COMBINATION STIRRER

Abstract

The present patent application describes a stirrer comprising a combination of at least one axially-conveying element and at least one radially-conveying element relative to the rotary shaft of the stirrer wherein the largest diameter of the at least one axially-conveying element is equal to or less than the inner diameter d.sub.i of the radially-conveying element. In one embodiment the stirrer according to the invention is a combination of one anchor stirrer with at least one inclined-blade stirrer. Furthermore the use of the stirrer according to the invention for the culture of cells in a dialysis method is described.

Claims

1. Agitator, comprising one radially-conveying element comprising at least two stirrer blades, and one or more axially-conveying elements each comprising at least two stirrer blades, wherein the stirrer blades of the radially-conveying element are parallel to each other, wherein the outer diameter of all axially-conveying elements is equal to or less than the inner diameter of the radially-conveying element, wherein all axially-conveying elements are individually connected to the radially-conveying element, wherein all axially-conveying elements are located within the radially-conveying element, and wherein all conveying elements have a fixed spatial orientation relative to each other.

2. Agitator according to claim 1, characterized in that the number of axially-conveying elements is 1 or 2 or 3.

3. Agitator according to any one of the previous claims, characterized in that one axially-conveying element is located at a maximum distance of 80% from the top of the blades of the radially-conveying element and/or one axially-conveying element is located at a maximum distance of 20% from the top of the blades of the radially-conveying element.

4. Agitator according to any one of the previous claims, characterized in that opposing stirrer blades of the radially-conveying element are linked to each other by two opposite stirrer blades of an axially-conveying element.

5. Agitator according to any one of the previous claims, characterized in that the radially-conveying element is an anchor impeller.

6. Agitator according to any one of the previous claims, characterized in that the axially-conveying element is an inclined-blade stirrer.

7. Agitator according to claim 6, characterized in that the pitch of the stirrer blades of the inclined-blade stirrer is between 10° and 80° relative to the shaft axis of the agitator.

8. Agitator according to one of the previous claims, characterized in that the radially-conveying element has 1 to 8 stirrer blades.

9. Agitator according to one of the previous claims, characterized in that the one or more axially-conveying elements have independent of each other 1 to 10 stirrer blades.

10. Agitator according to any one of the preceding claims, characterized in that the radially-conveying element has a height of at least 200 mm.

11. Device comprising an agitator according to any one of claims 1 to 10 within a cultivation vessel.

12. Device according to claim 11, characterized in further comprising a dialysis module.

13. Device according to any one of claims 11 and 12, characterized in that cultivation vessel is a stirred tank reactor.

14. Device according to any one of claims 11 to 13, characterized in that the cultivation vessel is a submersed gassed stirred tank reactor.

15. Device according to any one of claims 11 to 14, characterized in that the ratio d/D of agitator diameter (d) to cultivation vessel diameter (D) is of from 0.2 to 0.8

16. Device according to any one of claims 11 to 15, characterized in that the ratio H/D of filling height of the cultivation vessel (H) to cultivation vessel width (D) is of from 1.0 to 2.5.

17. Use of an agitator according to any one of claims 1 to 10 or a device according to any one of claims 11 to 16 for cultivating a cell expressing a polypeptide.

18. Method for the production of a polypeptide comprising a) providing a cell comprising a nucleic acid encoding the polypeptide, b) providing a device as reported herein, c) cultivating the cell in the device in a cultivation medium wherein the agitator provides a turbulent flow within the cultivation vessel, and d) recovering the polypeptide from the cells or the cultivation medium and thereby producing a polypeptide.

19. Use according to claim 17 or method according to claim 18, characterized in that the cultivating is a dialysis.

20. Use according to any one of claim 17 or 19 or method according to any one of claims 18 to 19, characterized that the cell is a mammalian cell.

21. Use or method according to claim 20, characterized in that the mammalian cell is selected from a CHO cell, a BHK cell, an NS0 cell, a COS cell, a PER.C6 cell, a Sp2/0 cell, or a HEK 293 cell.

22. Use according to any one of claims 17 and 19 to 21 or method according to any one of claims 18 to 21, characterized in that the polypeptide is an antibody.

Description

DESCRIPTION OF THE FIGURES

[0099] FIG. 1 Schematic diagram of various embodiments of the combination stirrer according to the invention; b: width of the stirrer blade; d: stirrer diameter; d.sub.w: diameter of the rotary shaft; h: height of the stirrer blade of the radially-conveying stirrer; h.sub.m: height of the fastening sleeve; h.sub.SB: height of the axially-conveying stirrer; h.sub.u: height of the reducer; l: length of the stirrer blade of the axially-conveying stirrer; α: blade pitch of the axially-conveying stirrer; z: number of stirrer blades per stirrer; d.sub.i: inner distance between the stirrer blades of the radially-conveying stirrer; h.sub.4/5: ⅘ height from above of h; K: stirrer head; a) general scheme of the agitator as reported herein; b) to f) embodiments of the agitator as reported herein; g) scheme of a radially conveying element seen from the top along the shaft axis; h) scheme of an axially conveying element seen from the top along the shaft axis; i) scheme of an embodiment of the agitator as reported herein seen from the top along the shaft axis showing the connection of opposing stirrer blades of the radially-conveying element via connecting stirrer blades of the axially-conveying element; j) scheme of an embodiment of the agitator as reported herein seen from the top along the shaft axis showing the connection of opposing stirrer blades of the radially-conveying element via connecting stirrer blades of the axially-conveying element wherein the diameter of the axially-conveying element is less than the inner diameter of the radially-conveying element and the spatial distance is bridged by a connector.

[0100] FIG. 2 Schematic diagram of a device for dialysis cultivation.

[0101] FIG. 3 Schematic diagram of the concentration gradients on the hollow fibers of the dialysis module.

[0102] FIG. 4 Typical concentration time course in the reactor (C.sub.R) and storage container (C.sub.V).

[0103] FIG. 5 Comparison of the mixing coefficients C.sub.H for different stirrer as a function of the Reynolds's number (Re); KR=combination stirrer as reported herein; SSR=standard disk stirrer; SBR=inclined-blade stirrer.

[0104] FIG. 6 Reference flake diameter d.sub.VF as a function of the volume-specific power input and stirrer configuration; KR=combination stirrer as reported herein; SSR=standard disk stirrer; SBR=inclined-blade stirrer; Pohlscheidt, M., et al.=Chem. Ing. Tec. 80 (2008) 821-830.

[0105] FIG. 7 Diagram of the power coefficient Ne of different stirrer as a function of the Reynolds's number (Re); KR=combination stirrer as reported herein; SSR=standard disk stirrer; SBR=inclined-blade stirrer.

[0106] FIG. 8 Mass transfer coefficient k.sub.a in the dialysis as a function of the specific power input; KR=combination stirrer as reported herein; SBR=inclined-blade stirrer.

EXAMPLE 1

[0107] Cultivation Vessel

[0108] All investigations were carried out in a 100 l Plexiglas® model container (referred to as DN 440 in the following).

EXAMPLE 2

[0109] Power Input

[0110] The power input of different stirrer was determined by measuring the torque on the rotary shaft. A data processing system model GMV2 together with the torque sensor model DRFL-II-5-A (both from the “ETH Messtechnik” Company, Gschwend, Germany) were used to record the torque. For each stirrer system the torque was firstly recorded at various revolution speeds in the unfilled state (M.sub.empty) and subsequently by means of a triplicate determination in the filled state (M.sub.load) according to equation 9:

[00009]$\begin{array}{cc}M=M_{\mathrm{load}}-M_{\mathrm{empty}}.& \left(\mathrm{Equation}9\right)\end{array}$

[0111] Afterwards the corresponding Newton number (Ne number) and the Reynolds's number (Re number) were calculated for each point. Since the Newton numbers for a stirrer system become constant in the turbulent flow region, the calculated Newton numbers were subsequently averaged in this region (Uhl, V. W. and Gray, J. B., Mixing Theory and Practice, Academic Press, 1966). This mean represents the total Newton number of the respective stirrer.

EXAMPLE 3

[0112] Homogenization

[0113] The homogenization was determined using the color change method as well as using the conductivity method.

[0114] The color change method is based on the decolorization of a starch solution stained with iodine-potassium iodide by addition of sodium thiosulfate (I, KI, starch, Na.sub.2S.sub.2O.sub.3 obtained from the Carl Roth GmbH & Co KG Company, Karlsruhe, Germany). A one molar sodium thiosulfate solution and a one molar iodine-potassium iodide solution (Lugol's solution) as well as a starch solution at a concentration of 10 g/l were used as the starting solutions. In correspondence with the conductivity experiments, at least four speed steps were examined per stirrer (quadruplicate determinations per speed step) in which a maximum of four experiments were carried out per container filling amount. In each case the starch solution was added once per filling of the container. For each individual measurement the corresponding volume of the iodine-potassium iodide solution was firstly added and subsequently the sodium thiosulfate was added. The mixing time has been determined manually from the time point at which the sodium thiosulfate was added and one second was subtracted in each case in order to take the addition time into consideration. After completion of the measurement the container filling volume was titrated (neutralized) with iodine-potassium iodide in order to compensate for the excess of the previously added sodium thiosulfate.

[0115] In the conductivity method the mixing time is defined as the time from addition of an electrolyte solution to the time at which the measured conductivity fluctuations for the last time exceed a tolerance range of ±5% around the conductivity values which are reached in a stationary state. If several probes are used, the longest detected mixing time in each case is regarded as representative for the entire system.

[0116] A 30% (w/v) NaCl solution (NaCl crystalline, Merck KGaA Company, Darmstadt, Germany) was used as an electrolyte solution to determine the mixing time by the conductivity method. This was added in pulses onto the liquid surface at the rotary shaft of the stirrer and the volume per addition was selected such that the jumps in conductivity which resulted in a stationary state did not exceed 200 mS/cm.

[0117] For each stirrer at least four speed steps were examined. The mixing time was determined at least eight times per speed step and these eight values were averaged. The mixing coefficient of the respective stirrer systems is given as the mean of the mixing coefficients averaged per speed step. The conductivity was in each case measured by three 4-pole conductivity probes (TetraCon, WTW Company, Weilheim) at various radial or axial positions in the container. The conductivity signals were read out online via the measuring amplifier that was used (Cond813, Knick “Elektronische Messgeräte GmbH &. Co, KG” Company, Berlin, Germany). The measured values were stored online and simultaneously for all probes by means of the software Paraly SW 109 (Knick “Elektronische Messgeräte GmbH & Co, KG” Company, Berlin, Germany) at a sampling rate of 5 seconds. After the series of measurements was completed the data were evaluated separately for each probe.

EXAMPLE 4

[0118] Shear Stress

[0119] A model particle system, the blue clay polymer flake system, was used to determine shear stress. This is a model particle system consisting of a cationic polymer (Praestol BC 650) and a clay mineral (blue clay) which is placed in the vessel. A flocculation reaction is started by adding Praestol BC 650 which generates flakes of a defined size. These flakes are subsequently broken up by the mechanical and hydrodynamic stress of the stirrer system. In the case of bubble-gassed systems they are additionally broken up by the energy dissipation when the bubbles are formed and burst. The average particle diameter of the model particle system was used as a measured variable to characterize the shear stress. In this case the change in the particle size was measured in situ by a Focused Beam Reflectance Measurement Probe from the Mettler Toledo Company (referred to as FBRM® in the following). The rates of change in particle size that were determined are a measure for the shear stress prevailing in the model system. The gradient of the rate of change of particle size becomes smaller during the course of the experiment but no equilibrium state is formed (particle comminution down to a diameter of the blue clay primary particles of ≈15 μm). For this reason an end flake diameter d.sub.P50′ for the blue clay polymer flake system was determined according to the following criterion (equation 10):

[00010]$\begin{array}{cc}\frac{d\left(d_{P50}\right)}{dt}\le 0.0055\left[\mathrm{\mu m}/s\right].fwdarw.d_{P50}=d_{P50}^{\prime }.& \left(\mathrm{Equation}10\right)\end{array}$

[0120] In order to ensure comparability of the end flake diameters at different power inputs and between different stirrers, the reference flake diameter was calculated as follows (equations 11 to 13):

[00011]$\begin{array}{cc}{d}_{\mathrm{VF}}=m.Math.d_{P50}^{\prime }-b.& \left(\mathrm{Equation}11\right)\\ m=1.3.Math.{10}^{-6}.Math.{\left(\frac{P}{V}\right)}^2+1.37.Math.{10}^{-3}.Math.\left(\frac{P}{V}\right)+2.46.& \left(\mathrm{Equation}12\right)\\ b=8.12.Math.{10}^{-5}.Math.{\left(\frac{P}{V}\right)}^2+6.48.Math.{10}^{-3}.Math.\left(\frac{P}{V}\right)+76.9.& \left(\mathrm{Equation}13\right)\end{array}$

TABLE-US-00002 TABLE 2 Substances used to determine the particle stress (concentrations are based on the container fill volume). Ingredient Concentration Manufacturer Wittschlicker blue clay 5 g/l Braun Tonbergbau Co., Germany Praestol 650 BC 5 ml/l Stockhaus GmbH & Co. (solution 2 g/l) KG, Krefeld, Germany NaCl 1 g/l Merck KGaA, Darmstadt, Germany CaCl.sub.2 — Carl Roth GmbH & Co. (solution 30 g/l) KG, Karlsruhe, Germany

[0121] Firstly the 100 l model container was filled with a corresponding volume (H/D ratio) of completely demineralized water (VE water) and maintained at a temperature of 20° C. Subsequently the conductivity was adjusted to a value of 1000 μS/cm by titration with a CaCl.sub.2 solution. The conductivity was measured by a 4-pole conductivity probe (probe: TetraCon, WTW Co. Weilheim; measuring amplifier: Cond813, Knick “Elektronische Messgeräte GmbH & Co, KG” Company, Berlin, Germany). Afterwards the blue clay and the NaCl were added in appropriate amounts to the solution. Subsequently a homogenization phase took place at the highest speed with a duration of at least 20 minutes. The FBRM® probe (FBRM® Lasentec® D600L, Mettler-Toledo GmbH Co., Giessen, Germany) was mounted in the container perpendicular from above (immersion depth 300 mm) at a radial distance of 70 mm to the wall. The flocculation reaction was subsequently started by adding Praestol 650 BS at a defined speed. The measured values were recorded online by means of the program data acquisition control interface version 6.7.0 (Mettler-Toledo GmbH, Giessen, Germany). The reference flake diameter was determined from the measurement data. At least three power inputs were measured for each stirrer. In each case three measurements were carried out per power input.

EXAMPLE 5

[0122] Dialysis (Mass Transfer Liquid-Liquid)

[0123] A NaCl solution (NaCl crystalline, Merck KGaA Company, Darmstadt, Germany) was used as a tracer substance to determine the concentration half-life of the module (DIADYN-DP 070 Fl OL; MICRODYN-NADIR GmbH Company, Wiesbaden, Germany) in relation to the stirrer system that was used and the volume-specific power input. The tracer substance was adjusted in the storage container at the start of each experimental run to a base-line conductivity of 1500 μS/cm. The reactor was filled with completely demineralized water for each experimental run. The filling volume of the reactor was 100 l (H/D=1.6) and that of the storage container was 400 l (H/D=2.0) and both containers were maintained at a temperature of 20° C. at the start of each experiment. The conductivity in both containers was measured by a 4-pole conductivity probe (probe: TetraCon, WTW Co. Weilheim, Germany; measuring amplifier: Cond813, Knick “Elektronische Messgeräte GmbH & Co, KG” Company, Berlin, Germany). The sampling rate of the measurement amplifiers that were used was 5 seconds and the measurement values were stored online and simultaneously for all probes by means of the software Paraly SW 109 (Knick “Elektronische Messgeräte GmbH & Co, KG” Company, Berlin, Germany). The NaCl solution was circulated by means of a peristaltic pump between supply container and dialysis module (housing pump 520 U, Watson-Marlow GmbH, Company, Rommerskirchen, Germany) at a constant flow rate of 2.1 l/min. For the evaluation, probe 1 was used as a reference probe for the reactor and probe 3 was used as a reference probe for the storage container. The data of these two probes were evaluated by an evaluation routine. In each case at least six different power inputs in the reactor were investigated per stirrer.

[0124] In order to compare the mass transfer characteristics determined by means of the NaCl solution, additional measurements were carried out with a glucose solution as tracer substance. The experimental setup was not changed for this. A defined glucose concentration (glucose solid, Merck KGaA Company, Darmstadt, Germany) at a concentration of 3 g/l was provided in the storage container. The glucose concentration was determined manually and simultaneously for the storage container and reactor at a time interval of 10 minutes by means of a blood sugar measuring instrument (ACCU-CHEK® Aviva, Roche Diagnostics GmbH Company, Mannheim, Germany).