Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
  • C12M29/08—Air lift

Definitions

• the invention relates to a bioreactor, the use of the bioreactor for the cultivation of microorganisms or cell cultures and a method for the cultivation of microorganisms or cell cultures.
• the air-lift bioreactor in particular has become established.
• gas such as air
• an upwardly directed portion of the bioreactor also known in the art as a riser.
• a fine bubble gassing takes place.
• the riser communicates at its upper and lower ends with the upper and lower ends of another upwardly directed portion of the bioreactor, known in the art as a downcomer.
• One widely used variant of the substantially cylindrical air lift bioreactor includes a centrally located cylindrical guide tube which directs the air lift bioreactor into a riser within the draft tube and a downcomer in the annulus between the draft tube and the container outer wall of the Air-Lift bioreactor.
• the buoyancy part in the annular space between the guide tube and the container outer wall and the driven part can be located within the guide tube.
• the supply of, for example, oxygen-enriched gas at the bottom of the riser reduces the average density of the suspension culture in the riser, resulting in an upward liquid flow in the riser, which subsequently replaces the liquid content of the downcomer which in turn descends to the bottom of the riser Risers are flowing.
• this streamlined design means that the Air-Lift bioreactors can achieve construction heights of several meters at typical working volumes of several hundred liters up to several cubic meters. For example, 12 m 3 of work volume corresponds to a height of 14.4 m with a H / D ratio of 14.
• Such air-lift bioreactors must therefore be placed in rooms with large ceiling heights or with openings over several floors. This requires a complex steel framework construction.
• the air-lift bioreactors must be steam sterilized in situ and can no longer be steam sterilized in an autoclave, along with the peripherals necessary for cell culture.
• conventional bioreactors with common H / D ratios of 2 can be transported in autoclaves and steam-sterilized there.
• the subject of the present invention is thus an air-lift bioreactor with a ratio H / D from height H to diameter D, which is smaller than 6.
• Air-lift bioreactors are reactors which have a riser, a downcomer and a gassing unit. Risers and downcomers are preferably formed via a cylindrical vessel into which a cylindrical tube is arranged (see, for example, FIG. 1). In a preferred embodiment, the cross-sectional areas of the riser and the downcomer differ by a maximum of 10%, more preferably they are the same size (see, eg, FIG. 2).
• the cylindrical vessel and the cylindrical tube preferably have the same cross-sectional geometry. They are preferably elliptical or round.
• the gassing unit is arranged either within the cylindrical guide tube or between the outer wall of the guide tube and the inner wall of the vessel.
• the riser is within the draft tube and the downcomer between the outer wall of the draft tube and the inner wall of the vessel; in the second case, the downcomer lies inside the guide tube and the riser between the outer wall of the guide tube and the inner wall of the vessel.
• gas e.g. Oxygen
• Carbon dioxide provides the gassing unit for a circulating flow between riser and downcomer.
• a gassing unit is used which produces bubbles with a diameter of less than 2 mm.
• the gassing unit is designed as a microbubble aerator.
• Mikroblasbegaser be understood bodies that can bring gas, especially oxygen in the form of fine bubbles in a liquid.
• fine gas bubbles gas bubbles which have a low tendency to coalesce in the culture medium used, for example special sintered bodies of metallic or ceramic materials, filter plates or laser-perforated plates, the pores or holes having a diameter of generally suitable
• the gassing unit is preferably designed as a hollow body, for example as a tube, through which gas can flow.With small gas empty tube velocities of less than 0.5 mh -1 , very fine gas bubbles are generated which in the media normally used in cell culture have a low tendency to coalescence.
• Membrane tubes are also suitable as microbubble aerators.
• Membrane hoses are understood to be flexible tubular structures which are permeable to gases such as oxygen and carbon dioxide.
• gases such as oxygen and carbon dioxide.
• membrane hollow filaments of microporous polypropylene may be mentioned, as exemplified in Chem. Ing. Tech. 62 (1990), No. 5, pp. 393-395 by H. Büntemeyer et al. to be discribed.
• the gassing unit is preferably arranged near the lower edge of the guide tube.
• the gassing unit is preferably designed annular or spiral, so that it reduces the flow cross-section only insignificantly. Plate-shaped gassing units lead to increased flow resistance.
• the resulting pressure loss must be compensated by a higher gas flow rate to maintain the circulation flow between the riser and downcomer.
• a higher gas flow rate results in an increased shear rate, which can be destructive to sensitive cells and thus should be avoided.
• the diameter of the preferably annular or spiral gassing unit should be designed so suitably for the cross section of the riser, that the cross section is applied as evenly as possible with gas bubbles. Accordingly, it would be necessary to avoid a gassing unit which is arranged with a small annular diameter in the middle of the riser, with the remaining (outer) riser cross-section being insufficiently supplied with the resulting gas bubbles. It is also conceivable to form the gassing unit meandering. Other forms are conceivable.
• all corners and edges within the bioreactor according to the invention are rounded, in particular the edges of the guide tube, in order to avoid eddies, which likewise lead to a loss of pressure and to increased shear.
• the bioreactor according to the invention preferably has means for guiding the flow, which favor a loop flow between the riser and the downcomer and keep pressure losses and shears low.
• the bottom of the bioreactor has an elevation which deflects the liquid flowing to the reactor bottom upwards.
• the flow cross sections in the lower and upper region of the bioreactor, in which the direction of flow reversal takes place and the medium passes from the riser into the downcomer or into the riser from the downcomer are of equal size and correspond in size to the flow cross sections of the riser and downcomer ,
• the materials commonly used in biotechnology for the cultivation of microorganisms and cells such. VA steel or glass.
• the guide tube is held within the vessel via supports. These may be attached to the bottom of the vessel, to the lid of the vessel or to the inner wall of the vessel. In a preferred embodiment, the guide tube hangs on supports which are attached to the lid of the vessel. Over the lid, the bioreactor is usually supplied with media, nutrients, additives (such as antifoam and buffer) and gases.
• the bioreactor according to the invention is suitable for the cultivation of microorganisms and cells (plant, animal, human) of all kinds.
• the use of the bioreactor according to the invention for Cultivation of microorganisms or plant, animal or human cells is the subject of the present invention.
• the present invention furthermore relates to a process for the cultivation of microorganisms or cell cultures.
• the method is characterized in that in a bioreactor with a ratio H / D from height H to diameter D is less than 6, preferably between 2 and 6, a loop flow (circulating flow) between an inner guide tube and the area between the outer wall of the guide tube and the inner wall of the bioreactor is produced by means of a gassing unit.
• the gassing unit is preferably a unit which produces bubbles with a diameter of less than 2 mm, more preferably the unit is a microbubble aerator.
• the gas volume flow is chosen so that the loop flow is maintained and the cells are adequately filled with gas, e.g. Oxygen, supplied and unwanted gas, e.g. Carbon dioxide, are released, the shear rate is kept to a minimum, to avoid destruction of sensitive cells. Furthermore, the gas volume flow is chosen so that a suspension of the cells is ensured, so sedimentation is prevented. Other (secondary) criteria are a sufficiently short mixing time and the lowest possible foaming.
• the gas bubbles can lead to the formation of foam. Foaming is to be avoided, however, as cells tend to bloom with the foam. In the foam layer, they do not find adequate cultivation conditions. The use of anti-foaming agents can be known to remedy this situation.
• the inventive method is operated so that the lateral surfaces above and below the guide tube differ by a maximum of 10%; they are preferably the same size. Furthermore, in a preferred embodiment, the size of the lateral surface between the guide tube and the liquid surface and / or the lateral surface between the guide tube and the bottom of the bioreactor differs by a maximum of 10% of the size of the cross-sectional area of the riser and / or downcomer. In a particularly preferred embodiment of the method according to the invention, the flow cross-section for the circulating flow in all areas of the reactor is almost the same or the same size in order to reduce pressure losses.
• the lateral surface between the guide tube and the bottom of the bioreactor is smaller than the cross-sectional areas of riser and downcomer.
• an increased flow rate is generated in the bottom area, which effectively prevents sedimentation of cells or microorganisms.
• the lateral surface between the guide tube and bottom of the bioreactor by at least 5% and by a maximum of 50% smaller, more preferably by at least 5% smaller and by a maximum of 30% smaller.
• microorganisms as well as animal, plant and human cells can be used in the process according to the invention.
• Air-lift bioreactors with low height-to-diameter ratio have greater homogeneity in dissolved oxygen, dissolved carbon dioxide and pH, not least due to a less pronounced hydrostatic pressure profile. (Thus, tall, slim bioreactors are susceptible to local (altitude-dependent) carbon dioxide partial pressures, each affecting pH.) The likelihood of undersupply of cells in the downcomer of the Air-Lift bioreactor decreases with dissolved oxygen. The generally better axial mixing also leads to better homogeneity in substrate concentrations. Air-lift bioreactors are often fumigated with macrobubbles. The gassing with microbubbles leads to high volume-specific phase interfaces and thus allows a significant reduction in the volume of gas required to drive the liquid flow. This is accompanied by a significant reduction in the shear stress of cells compared to coarse-bubble fumigation.
• FIG 1 shows schematically a bioreactor according to the invention (a) in cross section from the side and (b) in cross section from above.
• the bioreactor according to the invention comprises a cylindrical vessel (1), in which preferably centrally centered a likewise cylindrical guide tube (2) is introduced.
• an annular gassing unit (3) is introduced in the guide tube near the lower edge of the guide tube.
• the ratio H / D of height H to diameter D is between 1 and 6, preferably between 2 and 6.
• Figure 2 shows schematically a preferred embodiment of the bioreactor according to the invention in cross-section from above, in which the cross-sectional area A within the guide tube and the surface B between the outside of the guide tube (2) and the inner wall of the vessel (1) are the same size, ie riser and Downcomers preferably have the same size of the flow cross-section.
• FIG 3 shows schematically a preferred embodiment of the bioreactor according to the invention in cross-section from the side.
• the vessel (1) preferably has deflecting devices (9) on the reactor bottom.
• the guide tube (2) is attached to supports (5) on the lid (4) of the bioreactor. It has rounded edges to avoid pressure losses due to whirling and shearing.
• the preferred annular gassing unit is mounted in the present example of Figure 3 within the guide tube near the lower edge of the guide tube, so that the riser is located within the guide tube and the downcomer between the guide tube and vessel.
• gas supply passages (6) and supply of medium and / or buffers and / or additives are attached to the lid of the reactor (7).
• the bioreactor has means for heating and / or cooling and sensors for measuring e.g. Temperature, pH, dissolved oxygen concentration, dissolved carbon dioxide concentration, etc., which are not shown in the present case.
• the liquid level (8) in the reactor is so high that the flow cross sections in the deflection areas and in the riser and downcomer are the same size.
• FIG. 4 shows a photograph of a preferred embodiment of the bioreactor according to the invention.
• the bioreactor shown comprises a glass container with a double jacket, a lid, a bottom valve and a guide tube, which can be attached to the lid.
• FIG. 5 schematically shows the principle of surface equivalence: the cross-sectional areas of the riser and downcomer and the lateral surfaces above and below the guide tube are preferably the same size.
• FIG. 6 shows by way of example a gassing unit for the bioreactor according to the invention in the form of an annular micro-sparger.
• FIG. 7 shows a graphic representation of the results of the fermentation of BHK-21 cells from Example 2 in the bioreactor from Example 1.
• FIG. 8 serves to explain the information in Table 1.
• Deflectors A Cross-sectional area of the riser / downcomer
• FIG. 4 shows a preferred embodiment of a bioreactor according to the invention.
• the bioreactor shown comprises a glass container with a double jacket, a lid, a bottom valve and a guide tube, which can be attached to the lid.
• the cover holes are suitable for standard accessories. All components important for the subsequent fermentation can be applied in this way.
• the pipe which serves as a supply air line for the gassing unit ((micro) sparger), can also be attached to the lid in a height-adjustable manner.
• the sparger is installed in the middle of the lower part of the guide tube. As a result, inside the rise, outside the descent of the liquid flow takes place.
• the guide tube consists of a hollow double-walled cylinder. This not only serves the flow guidance; the guide tube is designed so that the installation of an internal cell separator is possible. This reduces the working volume from 15 L to 10 L.
• For tempering the bioreactor in the later fermentation operation serves a double jacket. Draining the liquid is made possible by a bottom valve.
• Table 1 The essential data are shown in Table 1.
• Table 1 Design of a preferred embodiment of a bioreactor according to the invention. There is areal equivalency between the cross-sectional areas of riser and downcomer as well as between the lateral surfaces above and below the riser. The difference between maximum and actual working volume is created by the guide tube, whose dimensions serve as placeholders for a possible internal cell separator.
• the drawing shows the glass container with guide tube.
• the bioreactor was designed with a H / D ratio of 2.
• the types of airlift fermenters are slimmer - ie with higher H / D ratios.
• H / D 2.
• Table 1 also shows the area equivalence between the cross-sectional areas of riser and downcomer and between the shell surfaces above and below of the riser. This results in an equal flow rate in all parts of the reactor. Pressure losses and the acceleration or deceleration of the liquid can be avoided.
• the principle of area equivalence is shown schematically in FIG.
• Example 2 Fermentation for biological characterization
• BHK cells (baby hamster kidney cells) are immortalized cells derived from the kidneys of a day old golden hamster. They are fibroblasts that were originally grown adherently. However, a large number of different BHK cell lines exist, which were mostly adapted to suspension culture.
• the starting cell density was 4 * 10 5 cells mL "1 with a vitality of 92% and the sparger fumigation rate of 15 L / h was initially maintained but increased to 17.5 L / h after one day.
• the cell density immediately increased slightly. Within a day, the cell density was doubled.
• foam was not a significant problem.
• the foam reached a maximum height of about 30 mm with occasional Antifoam C addition.
• the concentration of antifoam at the end of the fermentation was about 40 ppm, which is an acceptable amount.
• concentrations up to 500 ppm have been investigated for this cell line and considered to be uncritical. Due to the increased fumigation rate arise no foam problems. The fumigation rate should be chosen as low as possible for this reason alone. Since the results indicate that the foaming does not exceed a tolerable level, can be fumigated with 17.5 L / h.

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• 2008
  • 2008-12-20 DE DE102008064279A patent/DE102008064279A1/de not_active Withdrawn
• 2009
  • 2009-12-08 CN CN2009801516611A patent/CN102325870A/zh active Pending
  • 2009-12-08 EP EP09764744A patent/EP2379694A1/de not_active Withdrawn
  • 2009-12-08 WO PCT/EP2009/008733 patent/WO2010069492A1/de active Application Filing
  • 2009-12-08 US US13/140,464 patent/US20110256624A1/en not_active Abandoned

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