slip at the bottom of the channels against the direction of flow of the lower channel ends against. The cell culture solution freed from the cells is discharged through the cannula into a first chamber 56 located above the channels and finally transported out of the vessel.
In einem polyeder- bzw. In a polyhedral or cone-shaped separator, d is the width or the diameter of the cross section of the maximum separation surface. On the plastic walls there is a lower flow velocity than in the channel centers. The cells slide against the flow direction towards the solids collecting area The cell-culture solution freed from the cells rises into a crop stream collecting area For this purpose, the cell culture solution is transported with cells from the bioreactor in the cell separator according to the invention.
Preferably, the cells are cooled before entering the separator in an external vessel in order to slow down the metabolism and thus counteract a productivity-reducing shortage of Zel len. In cooled suspension, oxygen supply to the sedimenting cells is not required.
In most cases, a cooling of the cell culture solution to the ambient temperature of the separator is completely sufficient, so that in addition to the desired metabolism eleff effect convection currents are reliably avoided. To monitor the sufficient supply of the cells, the separator can be equipped with at least one disposable sensor z: B.
An accommodation of the sensors is possible both in the walls as well as the connecting line to the bioreactor or the harvesting vessels. The inventive method allows the effective retention and return of cells in a continuous flow sterile plastic bag. B of the retention and. Repatriation act on the lines only moderate shear forces, which are usually well tolerated by the cells. The cells are kept in the separator at fermentation temperature or a lowered temperature level and the supply of nutrients is given.
Exemplary embodiments of the invention will be explained in more detail below with reference to the drawings, without the invention being restricted thereto. Fig 1. Schematic representation of the disposable solids separator according to the invention with plate pack.
Fig Schematic 3-dimensional representation of the invented prosthesis in the disphenoid embodiment. Schematic longitudinal section of the solids separator according to the invention in the disphenoidal embodiment with flow inverter Andere Verschattungen, z. In order to reduce the respiratory activity of the cells in the bioreactor outlet, its temperature is lowered to a lower level as soon as possible after withdrawal in a cooling device. In this way, the cells in the cell separator are prevented from lingering in an acid-limited state for too long, which could physiologically damage the cells.
In the example shown, the separator consists of a separation bag and an integrated cooling device The liquid flows between bioreactor and separator are adjusted by the low-shear pumps and Other shadows, eg the positioning of one of the two pumps and in the bioreactor outlet, are also conceivable.
Bezugszeichen: Reference numerals:. Horizontal distributor or flow distributor with downwardly directed inlet flows. Profile eines Lamellenpakets Profiles of a lamella package. In the following, investigations of the applicability of the devices according to the invention are described without being limited thereto. Partikelsystem particle system. The polymer which is insoluble in water is mainly used in the clothing industry for the production of fibers, hereinafter an excerpt from the product data sheet of the manufacturer Dralon imbl I.
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