Patent Application
20150218501
The invention relates to a reactor tank designed as a disposable element having a cover and/or sensor patches readable opto-electronically fixed in the interior, a reactor comprising the reactor tank and reactor tank receiving peripherals comprising a reactor tank holder and optionally an opto-electronic measuring system for reading the sensor patches, wherein the reactor tank holder is coupled to a drive unit for generating a rotating-oscillation motion of the reactor tank around the central vertical axis thereof, and also the use of this device for culturing cells and/or microorganisms.
In the case of the highly regulated pharmaceutical production, a great expenditure in terms of time, technical expenditure and expenditure on staff is accounted for by the provision of cleaned and sterilized bioreactors. In order to avoid reliably cross contamination during a product change in a multi-purpose plant or between two product batches, in addition to the cleaning, a highly complex cleaning validation is required which, in the case of a process adaption, may need to be repeated.
This applies not only to upstream processing, USP, as to say the production biological products in fermenters, but also to downstream processing, DSP, that is to say purification of the fermentation products.
In USP and DSP, pressure vessels are frequently used as agitation and reaction systems. Especially in the case of fermentation, an aseptic environment is essential for successful culturing. For sterilization of batch or fed-batch fermenters, generally the steam-in-place (SIP) technique is used. In order in the case of a continuous process procedure to ensure sufficient long term sterility, the autoclaving technique is also utilized which, however, requires laborious transport of the reactors to the autoclave, and is only useable at comparative small reactor scales. The risk of contamination during fermentation is particularly critical during sampling and at moving stirrer shafts. The latter are generally equipped with complex sealing systems (e.g.: sliding-ring seals). Technologies which manage without such penetrations of the fermentation casing are preferred because of their greater process robustness.
The down time of standard reactors caused by the preparation procedures can be in the order of magnitude of the reactor availability, in particular in the case of short utilization periods and frequent product change. The process steps affected are, in USP of the biotechnological production, e.g. the steps of media production and fermentation, and in DSP solubilisation, freezing, thawing, pH adjustment, precipitation, crystallization, buffer change and virus inactivation.
In order to meet the requirement of rapid and flexible re-charging of the production plant, while ensuring maximum cleanness and sterility, designs of disposable reactors are enjoying constantly increasing interest on the market.
WO 2007/121958 A1 and WO2010/127689 describe such a disposable reactor for culturing cells and microorganisms. In one preferred embodiment it consists of a stable, preferably multilayer, polymer material pouch. The deformable disposable reactor is received by a container which supports it. In this process it is preferably introduced into the container from the front. The container is connected to a drive unit. By way of the drive unit, the container including the disposable reactor is put into a rotating-oscillating motion about a stationary, preferably vertical, axis of the container. By way of an square design shape of the disposable reactor and/or internals in the disposable reactor, in the case of the oscillating-rotating motion, a high introduction of work into the reactor contents can be achieved, so the disposable reactor can be used as a fermenter with surface gas treatment for culturing cells and microorganisms. The internals for supplying and monitoring reactor are mounted at the side at the bottom of the reactor via a connecting plate. These reactors are predominantly used at reactor volumes of more than 10 L.
For smaller reactor volumes, the production of a reactor pouch including appropriate container is too complex.
The challenge in small disposable reactors is to achieve the sensor technology, the mixing technology, the temperature control and supplying of the reactor in a form that is as compact and inexpensive as possible.
Small agitated disposable reactors are known from the prior art.
Sartorius Stedim Biotech, in its Universel® SU (http://www.sartorius-stedim.com/Biotechnology/Fermentation_Technologies/Reusable_Bioreactors/Data_Sheets/Data_UniVessel_SU_SBI2033-e.pdf), offers an agitated disposable reactor in which the reactor tank is cylindrical. The disposable reactor, for the mixing, possesses an agitator, and for the gas supply from below, possesses an L-sparger below the agitator. Via the lid, the agitator drive is ensured by a top-driven drive axle, the sensor technology (temperature, pH (chemistry), oxygen (chemistry)), the gas supply and gas disposal for the gas space, and further supply and sampling via conduits. The lid is fastened to the reactor tank by a clamp connection, and is sealed in a sterile manner against the reactor tank by O ring. The agitator drive is sealed with 2 lip seals. The sensor technology for monitoring pH and oxygen content can also be achieved by means of optoelectronic sensor patches at the bottom of the reactor tank. For operation, the reactor tank is positioned fixed in a special container, wherein this container possesses a holder ring and a foot having an optoelectronic sensor system for reading the sensor patches.
The disadvantage of this and/or similar reactor systems available on the market is that these stirred systems require moving internals and also a complex sterile sealing system in the lid, and, in view of the high shear forces are less suitable for the culture of very sensitive cells, such as e.g. stem cells.
Proceeding from the prior art, the object in question is to provide a low-shear system for carrying out processes having high requirements of cleanliness and sterility which reduces the expenditure in terms of time, equipment and staff on the provision of cleaned and sterilized components. The system shall be useable for process volumes from 10 mL to 20 L, in particular 50 mL to 10 L, and particularly preferably 250 mL to 3 L working volume. It shall meet the high requirements of the pharmaceutical industry, be simple and intuitive to handle and be inexpensive. It shall reduce safety risks due to the escape of substances from the process chamber to a minimum. It shall permit sufficient mixing of the reactor contents, be suitable for the culture of microorganisms and cell cultures and, in the process, ensure sufficient supply and disposal of the culture medium with liquid nutrient medium and in particular gaseous substances. It shall be just as suitable for process development as for the production of cell products, in particular cell products such as, e.g., human or animal body cells: stem cells, blood cells, leucocytes such as, e.g., natural killer cells (NK cells), tissue cells or pharmaceutical active ingredients such as, e.g., monoclonal antibodies, proteins, enzymes in bioreactors.
According to the invention this object is achieved by the use of a dimensionally stable, angular plastics bottle for delimiting the reactor interior, wherein the plastics bottle has a bottom, walls, an interior and at least one access to the interior, and preferably a pyramidal inwards-dished bottom, a wide neck and/or one or more sensor patches mounted in the lower region of the bottle at a site defined by coordinates.
The present invention first therefore relates to the use of a dimensionally stable, angular plastics bottle as bioreactor tank for the culture of cells, in particular sensitive cells and cells growing on (micro)supports, such as, e.g., stem cells, blood cells or tissue cells, wherein the plastics bottle has a bottom, walls, an interior and at least one closable access to the interior, in particular a bottleneck. Usually, in the interior of the plastics bottle, one or more sensor patches are mounted on one or more walls in the lower region, at a site defined by coordinates.
The present invention further relates to a reactor tank comprising a dimensionally stable angular plastics bottle which has a bottom, walls, an interior and at least one closable access to the interior, comprising at least one bottleneck, in particular closable by a lid, and/or at least one passage, and wherein one or more sensor patches are mounted in the interior, on one or more walls in the lower region of the plastics bottle, at a site defined by coordinates. Preferably, passages are accommodated in the lid.
Support-fixed sensor patches made up of fluorescent coloured layers are available on the market (e.g. from Presens, YSI) which can be affixed, e.g., to a bottle wall. Usually, at least one pH sensor patch and one oxygen sensor patch are used.
Alternatively, the reactor tank or bioreactor has passages for electrochemical sensors, preferably disposable sensors, e.g. according to US 20120067724 A1, on a bottle wall or in the lid, preferably in the lid.
In order that the reactor can meet the sterility requirements of the pharmaceutical industry, the plastics bottle is usually produced from a gamma-sterilizable plastics material. The reactor tank according to the invention is preferably made from a single- or multilayer transparent polymer material which permits a view into the reactor tank during operations.
Plastics or glass are relatively inexpensive materials which may also be processed relatively inexpensively. The disposal of the used reactor tank and the use of a new disposable reactor tank are thus more economical than cleaning used reactor tanks, in particular, since when a new disposable reactor tank is used, complex cleaning and cleaning validation are omitted. The reactor tank according to the invention is produced or cleaned in a cleanroom and is preferably sterile-packed.
The reactor tank according to the invention is dimensionally stable. Suitable materials or material combinations for the reactor tank according to the invention are all cell biological compatible materials known to those skilled in the art, in particular glass, polyethylene, polypropylene, polyetherketone (PEEK), PVC, polyethylene terephthalate and polycarbonate. Wall thicknesses of 0.1 mm-5 mm are preferred, and of 0.5-2 mm are particularly preferred.
The bottle materials are usually brought into the desired form by means of stretch blow moulding methods known from the prior art.
The cross section of the reactor tank or of the plastics bottle preferably has the shape of an n-gon where n is in the range from 3 to 12, preferably in the range from 3 to 6, very particularly preferably in the range 3 to 4, most preferably, n is equal to 4.
Preferably, the side walls of the reactor tank according to the invention or the plastics bottle are formed at least in part as flat surfaces which meet at an angle of 45° to 120°. Preferably, the side walls of the reactor form a polyhedron, wherein the bottleneck is mounted on one of the surfaces.
Preferably, the reactor tank or the plastics bottle is cuboidal with edge lengths H, b and c, wherein H is the height, b is the width and c is the depth of the plastics bottle and b≦c≦H. The wide neck is typically mounted on one of the small surfaces and the surface opposite it serves as 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 from 0.5 to 4, preferably 1 to 3, particularly preferably 1.5 to 2.5. In the preferred embodiment, the reactor tank has a square bottle cross section edge length a=c=D.
For better mixing of the reactor and reduced starting volume, the reactor tank and/or the plastics bottle usually has an inwards-dished bottom. For the configuration of the bottom, the teaching of WO 2010/127689 is incorporated by reference. The bottom has, in particular, the shape of an inwards-directed tetrahedron, an inwards-directed pyramid, the shape of a paraboloid or a bell shape. Particularly preferably, the bottom is formed pyramidally. The height hw of the dishing is in the range of 0.01 times to 1 times the circular equivalent diameter Dk of the bottom cross section. Preferably, the height hw of the dishing to the circle-equivalent diameter Dk is 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 the outer walls thereof. In a preferred embodiment, on the outside of the bottom of the plastics bottle or the reactor tank, a disposable heating mat is applied with which, owing to the positive connection of heating surface and shell surface, very efficient heat transport can be achieved. In this manner, the heating surface can be reduced to the bottom surface. For this purpose, this heating mat is usually adhesively connected to the outside of the bottom. Generally, the reactor tank does not need additional cooling, since switching off the heating mat in reactors having a small volume and thus high specific heat exchange surface area, leads to sufficiently rapid cooling. Additional cooling would be applicable if required, e.g., in the case of microbial applications at relatively low fermentation temperature and relatively high heat of respiration, by mounting Peltier elements to the side surfaces of the reactor tank or of the tank holder.
The reactor tank according to the invention is preferably a chamber that can be sealed off from the outside for carrying out chemical, biological, biochemical and/or physical processes. In particular, the reactor tank serves for providing a sterile chamber 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 tightly closed by means of a lid, wherein the lid possesses at least passages and/or connections for the gas and liquid supply and removal for the reactor tank. According to the invention, the lid does not have a passage for a drive axle [
In addition, the lid, if necessary, can comprise further passages and/or connections for elements from the group comprising:
The reactor tank, depending on application, is appropriately fitted with one or more of said elements.
In a preferred embodiment of the invention, the lid is composed of a stopper and a retainer sleeve. The stopper is usually produced of plastics selected from the group of polyether ether ketones, thermoplastic or silicone. Usually, the stopper is made as a disposable stopper, in a particular embodiment, alternatively, reusable.
Preferably, the stopper is introduced into the neck of the reactor for closure, sealed against the inside of the bottleneck by means of an O-ring seal mounted on the periphery, and, with a separate locking means such as, e.g., a screw-mountable retainer nut, screwed onto the thread of the bottleneck or clamped with a clamping ring. Alternatively, the stopper introduced into the bottleneck can be sealed by means of a sealing lip applied on the bottle opening and clamped with a separate screw-mountable retainer sleeve and screwed onto the plastic bottle. A further alternative is a lid which contains the identical passages as the stopper which is screwed on the plastic bottle and sealed off from the bottleneck and/or the bottle opening by an O ring. Preferably, the stopper pushed into the bottleneck is used, which is sealed with an O-ring seal on the bottleneck and which is screwed firmly with a separate screw-mountable retainer nut on the plastics bottle [
The reactor tank together with lid is preferably constructed as a disposable element, i.e. it is preferably intended not to clean the complete reactor tank after use, but to dispose of it. Therefore, the reactor tank preferably comprises only the essential elements which are necessary for providing a sterile reaction chamber:
The plastics bottle is usually produced and used as a disposable article.
For the culture of sensitive cells, or the production of clinical cell products, the gas is preferably supplied exclusively via the surface. In this case, the lid has no passage for a bubbling gas introduction element and the reactor tank according to the invention has no internals for bubbling gas introduction. For applications in the context of process development with the focus on scale up to large fermenters, for perfusion methods having high cell densities and for microbial processes, an installation can be provided for additional microscale or macroscale gas introduction (e.g. supplied by flexible tubular lines from the top via the lid and a sintered body adhesively applied to a container wall). Preferably, the reactor according to the invention may be produced completely from inexpensive elements and hereby permits the use of the reactor as a disposable system. Alternatively, all high-value 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, for cell retention a cell separator in the reactor tank is used. According to the invention, the internal cell separator is formed by a central vertical separator tube and a separator head having a collector for removing by suction culture solution freed from cells, wherein the lid has a passage for the collector and the cell separator is either rotatably mounted or statically fixed to the lid. The tube and the separator head can have differing lengths, geometry (conical and straight) and diameters, and have diverse tube internals (conical and ring internals, flow aligners). Particular embodiments are shown in
The present invention therefore further relates to an internal vertical cell separator for bioreactors formed by a central vertical separator tube and a separator head having a collector for removing cell-free medium by suction, wherein the cell separator is fixed or rotatably mounted to a lid for a reactor tank and the lid has a passage for the collector.
If a cell separator is used, the reactor tank usually has a wide neck, in order that the prefabricated cell separator fastened to the lid can be introduced through the bottleneck. If the cell separator is moveably fixed (=rotatably mounted) to the lid, it is only minimally affected by the rotary motion of the reactor tank owing to its inertia. As a result, the circulation flow transmitted by the separator into the sedimentation chamber and interfering with the sedimentation process are avoided, and the retention considerably improved, as shown in
The cell separator is preferably designed in such a manner that inner and outer region of the cell separator are substantially separated from one another by corresponding constrictions. In this manner, a transmission of the flows interfering with the sedimentation from the well mixed outer chamber into the sedimentation zone are reduced. In other words, the internal volume of the cell separator shall be affected as little as possible by flows in the outer volume (=culture volume), but a back-transport of the retained cells into the mixed supplied reactor region must remain ensured.
Preferably, the internal cell separator, for a reactor tank having the dimensions of cross sectional edge length D=120 mm, H=235 mm, has a separator tube (310) having a separator length l (370) from 40 mm to 200 mm, in particular from 90 to 190 mm, preferably 190 mm (
The separator tube has a round cross section with a tube diameter d (350), wherein the ratio of tube diameter d to the bottle cross section edge length D is usually from 0.25 to 0.90, in particular from 0.5 to 0.85, preferably 0.83. The tube diameter d is of importance for the cell retention to achieve the separator surface area.
It is preferred to select the bottleneck and cell separator cross sections in such a manner that the cell separator can be readily introduced into the bottle. This is necessary, in particular, when a reusable autoclavable lid is to be used which is to be connected to the gamma-sterilized reactor tank under the cleanbench.
In a first embodiment of the bioreactor, the gas introduction proceeds solely via the surface (
Further parameters of the cell separator are the separation area (=clarification area) A defined as
and also the clarification area loading v=q/A, wherein q is the harvest stream.
At the separator head, there is situated the collector (320) for removing the cell-free culture solution by suction. Usually, the ratio dv/d of the collector diameter dv (360) to the tube diameter d is 0.1 to 0.8, preferably 0.3-0.5.
Preferably, the collector (320) has a conical shape. This shape has the advantage that more space is available for introducing further elements (sensors, sampling line, etc.) via the lid. Likewise, the gas-introduction area is more slightly reduced.
Preferably, the separator in the reactor tank is used with a ratio l/s of the separator length l to the bottom spacing s from the separation tube of 0.75 to 0.9.
Internals in the separation tube and collector (320) are preferably dispensed with in the cell separator.
In hydrodynamic studies with the model particle PAN-X, a tube statically built in at the lid having differing tube lengths geometry (conical/straight head) and diameters and also diverse tube internals (cone and ring internals, flow aligners etc.) was studied. According to the experiments available to date, the statically installed (=corotating), internal cell separator for solids in the range of clarifying area loading v of 0.025<v [m/h]<0.2 at a power input P/VK of 3 W/m3 has a comparable degree of retention to static external systems (e.g. plate separator, vertical flow sedimentation tank) (
In particular, the cell separator according to the invention is applicable for the culture of readily sedimentable particles such as, e.g., support-fixed cells which is useable, depending on the sink velocity of the support materials, in considerably higher power introduction ranges P/VK of >>3 W/m3.
In a particular embodiment of the invention, the reactor according to the invention, in addition, has an automatic sampling element.
In a first embodiment, the sampling element consists of a receiving line which is conducted through the lid (=lid sampling element, see
After the sample removal, the sampling element is sterilized with EtOH and dried. Preferably, filter elements for air and EtOH are built in in order to prevent contamination of the sample withdrawal element. Preferably, the sampling element is coupled to the BayChromat-Platform for automated analysis from Bayer Technology Services GmbH.
In a further embodiment, the reactor tank has, on a bottle wall, in particular on the wall opposite the sensors (sensor patch or electrochemical sensors), a passage and/or a connection in the region close to the bottom for mounting a sampling system. Examples of passages and/or insertions are, inter alia, standardized Ingold stubs—or PG13,5-thread stubs. A suitable sampling system is described, e.g., in DE102008033286 A1.
The mixing within the reactor tank according to the invention proceeds via the reactor tank rotation changing direction periodically, which in combination with the angular shape of the plastics bottle, causes inwardly directed wave-shaped flows to the surface of the reactor contents. For configuration of the reactor motion, the teaching of WO 2010/127689 is incorporated by reference.
All remaining elements which are required for operating a reactor, in particular for culturing cells and/or microorganisms, in particular a drive unit for generating the reactor tank rotation changing direction periodically and optoelectronic sensor system for reading the sensor patches are provided by peripherals and are reusable. The reactor, which in the prior art is usually one coherent unit, is therefore in the present case preferably divided into separate parts which are configured according to their functions.
A further element of the reactor according to the invention is therefore the peripherals. In particular, as peripherals, reactor tank receiving peripherals are used which have one or more reactor tank holders, wherein the reactor tank and the reactor tank holder are, as separate parts of an overall system, matched to one another in such a manner that the reactor tank can be introduced into the reactor tank holder and/or in particular clamped in there, and is supported thereby in the liquid-filled state.
The reactor tank receiving peripherals for receiving a reactor tank according to the invention is a further element of the reactor according to the invention and comprises at least:
According to the invention, the data transmission proceeds in a line-bound manner via differential serial interfaces and/or wirelessly by radio such as, for example, Bluetooth of WLAN. Preferably, the optoelectronic sensor system possesses the differential serial interface for symmetrical signal transfer of the EIA485/RS485 type, on account of the robust data transmission and high tolerance to electromagnetic interference. For improved data transmission, a stepper motor without gear with direct coupling of the motor and the drive was identified as particularly advantageous, because it permits a particularly interference-free data transmission.
Preferably, the footprint adapted to the reactor is exchangeable or adaptable in such a manner that the reactor frame is applicable to reactors of various sizes.
The present invention also relates to the use of the reactor according to the invention and reactor tank and also to a method for the culture of cells and/or microorganisms.
In the reactor tank, during operations, the ratio of liquid level to reactor tank width is preferably 0.05 to 2, and particularly preferably 0.1 to 1, wherein the liquid level can change as a consequence of supplemental feeding with the growth of the cells. In addition, the reactor tank, while maintaining the preferred hydrodynamic and processing properties thereof, is operated with a sufficient head space between reactor tank head and liquid level (=full level 180, HL) of at least 5% to 50% liquid height, preferably at least 25% liquid height, in order, in the case of foam formation, to ensure sufficient spacing to the gas discharge line equipped with a sterile filter. For control of the fill level, usually, a capacitive sensor for fill level control is used through the lid or on one of the container walls.
It has surprisingly been found that a comparatively small angular amplitude is sufficient for the rotating-oscillating motion of the reactor in order to achieve good mixing and/or sufficient intensification of transport processes. In particular, it is scarcely necessary to achieve 3600° rotations (that is equivalent to 10 rotations) of the reactor, and so there is no requirement for structurally complex solutions for linking the oscillating and rotating reactor to the static surroundings (e.g. for feeding and removing media and gases, electrical energy and electrical signals).
In the use according to the invention, the reactor tank is moved at an angular amplitude α in the range from 2°≦|α|≦3600°, preferably 20°≦|α|≦180°, particularly preferably 45°≦|α|≦90° in a rotating-oscillating manner, wherein deviations from ±5° may be present. In particular, |α|=60° is considered to be very particularly preferably in the use of particularly low-shear bioreactors supplied with gas via the surface. In total, therefore, the oscillating motion sweeps through an angle of 2|α|.
Experiments have found that when the power input is elevated, movement states can be established in the reactor in which gas bubbles are introduced into the reactor medium. Gas bubbles are drawn in from a power input of P/VK>10 W/m3. For cells and/or microorganisms which are not damaged by bubbling gas introduction, a very simple increase in gas supply can be achieved in this manner. Via an additional bubbling gas introduction via a sintered tube preferably installed in the bottom region, the mass transport can be considerably improved. The flow generated by the rotary oscillation ensures gentle detachment of the microbubbles from the sparger and thus a large phase interface a or a large mass transfer coefficient kLa.
The invention will be described in more detail hereinafter with reference to figures, but without restricting it to the embodiments shown.
A disposable plastics bottle having a square cross section and cross section edge length D=120 mm, a height H=235 mm and a round bottleneck 110 having a neck cross sectional diameter of 105 mm served as container 100. The container had rounded edges (
A cell separator 300 was built into the container 100 in order to operate the bioreactor as a perfusion system (
In order to dispense with moving seals, the suction tube 340 was integrated fixed into the lid 120b and therefore followed the rotary motion changing direction periodically (also termed oscillation movement) around the fixed axis (101) of the bioreactor (corotating embodiment). For comparative experiments, alternatively, the suction tube 340 was fastened to a stand; in these experiments, the cell separator 300 was then used statically.
In the perfusion operation, the separator tube 310 projected into the suspension situated in the vessel (degree of filling 390> spacing from the bottom s, 380).
By way of a perfusion pump (peristaltic pump from Watson & Marlow) attached at the harvest stream collector 320, the suspension was withdrawn by suction from the bottom into the separator volume VA of the separator tube 310. Within the separator tube 310 the suspension ascended and was clarified by sedimentation of the cells/particles (vertical separation). The particles fell against the direction of flow downwards out of the separator volume back into the culture volume VK (
The clarifying area A of the separator tube corresponds to the circular cross section thereof and is calculated according to Equation III.
The particle system PAN-X (polyacrylonitrile, spherical particles from Dralon GmbH) was used as model particle for study of separation performance of the reactor bottle according to the invention with integrated cell separator in cell culture.
For examining the identity of the physical properties, the particle size distribution and the particle falling velocity were compared, since they are the determining factors of sedimentation.
The particle size distribution was determined via laser diffraction method (Mastersizer 2000, measured according to the operating instructions). The results were plotted as particle volume in % based on the total volume, as a function against particle size in μm. The modal value XMod, states what particle size is most frequently represented in volume terms and was approximately 21 μm.
The falling velocity was analysed using a sedimentation balance. For this purpose a suspension was produced which has the same concentration as that used in the experiment. The PAN-X was suspended in desalinated water (=completely ion-free (CIF) water) and had a mass concentration of approximately 3 g/l or a volume concentration of 0.88 by volume. A temperature of 20° C. was selected for the analysis. Falling velocities vs, of 0.129 m/h to 0.137 m/h measured under the experimental conditions were determined in various PAN-X batches and correspond to the conditions of non-hindered sedimentation.
CHO cells have, for example, a sedimentation rate of 0.0145 m/h [Searles J A, Todd P, Kompala D S, Biotechnol Prog (1994) 10: 198-206] and are thus relatively slowly 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 production of the model suspension, 3 g of PAN-X were weighed out and suspended in 1000 ml of CIF water, using a magnetic stirrer. For sampling, the harvest stream was collected in a measuring cylinder, while the volume taken off was replaced by CIF water up to a fill level H/D=1 by means of a second peristaltic pump.
Where not stated otherwise, all experiments were carried out with the following standard parameters:
Gravimetric determination of the particle concentration: the particle concentration in the harvest stream was determined gravimetrically by filtering off (filtration by suction) a defined volume of harvest stream and subsequently drying and weighing the filter by means of a drying balance.
The effect of acceleration or power input on the degree of retention at various separator lengths 1 (=370) was determined. For this purpose, experiments with a static separator and corotating separator were compared (for results see
Comparison of performance in the retention of the PAN model particles at various power inputs P/V of up to P/V=50 W/m3 and clarification area loading v=0.1 m/h showed that the degree of retention R decreased with increasing power input into the bioreactor with a corotating installation of the separator tube, with the degree of retention being considerably beneficially affected by increased length L of the separator.
In further experiments, the effect of various harvest stream collectors was studied for a separator length 1=108 mm and a power input of PN=11.12 W/m3 (a=1000°/s2) and v=0.1 m/h. The harvest stream collector with abrupt cross sectional constriction according to
Effect of the Distance from the Bottom:
For investigation of the distance from the bottom, a separator tube having a simple harvest stream collector according to
The clarification area loading v of the separator tube corresponds to the velocity of the vertically ascending medium and, according to Equation IV, has a direct effect on the particle retention. The effect of the clarification area loading was studied on the separator tube according to
A comparison of the performance of various separation systems is shown in
The studies which led to this invention were promoted according to the financial aid agreement “Bio.NRW: ProCell—Innovative platform technologies for integrated process development with cell cultures” in the context of the European Fund for Regional Development (EFRD).
| Number | Date | Country | Kind |
|---|---|---|---|
| 12006536.2 | Sep 2012 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/069024 | 9/13/2013 | WO | 00 |
