BIOREACTORS FOR ORBITALLY SHAKING CELL CULTURES, IN PARTICULAR SUSPENSION CULTURES

Abstract

The present invention relates to. a bioreactor vessel (1) having an outer vessel wall (2) and a bottom (3), further comprising an integrated internal structure (4) providing at least two additional surfaces (4a), (4b) to the internal reactor space of said vessel, said internal structure (4) being spaced apart from said outer vessel wall (2), as well as to a process for growing biological cells using said bioreactor vessel.

Claims

1-15. (canceled)

16. A bioreactor vessel having an outer vessel wall and a bottom, and further comprising at least one integrated internal structure providing at least two additional surfaces, an outer surface and an inner surface, to the inner bioreactor space of said vessel, said internal structure being spaced apart from said outer vessel wall.

17. The bioreactor vessel of claim 16, wherein said internal structure comprises a wall providing an outer surface and an inner surface, wherein said wall: (i) has at least one opening in the area closest to the bottom of the bioreactor vessel; or (ii) is spaced apart from the bottom of the bioreactor vessel; or (iii) has opening(s) and is spaced apart from the bottom of the bioreactor vessel.

18. The bioreactor vessel of claim 17, wherein said opening(s) and/or the space from the bottom of the bioreactor vessel have/has a dimension of at least 0.05 mm in any direction.

19. The bioreactor vessel of claim 17, wherein said opening(s) and/or the space from the bottom of the bioreactor vessel have/has a dimension of from 0.1 mm to 10 cm in any direction.

20. The bioreactor vessel of claim 17, wherein said opening(s) have a shape selected from the group consisting of: circular; half-circular; a circular sector; elliptical; half-elliptical; an oval sector; triangular; rectangular; polygonal; undulated; and any combination thereof.

21. The bioreactor vessel of claim 17, wherein said opening(s) is/are located at the lower end of the internal structure contacting the bottom of the bioreactor vessel.

22. The bioreactor vessel of claim 17, wherein said opening(s) leave intact at least 80% of the possible contact area between the lower end of the internal structure and bottom of the bioreactor vessel.

23. The bioreactor vessel of claim 16, wherein said outer bioreactor vessel wall and said internal structure have the same geometrical shape.

24. The bioreactor vessel of claim 23, wherein said outer bioreactor vessel wall and said internal structure have a cylindrical, conical, or elliptical shape.

25. The bioreactor vessel of claim 23, wherein said outer bioreactor vessel wall and said internal structure both have a cylindrical shape.

26. The bioreactor vessel of claim 16, comprising more than one internal structure, so that more than two additional surfaces are provided within the bioreactor vessel.

27. The bioreactor vessel of claim 16, wherein the outer surface of the internal structure is spaced apart from the inner surface of the bioreactor vessel wall by at least 1/15 of the cross section of the total bioreactor chamber, but not by more than ⅓ of the cross-section of the total inner space.

28. The bioreactor vessel of claim 16, wherein the outer surface of the internal structure is spaced apart from the inner surface of the bioreactor vessel wall by at least ⅒ of the cross section of the total bioreactor chamber, but not by more than ⅕ of the cross-section of the total inner space.

29. The bioreactor vessel of claim 16, wherein the outer surface of said internal structure is spaced apart from the inner surface of the bioreactor vessel wall in a distance such that the ratio of the cross section of the internal structure (CS.sub.in) to the cross section of the outer bioreactor vessel wall (CS.sub.out) is in the range from 0.95 to 0.4, wherein said cross sections are measured along the bottom of the bioreactor vessel in the space between the inner surfaces of the bioreactor wall and internal structure respectively.

30. The bioreactor vessel of claim 29, wherein the outer surface of said internal structure is spaced apart from the inner surface of the outer bioreactor vessel wall at a distance such that the ratio of the cross section of the internal structure (CS.sub.in) to the cross section of the outer vessel wall (CS.sub.out) is in the range from 0.85 to 0.6, wherein said cross sections are measured along the bottom of the bioreactor vessel in the space between the inner surfaces of the bioreactor wall and internal structure respectively.

31. The bioreactor vessel of claim 26, wherein each of the internal structures are spaced apart from each other or have a ratio of cross sections CS.sub.in to CS.sub.out to each other in the range from 0.95 to 0.4, wherein said cross sections are measured along the bottom of the bioreactor in the space between the inner surfaces.

32. The bioreactor vessel of claim 16, said bioreactor is a single reactor formed as a container, a flask, a bottle, a pipe, a tube, a cup or a bag, or is part of a multiarray with a plurality of individual vessels.

33. The bioreactor vessel of claim 16, further comprising a cover.

34. The bioreactor vessel of claim 16, further comprising an inlet and/or an outlet, allowing the addition or withdrawal of content of the vessel or the placement of a measuring device inside of the vessel.

35. A process for growing biological cells under defined gas conditions, wherein the bioreactor vessel of claim 16 is provided with cells for growing, is at least partially filled with a liquid, and is shaken so that the liquid is rotationally moved inside the vessel.

Description

FIGURES:

[0038] In FIG. 1 a bioreactor vessel (1) is shown in an inclined top view, comprising an outer vessel wall (2), with an outer surface (2a) and an inner surface (2b), a bottom (3) and an internal structure (4) having an outer surface (4a) and an inner surface (4b) and three openings (5) circumferentially distributed at the lower end of the internal structure (4). The bioreactor vessel is shown without any cover (6).

[0039] FIG. 2 shows a similar embodiment as provided in FIG. 1, wherein the openings have not a triangle shape as in FIG. 1, but a square shape and the internal structure includes only two openings. Further, in this Figure it is illustrated how to define the cross sections CS.sub.out and CS.sub.in of the vessel (1). The outer cross section CS.sub.out corresponds to the total cross section defined by the inner surface (2b) of the outer vessel wall (2), the inner cross section CS.sub.in corresponds to the cross section defined by the inner surface (4b) of the internal structure (4).

[0040] FIG. 3 shows a bioreactor vessel (1) in an inclined top view, wherein the internal structure (4) is spaced apart from the bottom (3) by holding structures (7).

[0041] FIG. 4 shows a bioreactor vessel in a cross-sectional view, comprising an outer vessel wall (2), and an internal structure (4) having two openings (5) close to the bottom. The outer wall (2) and the internal structure (4) of the bioreactor vessel have a different geometrical shape (outer wall “top opening” conical, internal structure cylindric).

[0042] FIG. 5 shows a bioreactor vessel in a cross-sectional view, comprising an outer vessel wall (2), and an internal structure (4) having one opening (5) close to the bottom. The outer wall (2) and the internal structure (4) of the bioreactor vessel have the same geometrical shape, both cylindric, however, the internal structure (4) is shorter than the outer wall (2).

[0043] FIG. 6 shows a bioreactor vessel in a cross-sectional view, comprising an outer vessel wall (2), and an internal structure (4) having two openings (5) close to the bottom. The outer wall (2) and the internal structure (4) of the bioreactor vessel have the same geometrical shape, both cylindric, with the same height.

[0044] FIG. 7 shows a bioreactor vessel in a cross-sectional view, comprising an outer vessel wall (2), and an internal structure (4) having one opening (5) close to the bottom. The outer wall (2) and the internal structure (4) of the bioreactor vessel have a different geometrical shape (outer wall cylindric, internal structure “top opening” conical).

[0045] FIG. 8 shows a bioreactor vessel in an inclined side view, comprising an outer vessel wall (2), and two internal structures (4) having both one opening close to the bottom (3). The outer wall (2) and the internal structures (4) of the bioreactor vessel have all the same geometrical shape.

[0046] FIG. 9 shows a graph of the transfer rate (capacity) of oxygen into different volumes of a liquid, shaken in bioreactor vessels according to the invention in comparison to a common bioreactor vessel (see Examples 1 and 2)

[0047] FIG. 10 shows a graph of the improvement of the oxygen transfer into a liquid, shaken in bioreactor vessels according to the invention (see Examples 1 and 2)

[0048] FIG. 11 shows a graph of the transfer rate (capacity) of oxygen into a liquid, shaken in bioreactor vessels according to the invention with different rotational speed. (see Examples 1 and 3).

EXAMPLES

Example 1 Experimental Design

[0049] TABLE-US-00001 Three cylindric bioreactor vessel types have been prepared of glass having the following dimensions Vessel Design A B C Vessel without IS Vessel with IS (tight) Vessel with IS (wide) Hight [mm] 120 120 120 Outer diameter of the vessel [mm] 60.6 60.6 60.6 Inner diameter of outer wall [mm] 55.6 55.6 55.6 Outer diameter of the IS [mm] - 44 38.1 Inner diameter of the IS [mm] - 40.5 34.4 Opening [mm in both directions]* - 2 2 IS = internal structure (cylindric) * 4 openings, each square-cut at the lower end of the internal structure

[0050] The bioreactor vessels A, B, C have been filled respectively with the same liquid volume (V.sub.L) as defined below and shaken under aerobic (air) conditions at 21° C. The oxygen transfer rate has been determined by the sulfite oxidation method as described in detail by Hermann R. et al.in the article “Optical method for the determination of the oxygen-transfer capacity of small bioreactors based on sulfite oxidation”, published in Biotechnology and Bioengineering, Vol 74, No. 5, Sep. 5, 2001 (John Wiley & Sons, Inc)

Experimental Setting

[0051] 0.5 M sulfite oxidation method with detection of color change: [0052] Shaker: Kühner Lab Shaker LSR-V-12.5, shaking diameter d.sub.0= 25 mm [0053] Sulfite system: 0.5 M NaSO.sub.3 (Bernd Kraft, Duisburg, Germany) in 12 mM degassed phosphate buffer (from 0.5 M Na.sub.2HPO.sub.4.Math.2H.sub.2O and 0.5 M NaH.sub.2PO.sub.4.Math.2H.sub.2O (Roth, Karlsruhe, Germany), 2.4*10.sup.-5 M bromothymol blue (Sigma-Aldrich, Steinheim Germany), pH 8 with 30 % H.sub.2SO.sub.4 (w/w) (Bernd Kraft, Duisburg, Germany), 10.sup.-7 M CoSO.sub.4.Math.7H.sub.2O (Sigma-Aldrich, Steinheim Germany) [0054] Sterile closure: Thomson Ultra Yield Flask AirOTop Enhanced Seal for Ultra Yield 2.5 L Flask (Thomson Instrument Company, Oceanside, USA) [0055] Camera: Samsung Galaxy A3 (2016) with App ,,IntervalCam″

[00001]$N{a}_{2}S{O}_3^2+\frac{1}{2}{O}_2\stackrel{C{o}^{2+}}{.fwdarw.}N{a}_{2}S{O}_4^2$

[00002]$OTR=c_{SO3}\ast \frac{v_{O2}}{t_{ox}}$

t.sub.ox = time till end of reaction (color change) v.sub.O2= stoechiometric coefficient of oxygen (0.5) c.sub.SO3=amount of sulfite used

[0056] In the diagrams (FIGS. 9 to 11) showing the results of the present examples the internal structure is named “tube”.

[0057] OTR.sub.max: maximum oxygen transfer rate (capacity)

Example 2: Determination of OTR.SUB.max Dependent From the Filling Volume V.SUB.L

[0058] In a first approach the oxygen transfer into the liquid is considered in dependency from the filling volume of the bioreactor vessels having the above described dimensions. Usually, the oxygen transfer capacity decreases when the volume increases, since the oxygen transfer occurs mainly at the liquid surface being in contact with the gas. The smaller the liquid volume, the greater is the ratio of liquid surface to liquid volume, if the liquid is provided in the same dimensioned vessel (as long as the whole bottom of the vessel is covered). Here, it is considered how the vessel design affects the oxygen transfer capacity.

[0059] The following samples have been tested (in vessel design A, B, C, respectively) [0060] 1. VL= 5 ml, shaken at 250 rpm rotation [0061] 2. VL= 10 ml, shaken at 250 rpm rotation [0062] 3. VL= 15 ml, shaken at 250 rpm rotation [0063] 4. VL= 10 ml, shaken at 300 rpm rotation [0064] 5. VL= 15 ml, shaken at 300 rpm rotation

[0065] FIG. 9 shows the results of the oxygen transfer capacity dependent from the filling volume of the vessels at two rotation speeds (FIG. 9A: 250 rpm, FIG. 9B: 300 rpm). As can be seen the oxygen transfer rate increases due to the presence of the internal structure (“tube”), wherein the tight gap between the outer wall of the vessel and the internal structure improves the OTR.sub.max noticeably stronger than the wider gap. Increasing the rotary speed increases the oxygen transfer rate noticeably (compare FIGS. 9A to 9B)

[0066] FIG. 10 shows the extent of improvement of the oxygen transfer obtainable by the inclusion of the internal structure (tube): In FIG. 10A it can be clearly seen that at 250 rpm the obtainable improvement of oxygen transfer is much stronger in a higher volume, in both the widely and the tightly gapped vessels. At 300 rpm the obtainable improvement is almost independent from the filling volume, see FIG. 10B. In both diagrams it is shown that the improvement obtainable with the smaller distance between outer wall and internal structure (tight gap) is noticeably higher than the improvement obtainable with the larger distance (wider gap). However, both designs have a clear positive effect to the oxygen transfer compared to the bioreactor without any internal structure.

Example 3: Determination of OTR.SUB.max Dependent From the Rotational Speed N

[0067] Further, the oxygen transfer into the liquid has been considered in dependency from the rotational speed (n) of the bioreactor vessels having the above described dimensions. It is basically known that the oxygen transfer capacity increases when the rotation speed increases, since the liquid is bouncing and flowing to a higher extent along the inner wall surface of the vessel, thus increasing the liquid surface area by forming a film on the inner surface of said wall. Since oxygen transfer occurs mainly at the liquid surface being in contact with the gas, the gas exchange increases when said surface is increased.

[0068] FIG. 11 A shows said improvement of oxygen transfer for samples 2 and 4, and FIG. 11 B for samples 3 and 5. As can be seen the provision of an internal structure providing additional surfaces increases the oxygen transfer into the liquid at both rotational speeds. The stronger effect of the tighter gap can be explained by the provision of a larger area of the additional inner surface (the inner surface of the internal structure) for forming a liquid film.

[0069] The dimensions and design of the bioreactor vessels shown in the above examples should be considered as illustrative. The particular sizes defined herein can be varied. Indeed, bioreactor vessels having larger or smaller dimensions can be prepared, however, it is particularly preferred that the proportions of the dimensions, especially the proportions of the walls, spaces, cross-sections and the size of the openings correspond essentially to the herein described dimensions.