EP2342315A2

EP2342315A2 - Method for reducing deposits during the cultivation of organisms

Info

Publication number: EP2342315A2
Application number: EP09778578A
Authority: EP
Inventors: Helmut Brod; Björn FRAHM; Joerg Kauling
Original assignee: Bayer Technology Services GmbH
Current assignee: Bayer Intellectual Property GmbH
Priority date: 2008-09-26
Filing date: 2009-09-17
Publication date: 2011-07-13
Also published as: CN102272285A; US20110165677A1; WO2010034428A3; DE102008049120A1; WO2010034428A2

Abstract

The invention relates to a method for reducing deposits during the cultivation of organisms, particularly cell cultures, which tend to agglomerate or adhere to the bioreactor and the elements thereof, or in which cell debris or substances easily agglomerate or adhere.

Description

Method for reducing deposits in the cultivation of organisms

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.

The cultivation of human, animal and plant cells has gained great importance in the production of biologically active substances and pharmaceutically active products. In particular, the cultivation of cells, which is often carried out in a nutrient medium in free suspension, is demanding because the cells, in contrast to microorganisms are very sensitive to mechanical shear stress and insufficient supply of oxygen and nutrients (see, for example, H.-J. Henzler 2000 Particle Stress in Bioreactors Adv. Biochem. Eng./Biotechnol. 67: 35-82 or JG Aunis, H.-J. Henzler 1993. Aeration in Cell Culture Bioreactors. In: Biotechnology, Second, Completely Revised Edition, Volume 3 : Bioprocessing: 219-281, VCH Wiley or Studies on Extracellular and Intracellular Oxygen Transfer, by the Faculty of Natural Sciences of the University of Hanover to obtain the degree of Dr. rer. Nat., Licensed dissertation by Oliver Schweder, 2006). In contrast to nutrients that are present in such a concentration in the nutrient medium that they do not have to be continuously replenished, the oxygen solubility of the nutrient medium is so low that without continuous oxygen supply, the cells would rapidly reach an oxygen limitation. In addition to the sufficient supply of oxygen, the removal of carbon dioxide is of similar importance.

Mostly human, animal and plant cell lines are cultivated intermittently. This has the disadvantage that an optimal supply of the cells as a result of constantly changing substrate, product and biomass concentrations is difficult to achieve. At the end of

Fermentation also accumulates by-products, e.g. the lysis products died

Cells, which usually have to be eliminated at great expense in the subsequent workup.

For the reasons mentioned, but especially in the production of unstable products which can be damaged for example by proteolytic attacks, therefore, preferably continuously operated 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.

The accumulation of debris in conventional systems is a major cause of the necessary reduction in cell density and the premature termination of the cultivation of corresponding cell lines. Furthermore, in conventional systems, the attachment of cells, debris and / or proteins to probes or other measurement / analytical devices will result in their malfunctioning or nonfunctioning, which usually can not be resolved or compensated for during ongoing operation of the bioreactor, thereby prematurely terminating the culture may be necessary.

Problems in (cell culture) fermentations with regard to deposits in the bioreactor and the adhesion of cells and Zelldebris, especially on the membrane tubes and the formation of cell agglomerates, have long been described in the literature.

To reduce deposits, 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. In addition, it is known (see, for example, EP0422149B1) that 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) is introduced near the outlet at the bottom of the vessel and generates a flow which is intended to completely capture the culture medium in the vessel and to achieve complete mixing of the components of the culture medium and possibly free-floating culture organisms. In a particular embodiment, 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. It is known (see, for example, EP0422149B1) that the formation and bursting of gas bubbles have high shear forces which can lead to cell damage. In addition, gas bubbles lead to the formation of foam. Foaming is to be avoided, however, as cells tend to float with the foam. In the foam layer, they do not find adequate cultivation conditions. The use of antifoam agents can lead to cell damage or yield losses in the workup or to an increased workup effort. In addition, a sufficient oxygen supply in shear-sensitive cells can be ensured with a coarse-bubble gassing method only up to relatively low cell densities (H.-J. HeΩzler: "Process engineering design documents for stirred containers as fermenters" Chem. Ing. Tech. 54 (1982) No. 5 p 461-476, H.-J. Henzler, J. Kauling: "Oxygenation of cell cultures" Bioprocess Engineering 9 (1993) pp. 61-75, tables and stirring "Edited by M. Kraume, WILEY-VCH 2003). Scaling of a coarse-bubble fumigation to a bioreactor on an industrial scale is difficult, and for these reasons, the culture device described in EP1935973A1 is therefore unsuitable for the industrial cultivation of a variety of organisms.

The bubble-free fumigation solves the problem by the gas exchange takes place over a submerged membrane surface. Here, the fumigation is carried out with closed or open-pore membranes. These are e.g. arranged in the liquid moved by a stirrer. For example, 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. 61-75, EP0172478B1, EP0240560B1) 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 However, 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) .To achieve high mass transfer rates, it is necessary . install a corresponding amount of membrane area in the bioreactor. However, 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. It is conceivable to increase the power input. Since the mass transfer coefficient depends on the input of power, an increase in the mass transfer rate can be achieved. However, the potential is limited by the resulting shear stress of the cells due to the higher power input.

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.

While an improvement of the oxygen supply and reduction of the shear forces compared to the static membranes described in EP0172478B1 and EP0240560B1, which are impinged by an agitator, is achieved by the rotationally oscillating movement of the membrane surface described in WO2007098850 (A1), WO2007098850 (A1) is to be feared in that particles are captured by the movement of the membrane tubes through the culture medium, which either settle on the membrane tubes or slide along the membrane tubes into the dead spaces between the membrane tubes and lead to deposits here.

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. In addition, 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.

Starting from the described prior art, 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.

It has surprisingly been found that the agglomeration and / or addition of cells, debris and / or proteins, in particular to the elements for gas transfer, e.g. For oxygen supply, but also on all other surfaces and probes can thereby significantly reduced or even prevented that emerges for gas supply a membrane surface in the culture medium, which performs a discontinuous movement in the culture medium.

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.

The agglomeration and / or attachment of cells, debris and / or proteins, especially on the membranes, but also on all other contact surfaces and probes, is significantly reduced or prevented by the discontinuous movement, creating a higher

Gas exchange across the membranes is possible and this also for a longer period. This increases cell density and thus product yield, and extends the maximum runtime of the process.

"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.An example of 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.

Preferably, 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. However, the second movement is preferably a mirror, point and / or rotationally symmetrical design for the first movement.

In a preferred embodiment of the method according to the invention, 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.

Particularly preferably, the membrane surface carries out a rotationally oscillating movement. In the case of 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. Subsequently, 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. Under a rotationally oscillating motion movement is to be understood, 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.

Preferably, the movement is carried out so that the membrane surface first rotates in one and after a predetermined time in the opposite direction.

In a preferred embodiment of the method according to the invention, 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. Preferably, 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.

In a preferred embodiment of the method according to the invention, 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. As an example 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. Likewise, 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,

WO07 / 098850A1.

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. ¹ In the inventive method, 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 .

In a preferred embodiment of the method according to the invention, 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. For this purpose, 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. The use of 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.

As a drive for generating a rotationally oscillating movement, the power provided by the motor must be sufficient to perform an oscillating movement with a given movement sequence with the rotor despite the mass moment of inertia of the rotor and the culture medium. For the design of the drive so both the moment of inertia of the rotor and the force of the culture medium on the rotor is crucial. With sufficient engine speed, a transmission provides the ability to provide the required torque. As drive configuration, for example, an eccentric drive comes into question. An eccentric drive converts the uniform rotation of a conventional drive motor into a rotationally oscillating motion on the output shaft. In addition come as a drive configuration for the device according to the invention also freely programmable positioning drives in question, such as a stepper motor. The advantage of such freely programmable drive systems is that the rotationally oscillating movement of the membrane surface can be adapted to the requirements of the process in a wide range, while an eccentric drive usually has only limited adjustment options. Parameters of the drive such as speed, torque and gear reduction are freely selectable for the respective application and depend on the scale. For applications in the field of biotechnology, the parameters are usually designed so that a volume-specific power input of 0.01 W per m ^"3 up to 4000 W per m ^{" 3} liquid volume, preferably by 1000 W per m ^"3 results.

For cell cultures, the volume-specific power input is usually between 0.01 and 100 W per m ³ .

Furthermore, 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.

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.

In a particular embodiment of the device, two winding arms form a rotor arm. On these winding arms, the membrane surface, preferably the membrane tubes, horizontally or vertically wound at regular or irregular intervals.

If now a rotation of the rotor, the membrane tubes are moved through the culture medium in the reactor and thereby flowed tangentially. Surprisingly, it has been found that, as the person skilled in the art would expect, the flow does not cause particles in the solution to be captured by the membrane surfaces and held or transported into (their) dead spaces, resulting in deposits. Surprisingly, it has been found that a discontinuous movement, preferably a rotationally oscillating movement, leads to a reduction of deposits in comparison to a statically arranged membrane surface, in which optionally an additional agitator causes a flow with culture medium. With regard to the flow of the membrane hoses, it should be noted that the flow at the same angular velocity 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. Preferably as many membrane hoses as far outside as possible are installed with good flow. 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). In addition, under the increasing number of 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. Here, 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. In 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.

Another advantage of the device with wound membrane tubes is that 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. Henzler, GN Jovanovic, KB Konstantinov:, Fixperimental and Theoretical Analysis of Tubular Membrane Aeration for Mammalian Cell Bioreactors "Biotechnology Progress 19 (2003) Pp. 1183-1189). With membrane hoses, the reduction of the hose tension leads to an increased deflection of the hoses during the movement. A greater deflection of the hoses improves their flow around and thus the mass transfer coefficient. Depending on the type of application, 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. Furthermore, 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. Furthermore, 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.

Further embodiments of 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.

It is preferred in the cultivation of cell cultures, which for agglomeration or

Attachment to the bioreactor and its elements tend, or where cells, cell debris or

Substances easily agglomerate or adhere. Here it shows no adverse effects, e.g. on cell biology, e.g. regarding apoptosis and cell cycle. examples for

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.

With a discontinuous, in particular a rotationally oscillating movement of the membrane surface within the culture medium, three functionalities are combined:

1. 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.

2. 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.

3. 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.

It is conceivable, in addition to the discontinuous movement of the membrane surface, to also carry out a discontinuous movement of one or more probes (pH probe, thermometer, electrode for determining the oxygen content and similar probes) in the bioreactor. 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. Examples

The invention is explained in more detail below by means of examples, without, however, limiting them to these.

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). Preferably, 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. In this example, the distributor rings each have 16 connecting pieces, which allow the supply of the membrane hose segments on the up to 16 rotor arms. In this case, 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. For each rotor arm, 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. In order to prevent slippage of the membrane tubing made of silicone with 1.98 mm inner diameter and 3.18 mm outer diameter, parallel recesses are produced with a distance of 3.65 mm. The difference of 3.65 mm compared to 3.18 mm results from the motivation that the membrane hoses even in the pressurized state (up to 1.5 bar overpressure) have room to increase in volume ("puffing"), whereby the pressure loss in the hose deflection is minimized.

As 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.

In Table 1, examples of angular accelerations and maximum angular velocities as well as maximum speeds of the rotor arm ends, ie those points of the rotor which move fastest, are exemplarily listed for three scales in the specified configuration.

^* in the geometr. similar model systems measured Table 1

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.

In addition, 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.

Via the membrane surface (membrane hoses), 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. Through the headspace of the fermenter, 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.

According to the invention, 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 erfϊndungsgemäße method is hereinafter referred to as DMA method (Dynamic Membrane Aeration) and the corresponding device for carrying out the erfϊndungsgemäßen method as a DMA reactor.

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

Anchor stirrer equipped. The static membrane area comprised 49.6 m hose length

(corresponding to 41.3 m ² membrane surface per m ³ Reaktorfüllvolumen) of the above

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

Improvement of substance exchange (oxygen supply, carbon dioxide removal). 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 damage and undesired by-product formation was avoided, was required to avoid / limit cell agglomeration and deposition.

Information on the structure of the system for continuous cell culture operation and the cell separator can be found again in WO2003 / 020919A1.

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.

Daily samples from the bioreactor and harvest stream were taken from both the DMA and the reference methods based on cell density, vitality, aggregation rate, offline pH, dissolved oxygen concentration and dissolved carbon dioxide, concentration Glucose, lactate, glutamine, glutamate, ammonium, LDH and titer (coagulation factor VHI (rFVπi)).

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) In order to minimize the influence of cell agglomeration and to determine a cell density which is as representative as possible, a pipetting was carried out beforehand, whereby the cell agglomerates are largely dissolved by the shearing forces in the pipette In Fig. 3 (b) a decrease in the cell density after 53 days to about 10 × 10 ⁶ cells mL ^"1 to observe. Cause were deposits on the membrane tubes, which obviously led to a lower oxygen input. The DMA process did not show these deposits; Here, a high cell density was maintained over the entire observation period.

After the first cell cultivation, 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].

In the DMA method, it was possible to reach and maintain a cell density of more than 15 × 10 ⁶ cells mL ^"1 after just under 7 days of culturing In the reference method, such a cell density was not achieved and the production rate was correspondingly lower

In both cell cultures, 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.

In the example, the following advantages of the method according to the invention compared to the described reference method were summarized:

- increased oxygen input; the cell density was in the DMA process throughout

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.

- In the DMA process, comparable flow conditions could be achieved with approximately one third of the power input of the reference system (relative to the shear rate).

- The DMA method showed no adverse effects on cell biology (apoptosis and cell cycle).

pictures

Fig. 1: Schematic representation of a rotationally oscillating movement for loading and

Degassing of liquids by means of a membrane surface in a container.

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

(a) in the DMA process 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 ] is plotted against the time t in the unit [days].

4: Graphical representation of the temporal evolution of the density of living cells

(a) in the DMA process and (b) in the reference process in a 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].

Reference numerals:

1 membrane hoses

2 rotor shaft

3 Rotation direction 4 bioreactor

5 fluid level

Claims

claims

1. A method for reducing deposits in the cultivation of cells and organisms, characterized in that a gas exchange in the culture medium immersed membrane surface carries out a discontinuous movement.

2. The method according to claim 1, characterized in that the membrane surface performs a movement with reversal of movement.

3. The method according to claim 1 or 2, characterized in that the membrane surface ausfuhrt a rotationally oscillating movement.

4. The method according to any one of claims 1 to 3, characterized in that the movement comprises a periodic sequence of acceleration and deceleration between two movement reversal points.

5. The method according to any one of claims 1 to 4, characterized in that the membrane surface is formed from one or more membrane tubes.

6. The method according to claim 5, characterized in that the membrane surface is mounted in the form of one or more membrane tubes to star-shaped mounted on a rotor shaft rotor arms, and is flowed tangentially due to the discontinuous movement.

7. The method according to any one of claims 1 to 6, characterized in that the membrane surface is connected via a common holder with one or more probes.

8. Use of the method according to any one of claims 1 to 7 for the cultivation of cells or organisms.