EP2342315A2 - Verfahren zur reduzierung von ablagerungen bei der kultivierung von organismen - Google Patents
Verfahren zur reduzierung von ablagerungen bei der kultivierung von organismenInfo
- Publication number
- EP2342315A2 EP2342315A2 EP09778578A EP09778578A EP2342315A2 EP 2342315 A2 EP2342315 A2 EP 2342315A2 EP 09778578 A EP09778578 A EP 09778578A EP 09778578 A EP09778578 A EP 09778578A EP 2342315 A2 EP2342315 A2 EP 2342315A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- membrane
- movement
- membrane surface
- cell
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
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- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/24—Gas permeable parts
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
- B01F23/20—Mixing gases with liquids
- B01F23/23—Mixing gases with liquids by introducing gases into liquid media, e.g. for producing aerated liquids
- B01F23/231—Mixing gases with liquids by introducing gases into liquid media, e.g. for producing aerated liquids by bubbling
- B01F23/23105—Arrangement or manipulation of the gas bubbling devices
- B01F23/2312—Diffusers
- B01F23/23124—Diffusers consisting of flexible porous or perforated material, e.g. fabric
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
- B01F23/20—Mixing gases with liquids
- B01F23/23—Mixing gases with liquids by introducing gases into liquid media, e.g. for producing aerated liquids
- B01F23/231—Mixing gases with liquids by introducing gases into liquid media, e.g. for producing aerated liquids by bubbling
- B01F23/23105—Arrangement or manipulation of the gas bubbling devices
- B01F23/2312—Diffusers
- B01F23/23126—Diffusers characterised by the shape of the diffuser element
- B01F23/231265—Diffusers characterised by the shape of the diffuser element being tubes, tubular elements, cylindrical elements or set of tubes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
- B01F31/44—Mixers with shaking, oscillating, or vibrating mechanisms with stirrers performing an oscillatory, vibratory or shaking movement
- B01F31/445—Mixers with shaking, oscillating, or vibrating mechanisms with stirrers performing an oscillatory, vibratory or shaking movement performing an oscillatory movement about an axis
-
- 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
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
- C12M27/04—Stirrer or mobile mixing elements with introduction of gas through the stirrer or mixing element
-
- 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
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
-
- 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
- C12M39/00—Means for cleaning the apparatus or avoiding unwanted deposits of microorganisms
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
- B01F23/20—Mixing gases with liquids
- B01F23/23—Mixing gases with liquids by introducing gases into liquid media, e.g. for producing aerated liquids
- B01F23/231—Mixing gases with liquids by introducing gases into liquid media, e.g. for producing aerated liquids by bubbling
- B01F23/23105—Arrangement or manipulation of the gas bubbling devices
- B01F23/2312—Diffusers
- B01F23/23124—Diffusers consisting of flexible porous or perforated material, e.g. fabric
- B01F23/231244—Dissolving, hollow fiber membranes
Definitions
- the invention relates to a method for reducing deposits in the cultivation of cells or organisms, in particular of cell cultures, which tend to agglomerate or adhere to the bioreactor and its elements, or in which cells, Zelldebris or substances easily agglomerate or adhere.
- Fermentation also accumulates by-products, e.g. the lysis products died
- bioreactors are used. With continuous bioreactors, higher cell densities and associated higher productivity can be achieved compared to batch culture.
- Some cell lines have the property of preferentially forming agglomerates and / or of adhering to the inner regions of a culture vessel / bioreactor or causing / promoting an attachment of cell debris or substances (eg proteins) to the inner regions of the culture vessel (see, for example, EP0242984B1). , This is generally disadvantageous because the functionality of elements in the bioreactor, such as membranes for gas transfer or probes, sometimes considerably limited or even repealed.
- a double-walled fermenter is provided in EP0242984B1 with a spiral stirrer whose stirring blades are designed to just before an inner (semipermeable) wall of the fermenter.
- the movement of the stirring blades in the vicinity of the inner wall causes turbulence, which should prevent the deposition of cells / cell residues / cell products on the inner wall and thus fouling.
- a disadvantage of the fermenter described is that cell cultures can be damaged by the turbulence.
- agitators, in particular the stirring blades described in EP0242984B1 exert high shear forces which can damage cell membranes, in particular of cell-wall-less cells.
- EP1935973A1 describes a culture device for aquatic organisms, which manages without stirrer.
- Gas oxygen to supply the organisms
- a single nozzle is arranged centrally in the outlet and a separating disk is introduced in the vessel so that a clearly directed flow consists of upward and downward movement of the nutrient liquid around the separating disk as a result of the inflow of gases results.
- a clearly directed flow consists of upward and downward movement of the nutrient liquid around the separating disk as a result of the inflow of gases results.
- the bubble-free fumigation solves the problem by the gas exchange takes place over a submerged membrane surface.
- the fumigation is carried out with closed or open-pore membranes. These are e.g. arranged in the liquid moved by a stirrer.
- membranes can be wound up as tubes on cylindrical basket stators (H. -J. Henzler, J. Kauling: "Oxygenation of cell cultures” Bioprocess Engineering 9 (1993) pp.
- Silicone is the preferred tubing for porous polymers because of its high gas permeability, high thermal stability and the hose properties distributed homogeneously over the length of the tubing segments of up to approx
- dead spaces between the hoses and between the stator and the hoses, in which deposits can easily form are problematic.
- the progressive deposition of substances on the silicone hoses themselves leads to an increasingly poorer gas transfer, eg for the supply of cell n with oxygen or when removing carbon dioxide. In general, the silicone tube is discarded after a single use.
- a disadvantage of the described membrane gassing is, in addition, the comparatively low mass transport coefficient (H.-J. Henzler, J. Kauling: "Oxygenation of cell cultures” Bioprocess Engineering 9 (1993) pp. 61-75) .
- H.-J. Henzler, J. Kauling: "Oxygenation of cell cultures” Bioprocess Engineering 9 (1993) pp. 61-75) To achieve high mass transfer rates, it is necessary . install a corresponding amount of membrane area in the bioreactor.
- this is complicated in terms of construction and handling (assembly, sterilization, cleaning, generation of insufficiently mixed areas, etc.) and leads to the increase of dead spaces.
- WO2007098850 (A1) describes a method and a device for the gassing of liquids, in particular of liquids used in biotechnology and especially of cell cultures, in which the gas exchange takes place via one or more submersed membrane surfaces (eg hoses) , wherein the membrane surface performs any rotationally oscillating motion in the liquid.
- the movement can be optimized so that the flow of the membrane surface is optimal. Since the mass transport coefficient depends on the flow of the membrane surface, an improved oxygen supply can be achieved.
- Another advantage of the rotationally oscillating movement of the membrane surface is the fact that a separate stirring or mixing element for generating an inflow of the membrane surface is eliminated.
- WO86 / 07604A1 describes an air lift fermenter. It is proposed to use flocculants in the cultivation of animal cells to separate cell debris from the product stream. The flocculated heavy particles sink into a turbulence-free zone of the Air-Lift fermenter and can be removed here.
- the use of flocculants can reduce deposits but not permanently prevent them.
- the use of flocculants in the use of rotationally oscillating membrane surfaces for the supply of oxygen entails the risk that flocculated particles are captured and transported to dead zones, where they can settle permanently.
- the use of flocculants is not in the Cultivation of all cell lines attached, since flocculants can exert negative influence on the physiology of the cells and excess flocculant may need to be removed from the product.
- the object is to provide a method for reducing deposits in the cultivation of cells or organisms, in particular in the cultivation of cell cultures that are prone to agglomeration or adhesion to the bioreactor and its elements, or in which Cells, cell debris or substances easily agglomerate or adhere.
- the process sought should provide optimal nutrition to organisms with nutrients, in particular with respect to gas transfer, e.g. with oxygen, ensure. It should do without the use of additional shear forces, which leads to the destruction of cells and thus to the reduction of productivity.
- the process sought should be able to do without the use of chemicals (e.g., flocculants) to avoid additional burden on the organisms and more effort in product isolation.
- the process sought should, in particular, reduce deposits which could reduce gas transfer, e.g. the oxygen supply, lead.
- the process sought should be simple to perform and inexpensive.
- the present invention is therefore a method for reducing deposits in the cultivation of cells and organisms, in particular cell cultures, which tend to agglomerate or adhere to the bioreactor and its elements, or in which cells, cell debris or substances easily agglomerate or Adhere, characterized in that a gas exchange into the culture medium immersed membrane surface carries out a discontinuous movement.
- “Movement” is generally understood to mean a process in which a moving body (here the membrane surface) undergoes a change in its arrangement in space, whereby the body can move as a whole (translation) or only parts of the body, eg by bending the body Body (vibration)
- the movement of the body can also consist of a rotation (rotation) and combinations of translation, vibration and rotation are possible.
- a “discontinuous motion” is understood to mean a movement that does not proceed uniformly over a given period of time.
- a discontinuous motion is the movement of a pendulum.
- the pendulum performs over the period of one period, eg, starting with a maximum deflection of the pendulum to the right first accelerate to the left until it reaches a maximum speed in the rest position, then slowly decelerate the pendulum until the pendulum comes to a halt for a moment in the maximum excursion to the left before the pendulum accelerates again, in the rest position the pendulum maximum speed is reached and decelerated again until it has returned to its initial position (maximum deflection to the right), in contrast, a continuous movement is understood to mean a movement that is uniform over a given period of time the rotation of a stirrer at a constant angular velocity about a fixed axis of rotation.
- the membrane surface performs a discontinuous motion with a reversal of motion, i. the membrane surface first carries out any kind of first movement in a first direction before the membrane surface comes to a standstill and then carries out any kind of second movement in another direction, preferably in the direction opposite to the first direction.
- the first movement and the second movement can be completely different from each other.
- the second movement is preferably a mirror, point and / or rotationally symmetrical design for the first movement.
- the membrane surface performs an oscillating movement.
- oscillating is understood to mean a regularly and uniformly repeating process, ie the method according to the invention is preferably characterized in that there is a period of time, hereinafter referred to as the period in which the membrane surface makes any first movement, and subsequent movements copies the first movement are characterized by the same temporal sequence of accelerations and speed as the first movement.
- the movement of a pendulum described above is an example of an oscillating motion.
- the membrane surface carries out a rotationally oscillating movement.
- a rotationally oscillating movement the membrane surface first moves (rotates) in one direction of rotation, wherein the movement can be configured as desired.
- An example is the acceleration of the membrane surface with a certain angular acceleration up to a certain angular velocity with which the membrane surface then moves for a certain time.
- the membrane surface is decelerated to a standstill with a fixed delay. It then follows, if necessary after a defined downtime, then the movement in the other direction of rotation. This movement can be mirror-image of the previously described or otherwise designed.
- a rotationally oscillating motion movement in which the membrane surface is first accelerated in one direction, a time t, which is greater than or equal to zero, rotated at a constant speed in this predetermined direction, then braked (the diaphragm surface but can also continue to rotate with a small angular velocity in the same direction), and then accelerated again in the same direction.
- the movement is carried out so that the membrane surface first rotates in one and after a predetermined time in the opposite direction.
- the movement of the membrane surface is rotationally oscillating with reversal of direction of rotation and with minimal downtime at the points of reversal of rotation.
- a minimal downtime is understood to mean that the reversal of the direction of rotation takes place without a technical / avoidable delay, ie, the diaphragm surface experiences an acceleration in the direction opposite to the previous direction immediately after reaching a point of reversal of the direction of rotation.
- the preferred embodiment is further characterized in that the membrane surface is accelerated from a point of reversal of direction over a definable period of time with constant angular acceleration and then, upon reaching a maximum velocity, the membrane surface is decelerated again with a constant angular delay until the membrane surface is the second point of reversal of direction reached (movement phase 1). Then, a movement phase 2 is mirrored to the movement phase 1.
- the constant angular acceleration and angular delay are equal in magnitude.
- the preferred Embodiment of the inventive method is characterized in that no movement phase occurs at a constant angular velocity.
- the membrane surface is flowed tangentially as a result of the discontinuous movement within the culture medium.
- the tangential flow ensures effective gas exchange between membrane surface and culture medium (oxygen supply, carbon dioxide removal).
- Membrane surface is understood to mean a surface through which a gas, in particular oxygen, in dissolved form or in the form of fine bubbles can be introduced into a liquid and / or a gas can be removed from the liquid Gas bubbles understood that have a low tendency to coalescence in the culture medium used.
- Suitable membrane surfaces are, for example, special sintered bodies of metallic and ceramic materials, filter plates or laser-perforated plates which have pores or holes with a diameter of generally smaller than 15 microns.
- the membrane surfaces are preferably designed as hollow bodies, eg tubes, through which gas can flow. At low gas blanket velocities of less than 0.5 mh -1 , very fine gas bubbles are produced, which have a low tendency to coalesce in the media normally used in cell culture.
- Membrane surfaces are also suitable membrane hoses.
- Membrane hoses are understood to be flexible tubular structures which are permeable to 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.
- silicone tubes can be used, as described by way of example in the following documents: H.-J. Henzler, J. Kauling: "Oxygenation of cell cultures” Bioprocess Engineering 9 (1993) pp 61-75, EP 1948780, WO07 / 051551A1,
- Non-porous silicone tubes are preferably used as membrane surfaces. Preferably, these are in a range of inner diameter ⁇ 1 mm with an outer diameter of ⁇ 1.4 mm to an inner diameter of ⁇ 2 mm with an outer diameter of ⁇ 3 mm.
- the parameters hose diameter and hose total length should be selected so that a sufficient mass transport for the application is guaranteed.
- the mass transfer is among others the ratio of membrane surface to reactor liquid volume determined (volume-specific mass transfer area). Here, " 'in use for animal cell cultures.
- the volume-specific mass transfer surface area values between 0.1 m reached 1 and 150 1 m" are preferably 1 m is 1 to 45 m "' values of 25 m from 1 to 100 m ' 1 and more preferably 5 m "1 to 75 m '1 .
- the membrane surface is attached to a rotatably mounted rotor mounted inside a container, e.g. a bioreactor, can be moved.
- the rotor is designed so that it can carry at least one membrane surface such as hoses, cylinders, modules, etc. inside the bioreactor.
- the rotor is preferably used for carrying out a rotationally oscillating movement.
- the rotatably mounted rotor may e.g. be offset from outside the bioreactor by a drive in a rotational oscillating movement.
- the transmission of the required drive torque from the drive to the rotor inside the reactor can be done either via a magnetic coupling, or the rotor shaft is passed through a rotating seal through the housing of the bioreactor and coupled directly to the drive.
- a magnetic coupling is particularly advantageous from a sterile technical point of view because it clearly separates sterile and non-sterile spaces from one another without rotating seal.
- the volume-specific power input is usually between 0.01 and 100 W per m 3 .
- the parameter design should be such that maximum relative velocities between rotor and culture medium of 1 ms.sup.- 1 result for the cell culture application.
- the transmission In order to absorb the stresses from the transmission and rotor connection, the transmission is usually connected to the rotor via any torsionally rigid coupling which absorbs small shaft misalignment or low shaft misalignment.
- the device for attaching one or more membrane surfaces can advantageously be easily adapted in its design to the particular conditions in cell cultures, e.g. Cell agglomeration, to be adjusted. This can be done for example by the nature and arrangement of the membrane surfaces.
- the rotor preferably has 1 to 64, preferably 2 to 32 and particularly preferably 4 to 16 rotor arms, to which one or more membrane surfaces can be attached.
- two winding arms form a rotor arm.
- the membrane surface preferably the membrane tubes, horizontally or vertically wound at regular or irregular intervals.
- the flow of the membrane hoses generally improves with increasing radial distance from the rotor shaft, depending on the position of the membrane hose.
- the reason for this is the equally increasing peripheral speed.
- One way to meet this requirement is to increase the number of rotor arms around the shaft. Negatively, however, an increase in the number of arms affects both the mixing and the flow to the membrane (creation of less mixed compartments between the arms).
- the handling of the rotor suffers during the winding and unwinding of the hoses and during installation and removal. The attachment of the arms to the shaft designed with larger numbers of arms for reasons of space increasingly difficult.
- the supply of the discontinuously moved membrane surface for the supply and removal of gas preferably takes place from the non-agitated environment, e.g. the reactor lid, with a rotary seal or with the help of flexible hoses.
- Rotary seals are usually undesirable in cell culture technology because they can cause difficulties in cleaning and sterilization.
- the method according to the invention with reversal of movement offers a clear advantage over a method without reversal of the direction of movement: Without reversing the direction of movement, the tubes would twist more and more with increasing rotation and finally tear off.
- a motion reversal movement e.g. in the case of rotationally oscillating membrane surfaces, there is no net torsion of the flexible tubes due to the reciprocation.
- the prerequisite is of course the design of the reciprocating motion such that the membrane surface is at the start of the movement after completion of a period of movement.
- the tension of the membrane surface such as the membrane tubes can be varied.
- the optimum stress results, inter alia, from the parameters pressure of the gas or gas mixture flowing into the space inside the membrane surface, pressure of the gas or gas mixture flowing out of the space inside the membrane surface, and geometry, flow resistance and deformation of the space within the membrane surface (eg Inlet pressure, outlet pressure, inside diameter, number and geometry of the membrane tube curvatures, as well as the deformation of the curves) (HN Qi, CT Goudar, JD Michaels, H.-J.
- the voltage should be selected such that the membrane hoses are fastened on a long-term stable basis, but on the other hand can preferably move in the flow and deflect, for example, a few millimeters.
- the reduction of the hose tension results in the problem of fixing the membrane hoses on the winding arms.
- a large force on the membrane hoses could lead to slipping of the membrane hoses from the winding arms with less hose tension.
- To address this problem e.g. provide the surface of the winding arms with an external thread.
- e.g. Webs are provided outside of the winding arms, which prevent slipping of the hoses outside of the arms. It is important to ensure that the wound membrane hoses are not damaged by any burrs of the thread.
- the external thread on the winding arms of a star holder offers the possibility to vary the hose winding. When rewinding the tubes, e.g. Only every second or third thread recess can be used. As a result, the setting of a defined distance between the individual diaphragm hoses is possible.
- membrane surfaces in the form of hoses which are attached to the arms of a rotor and configured to carry out a discontinuous movement, can be found in the application WO2007098850 (A1).
- the membrane surface which carries out a discontinuous movement can be completely or partially immersed in the culture medium. It is also conceivable to vary the immersion depth during the discontinuous movement.
- the inventive method can be used in many ways, e.g. in the cultivation of organisms, human, animal or plant cells, in the processing of
- Waste water or any other process in which deposits may form.
- Cell cultures are eg BHK cells (Baby Hamster Kidney) for the production of Coagulation factors or CHO cells (Chinese Hamster Ovary) for obtaining therapeutic antibodies.
- the membrane surface provides for the necessary gas exchange and thus for the necessary supply of the organisms with e.g. Oxygen, as well as the necessary removal of gaseous metabolites (especially carbon dioxide) of the organisms.
- the oscillating movement improves the mass transfer significantly compared to a statically arranged membrane surface, which is impinged by an additional agitator. An additional agitator is not necessary.
- the oscillatory motion has, surprisingly, a reducing effect on the formation of deposits and agglomerates, both on deposits and agglomerates settling on the membrane surface, and on deposits and agglomerates settling on other elements / surfaces within the bioreactor.
- probes pH probe, thermometer, electrode for determining the oxygen content and similar probes
- One or more probes are preferably connected to the membrane surface, possibly via a common holder, so that the membrane surface and probe (s) are caused to make a joint / coupled movement. In this way, deposits on the probes are more effectively avoided.
- Example 1 Device for carrying out the method according to the invention
- FIG. 1 schematically shows an example of a device for carrying out the method according to the invention.
- the membrane surface is formed by membrane tubes (1) which are arranged on a rotor shaft (2) vertically transversely to the direction of rotation (3). Through the membrane hoses oxygen-containing gas can be pumped to supply organisms.
- the device is preferably operated within a bioreactor (4).
- the membrane surface is fully immersed in the culture medium, i. the liquid surface (5) is in operation above the membrane surface.
- the device can perform a rotary movement about the rotor shaft (2). Preferably, it performs a rotationally oscillating motion. This movement leads to an improved supply of the organisms in the bioreactor and to a significantly reduced tendency to form deposits and agglomerates (compared to a static membrane surface, which is impinged by a stirrer).
- FIG. 2 shows the photograph of a device for receiving membrane tubes.
- the device comprises at the top two concentric distribution rings for the supply and removal of gas. Most of the external gas supply is used so that the oxygen-rich gas first enters the tube sections, which are farthest from the rotor shaft and are best flowed to it.
- the distributor rings each have 16 connecting pieces, which allow the supply of the membrane hose segments on the up to 16 rotor arms.
- the photo shows the rotor with only 8 mounted rotor arms, whereby the 8 remaining possible rotor arms would each be mounted between the present ones.
- a membrane hose segment of 57 m in length is wound up in this example. Now, if a rotation of the rotor, the membrane tubes are moved by the fluid in the reactor and thereby flowed tangentially.
- the apparatus shown in FIG. 2 for carrying out the method according to the invention is used for gassing a cell culture bioreactor with a liquid volume of up to about 200 l, the internal reactor diameter being 510 mm and the height to diameter ratio being 2: 1.
- the centric rotor shaft has a diameter of 20 mm and the rotor an outer diameter of 409 mm.
- the rotor arms have in the recess in which the Membrane tube is guided, a radius of 7.7 mm.
- parallel recesses are produced with a distance of 3.65 mm.
- a rotor drive for example, a stepper motor with a maximum speed of 2500 min '1 , a standstill torque of 5.8 Nm and a gear ratio of 1: 12 can be used.
- Example 2 Use of the method according to the invention for cultivating a sticky human hybrid cell line HKB-11
- the process according to the invention has been described by way of example in the cultivation of the human cell line HKB-I 1 for the production of blood coagulation factor VHI (Mei, Baisong et al., "Expression of Human Coagulation Factor VJII in a Human Hybrid Cell Line", HKBI 1, Molecular Biotechnology. 34 (2): 165-178, October 2006).
- This cell line has a very high tendency to aggregate formation.
- the same cell line was cultured in a reference method (not according to the invention) in order to be able to compare the methods.
- the process according to the invention was carried out in a 15 L bioreactor from Applikon.
- the bioreactor was equipped with a rotor having a membrane surface in the form of silicone tubing (SILASTIC RX 50 Medical Grade Tubing Special, 0.078 in. (1.98 mm) ID x 0.125 in. (3.18 mm) OD (500 ft roll, Dow Corning). ) was attached.
- the membrane hoses were mounted on the 8 arms of the rotor, which were mounted in a star shape around a rotor shaft.
- the total length of membrane tubing was 58.7 m (48.8 m 2 membrane surface area per m 3 reactor volume at 12 L filling volume), with the two innermost rows of rotor arms not wound.
- the complete winding would have corresponded to a total length of membrane tube of 65 m (54.1 m 2 membrane surface area per m 3 reactor volume at 12 L filling volume).
- the rotor could be put into a discontinuous motion by a servomotor (Model No. 23S21, Jenaer Antriebstechnik, Jena, Germany) with a standstill torque of 0.9 Nm, to which a planetary gear with a reduction of 1:12 was flanged.
- Information on the human HKB cell line used can be found in the following literature: Mei, Baisong et al., "Expression of Human Coagulation Factor VIII in a Human Hybrid Cell Line", HKBI, Molecular Biotechnology. 34 (2): 165-178, October 2006.
- the cells were supplied with oxygen and freed of carbon dioxide.
- the gas flow rate was 1 standard liter per hour.
- the gas flow through the membrane hoses of the 8 rotor arms is brought together again at the end of the membrane hoses and led through a flexible hose to the bioreactor lid. There, the backpressure at the gas outlet is varied between 5 and 15 psig. This offers the possibility to specifically influence the gas transfer properties.
- an air stream of 1 standard liter per hour was continuously passed through the supply air and exhaust air connection during the cultivation. Information on the structure of the plant for continuous cell culture operation can be found in WO2003 / 020919A1.
- the membrane surface within the culture medium has been set in a rotationally oscillating motion.
- the sequence of movement was as follows: starting from one of the points of reversal of the direction of rotation, the membrane surface became a constant Angular acceleration of 11 rads "2 accelerated for a duration of 400 ms and then delayed for the same period with one of the same angular acceleration, so that it again came to a standstill after 800 ms
- the swept angle is 90 ° to about 56 W m "3 .
- the maximum speed of the rotor ends is approx. 0.44 ms "1 .
- the erf ⁇ ndungshiele method is hereinafter referred to as DMA method (Dynamic Membrane Aeration) and the corresponding device for carrying out the erf ⁇ ndungshielen method as a DMA reactor.
- DMA method Dynamic Membrane Aeration
- the reference method was also carried out in a structurally identical 15 L bioreactor (reference reactor) from Applikon. This was with a static membrane surface and a
- the static membrane area comprised 49.6 m hose length
- Silicone hose (for DMA and reference system the same silicone hose make was used).
- the flow rate through the membrane tubes was 0.5 standard liters per hour.
- the internally designed anchor stirrer was used for the flow of the membrane surface to the
- the anchor stirrer was operated at a constant speed of 150 rpm (corresponding to approx. 165 W m ⁇ 3 ). This high stirrer speed or high power input, which otherwise for reasons of
- cell inoculum For inoculating the reference system cell inoculum was used, which was bred in advance in shake flasks in an adequate amount. Inoculation of the 15 L DMA reactor was carried out with cells from the 15 L reference system, whereby the comparability of both systems with respect to common cell source and up to the small time difference is also given in terms of equal cell age. Examples of the animf cell densities are shown in FIGS. 3 and 4.
- Figure 3 shows the temporal evolution of the density of living cells (a) in the DMA method and (b) in the reference method in a first cell culture.
- the density cd of living cells in the unit [10 6 cells mL "1 ] versus time / unit [days] is plotted in each case
- the cell density was determined using a CEDEX system (Innovatis GmbH, Bielefeld, Germany)
- a pipetting was carried out beforehand, whereby the cell agglomerates are largely dissolved by the shearing forces in the pipette
- the bioreactor of the DMA method was washed only with medium (medium formulation subject to secrecy). Deposits on the sensors and the membrane surface remained. Subsequently, a second cell cultivation was carried out with freshly grown cells. The procedure was used to simulate long-term cultivation.
- Figure 4 shows the time evolution of the density of living cells (a) in the DMA method and (b) in the reference method in the second cell culture.
- the density cd of living cells in the unit [10 6 cells mL '1 ] is plotted against the time t in the unit [days].
- the DMA method thus showed a higher cell density and thus a higher production rate than the reference method.
- the cause was demonstrably the reduced tendency to form deposits in the DMA process compared to the reference method.
- Cultivation time on average higher than the reference method. - In the DMA process, less deposits were observed on both the membrane tubing and the stationary parts of the bioreactor and on the probes.
- the DMA method showed no adverse effects on cell biology (apoptosis and cell cycle).
- Fig. 1 Schematic representation of a rotationally oscillating movement for loading
- Fig. 2 Photograph of a device for receiving a membrane surface: membrane hoses are wound on the star-shaped arms of a rotor.
- Fig. 3 Graph showing the temporal evolution of the density of living cells
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Abstract
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DE102008049120A DE102008049120A1 (de) | 2008-09-26 | 2008-09-26 | Verfahren zur Reduzierung von Ablagerungen bei der Kultivierung von Organismen |
PCT/EP2009/006722 WO2010034428A2 (de) | 2008-09-26 | 2009-09-17 | Verfahren zur reduzierung von ablagerungen bei der kultivierung von organismen |
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EP2342315A2 true EP2342315A2 (de) | 2011-07-13 |
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EP09778578A Withdrawn EP2342315A2 (de) | 2008-09-26 | 2009-09-17 | Verfahren zur reduzierung von ablagerungen bei der kultivierung von organismen |
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US (1) | US20110165677A1 (de) |
EP (1) | EP2342315A2 (de) |
CN (1) | CN102272285A (de) |
DE (1) | DE102008049120A1 (de) |
WO (1) | WO2010034428A2 (de) |
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US20120282677A1 (en) | 2011-05-03 | 2012-11-08 | Bayer Intellectual Property Gmbh | Photobioreactor comprising rotationally oscillating light sources |
DK3626737T3 (da) | 2011-05-13 | 2024-02-05 | Octapharma Ag | Fremgangsmåde til at forøge produktiviteten af eukaryote celler i produktionen af rekombinant fviii |
US9884295B2 (en) | 2012-10-08 | 2018-02-06 | Doosan Heavy Industries & Construction Co., Ltd. | Membrane bioreactor system using reciprocating membrane |
CN104403937B (zh) * | 2014-12-17 | 2016-08-17 | 大连大学 | 旋转内循环气升式膜生物反应器及其工艺系统 |
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DE3574397D1 (en) | 1984-08-03 | 1989-12-28 | Biotechnolog Forschung Gmbh | Process and apparatus for the bubble-free aeration of liquids, in particular culture media for the propagation of tissue culture |
GB8515636D0 (en) | 1985-06-20 | 1985-07-24 | Celltech Ltd | Fermenter |
DE3535183A1 (de) * | 1985-10-02 | 1987-04-16 | Biotechnolog Forschung Gmbh | Vorrichtung und verfahren zur blasenfreien begasung von fluessigkeiten, insbesondere von kulturmedien zur vermehrung von gewebekulturen |
GB8607046D0 (en) | 1986-03-21 | 1986-04-30 | Fisons Plc | Culture device |
US4960706A (en) | 1989-03-27 | 1990-10-02 | Baxter International, Inc. | Static oxygenator for suspension culture of animal cells |
DE9215153U1 (de) * | 1992-11-06 | 1993-01-07 | B. Braun Biotech International Gmbh, 3508 Melsungen, De | |
PT1451290E (pt) | 2001-08-31 | 2011-03-09 | Bayer Schering Pharma Ag | Uma unidade e um processo para levar a cabo a fermentação com elevada densidade celular |
CN1155690C (zh) * | 2002-07-04 | 2004-06-30 | 中国人民解放军第三军医大学第一附属医院 | 循环和生理应力模拟工程化组织三维培养装置 |
CN1249217C (zh) * | 2003-01-27 | 2006-04-05 | 英科新创(厦门)科技有限公司 | 细胞培养方法和装置 |
US7122121B1 (en) * | 2004-05-28 | 2006-10-17 | Jiang Ji | Advanced submerged membrane modules, systems and processes |
DE102005053334A1 (de) | 2005-11-07 | 2007-05-24 | Bayer Technology Services Gmbh | Module zur Membranbegasung |
DE102006008687A1 (de) | 2006-02-24 | 2007-08-30 | Bayer Technology Services Gmbh | Verfahren und Vorrichtung zur Be- und Entgasung von Flüssigkeiten |
DE102006062634A1 (de) | 2006-12-23 | 2008-06-26 | Stiftung Alfred-Wegener-Institut für Polar- und Meeresforschung Stiftung des öffentlichen Rechts | Kulturvorrichtung für aquatische Organismen |
-
2008
- 2008-09-26 DE DE102008049120A patent/DE102008049120A1/de not_active Withdrawn
-
2009
- 2009-09-17 US US13/062,556 patent/US20110165677A1/en not_active Abandoned
- 2009-09-17 WO PCT/EP2009/006722 patent/WO2010034428A2/de active Application Filing
- 2009-09-17 CN CN2009801377765A patent/CN102272285A/zh active Pending
- 2009-09-17 EP EP09778578A patent/EP2342315A2/de not_active Withdrawn
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WO2010034428A2 (de) | 2010-04-01 |
CN102272285A (zh) | 2011-12-07 |
DE102008049120A1 (de) | 2010-04-01 |
WO2010034428A3 (de) | 2010-07-08 |
US20110165677A1 (en) | 2011-07-07 |
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