bioreactors or fermentors for the cultivation of suspended cells or microorganisms on an industrial scale

Abstract

The invention relates to a bioreactor or fermenter for cultivating cells or microorganisms in a liquid medium on an industrial scale, comprising: a vessel containing a culture in a liquid medium having a defined fill height; an agitator arranged in the vessel for agitating the liquid medium; a first sparger arranged in a bottom portion of the vessel; and a second sparger or optionally more spargers arranged above the first sparger for continuously supplying additional air bubbles and / or additional oxygen bubbles to the liquid medium, whereby the second sparger is located at a position above the first sparger in the bioreactor or fermenter at a predefined distance η. A method for independently managing dissolved CO2 and O2 by selecting a suitable correction gas flow rate q mod is also described.

Description

Technical Field

[0001] This invention relates to bioreactors or fermenters for culturing suspended cells or microorganisms on an industrial scale. Background Technology

[0002] In biopharmaceutical approaches, achieving industrial-scale production is always of particular significance. Often, increasing bioreactor volume leads to a decline in the cell performance of the cultured cells, as maintaining culture conditions frequently limits the possibility of large-scale culture. In addition to large-scale production, it is essential to meet the quality standards of the finished product and simultaneously provide a reliable supply to the market. Therefore, it is always desirable to reduce or eliminate the size dependence of cell culture performance to achieve consistently high product titers and high product yields. As expected, the increased volumetric utilization of the increased bioreactor or fermenter volume will lead to improved productivity.

[0003] For cell culture, ensuring ideal growth conditions is crucial. In this regard, maintaining a favorable physicochemical environment is important, such as desired dissolved oxygen levels, culture pH, temperature, etc. However, cells are known to exhibit metabolic responses to their environment. In particular, concentration gradients can inhibit cell growth in large-scale bioreactors. Furthermore, pH, for example, has a significant impact on the surrounding medium. In stirred bioreactors or fermenters, metabolically active cells secrete CO2 (which is dissolved in the surrounding liquid medium) and absorb O2 from their environment to complete cellular respiration. For example, the following reaction of CO2 in the liquid medium can be observed (pH < 8.0):

[0004]

[0005] Therefore, CO2 entering the liquid phase of a bioreactor or fermenter will create an acidic environment due to the decrease in pH. Conversely, CO2 degassing will increase the pH. Therefore, a common practice is to supply oxygen to the bioreactor or fermenter via a gas supply or gas supply unit (called a "bubbler").

[0006] Therefore, to achieve high product quality and efficiency, it is often necessary to ensure a constant oxygen supply and a clearly defined consumption of dissolved carbon dioxide through so-called (CO2-) stripping. Reliable scale-up of any cell or microorganism to be cultured should be possible for any size bioreactor or fermenter.

[0007] Conventional gas supply units in bioreactors or fermenters cause bubbles to enter the liquid phase (which initially consists of pure O2 if pure O2 is used as the gas supply). During the residence and rise of bubbles in a reactor containing cells or microorganisms, O2 crosses from the gas phase into the liquid phase (reaction (1)) and vice versa, CO2 formed in metabolic reactions crosses from the liquid phase into the gas phase, i.e., into the bubbles (reaction (2)). Due to Henry's Law constant (defined, for example, by Christian Sieblist et al. Insights into large-scale cell-culture reactors: II. Gas-phase blend and CO2 stripping, Biotechnol. J. 2011, 6, 1547-1556), the transfer of O2 and CO2 occurs at different rates.

[0008] The processes and reactions discussed in the discussion are described in an illustrative manner, taking place in a bioreactor or fermenter, such as... Figure 1 As shown. Figure 1 The distribution of dissolved CO2 in an illustrative bioreactor or fermenter with a gas supply that provides, for example, pure O2 at the bottom and near a stirrer (not shown). Figure 1 The bubble 10 shown appears in three different states as it rises in the liquid: initially as a simple bubble 10.1, then as a bubble 10.2 in the middle, and finally as a bubble 10.3 at the top of the bioreactor or fermenter. Therefore, starting with the gas supply from the bottom of the bioreactor, O2 containing bubble 10.1 begins to rise to the surface of the liquid medium. Figure 1 The middle section schematically illustrates the process and reactions that occur: O2 gas crosses from bubble 10.2 into the liquid phase (reaction (1)), and CO2 gas crosses from the liquid phase into bubble 10.2 (reaction (2)). The Henry's law constants for O2 and CO2 are significantly different (Sieblist et al.; in the above citation). When measured under the same conditions, the values ​​for O2 are approximately 0.0013 mol / (kg*bar) and for CO2 are approximately 0.034 mol / (kg*bar); that is, about 25 times higher, i.e., the values ​​for CO2 are much larger, causing accelerated diffusion. Therefore, the rate of reaction (2) is much faster than that of reaction (1) (its rate is much higher than that of reaction (2)). Figure 1 (Illustrated by arrows of varying thicknesses). Because the Henry's constant for CO2 is higher than the corresponding value for O2, the carbon dioxide concentration in the bubbles increases faster than it decreases during the journey through the reactor compared to the value for O2. As a result, the driving force for CO2 mass transfer decreases during bubble ascent.

[0009] In fact, bubble 10 can supply oxygen to the liquid phase for several minutes, but its carbon dioxide uptake stops within seconds due to CO2 saturation. Once the bubble is saturated with CO2, it no longer absorbs carbon dioxide. Figure 1 Bubble 10.3 is shown in the diagram. After a few seconds, although bubble 10.3 continues to supply oxygen to the culture, it no longer has the ability to absorb CO2. Therefore, the CO2 stripping activity of bubbles provided in the liquid medium of a bioreactor is only partial. Thus, after a specific rise to a certain height within the bioreactor or fermenter, the bubbles reach a CO2 saturation concentration. Therefore, in Figure 1 In the upper part, bubble 10.3 no longer absorbs CO2 gas, and only O2 gas passes through bubble 10.3 and enters the liquid phase. Therefore, from Figure 1 From the bottom to the top, from state 10.1 through state 10.2 to state 10.3, the delivery of O2 into the liquid medium decreases, while the CO2 concentration in the bubbles increases until a saturation concentration is reached. This is indicated by a triangular arrow. Figure 1 The right side is schematically shown, and the arrow (3) from the width to the arrowhead symbolizes that the relative delivery trend of O2 to the culture medium (i.e. from the gas phase to the liquid phase) is decreasing. Figure 1 Arrow (4) in the diagram symbolizes the increasing saturation of the bubbles with CO2 from the tip to the width, thus allowing CO2 to pass from the liquid phase into the gas phase much faster than the delivery of O2. Square (5) shows the region where it is no longer possible to absorb dissolved CO2 from the liquid phase into bubbles 10.3, as bubbles 10.3 have reached a CO2 saturation concentration.

[0010] Therefore, after a very short time, bubble 10.3 becomes saturated with CO2 gas and can no longer absorb more CO2 from the liquid phase, but bubble 10.3 can still release O2 gas into the liquid environment. In bioreactors or fermenters with a permanent gas supply, a series of bubbles are provided, which enter the liquid phase and contribute to a CO2 gradient within the liquid phase. At the bottom, the bubbles have the ability to absorb CO2, a capacity that is gradually lost as the bubbles rise to the top.

[0011] In particular, CO2 removal from the culture is known to be a major problem in large-scale production bioreactors or fermenters, because stripping is mainly affected by changes in the gas composition of the bubbles as they move from the gas supply system toward the top of the bioreactor or fermenter.

[0012] Therefore, as mentioned above, the management of O2- and CO2- concentrations is of particular significance in biopharmaceutical methods (especially large-scale biopharmaceutical methods). To develop a strategy that allows for improved control and adjustment of CO2 stripping in large-scale systems, the mass transfer performance of oxygen and carbon dioxide must be evaluated in detail. The parameter to be observed related to CO2 gas is the (volume) carbon dioxide mass transfer coefficient k. L a CO2 , where k L is the transport coefficient for CO2, and 'a' is the specific interfacial area; therefore, a = A / V L That is, per culture volume V L The total mass transfer cross section A (see Christian Sieblist et al., in the above citation). The parameter to be observed in relation to O2 gas is the (volume) oxygen mass transfer coefficient k. L a O2 The mass transfer coefficient can be based on volume, and then on volumetric mass transfer coefficient.

[0013] Furthermore, on the one hand, excessively high concentrations of dissolved CO2 must be avoided, and CO2 needs to be stripped away. Inadequate CO2 stripping in large bioreactors or fermenters (i.e., those with great height and therefore long distances for bubble rise) often leads to the accumulation of dissolved CO2, resulting in high CO2 concentrations in the liquid phase, which inhibits cell growth and product formation. On the other hand, nucleic acid synthesis requires carbon dioxide, and its quantity must not be insufficient. Therefore, it should be remembered that CO2 stripping should not have any negative impact on the cells or microorganisms being cultured.

[0014] Therefore, a strategy must be developed to enhance and allow for the control and adjustment of the carbon dioxide mass transfer coefficient k in large-scale systems. L a CO2 without significantly affecting the oxygen mass transfer coefficient k L a O2 .

[0015] To investigate and elucidate the relationship between CO2 and O2 mass transfer, we have conducted various studies to confirm the effects of different operating conditions. In particular, the mixing efficiency and mass transfer performance of CO2 have been examined in detail on both laboratory and industrial scales. The results are summarized in... Figure 2 and Figure 3 middle.

[0016] Figure 2 and Figure 3 The figure shows the manually given apparent gas velocities w in two different volumes, at laboratory and industrial scales, respectively. 0 g The volumetric mass transfer coefficient k of carbon dioxide L a CO2Dependence on the power input P / V of the volumetric mixer. Specifically, Figure 2 Experiments on a laboratory scale are shown in an aerated stirred bioreactor or fermenter with a volume of 2 L (height in the range of cm). Figure 3 An industrial-scale experiment is shown in an aerated stirred bioreactor or fermenter with a volume of 12,000 L (height in the m range).

[0017] As expected, Figure 2 and Figure 3 The volumetric mass transfer coefficient k of carbon dioxide is shown in two experiments, namely on a laboratory scale and on an industrial scale. L a CO2 The degree of increase is related to the apparent gas velocity w 0 g The degree of increase was the same. Furthermore, it was found that the mass transfer performance of carbon dioxide differed significantly at the laboratory scale compared to industrial-scale methods. However, unexpectedly, the power input of the volumetric stirrer had a significant impact on the laboratory scale but a very small impact on the industrial scale. From Figure 2 and Figure 3 It can be seen that, compared with laboratory-scale reactors, the volumetric mass transfer coefficient k of industrial-scale reactors is significantly higher. L a CO2 Up to 10 times lower.

[0018] To better illustrate the differences between laboratory-scale and industrial-scale experiments, refer to Figure 4 The figure shows the mass transfer coefficient k of carbon dioxide between laboratory and industrial scales. L a CO2 A comparison. Figure 4 This shows the relationship with a volumetric power input P / V = 21W / m. -3 The relative effect of specific power input on the volumetric carbon dioxide mass transfer coefficient compared to the measured volumetric mass transfer coefficient. The volumetric carbon dioxide mass transfer coefficient k between laboratory and industrial scales. L a CO2 In the comparison, it becomes apparent that, at the industrial scale, mass transfer performance does not significantly improve with increasing volumetric power input P / V, while at the laboratory scale, the mass transfer performance of CO2 increases from 21 W / m³. -3 Up to 168W m -3 This can increase the rate by up to 70%. Therefore, Figure 4 This demonstrates that in a laboratory-scale (2L system), k increases with increasing stirrer input. L a CO2 An increase of +70% was observed, while in industrial-scale (12000L system), k was observed. L a CO2 Only a +5% increase.

[0019] Therefore, it can be clearly seen that, based on Figures 2 to 4 For industrial-scale bioreactors or fermenters, the mass transfer performance of CO2 does not significantly improve with increasing volumetric power input (P / V), while at the laboratory scale, the mass transfer performance of CO2 can be improved by up to 70% (see [link to relevant documentation]). Figure 4 ).

[0020] The different behaviors of the two systems can be largely explained by the residence time in the gas phase within the systems, as explained above. On an industrial scale, the CO2 concentration equilibrium between the bubbles and the liquid is reached long before the bubbles reach the surface, whereas on a laboratory scale, the residence time is too short to reach equilibrium. Therefore, increasing the stirrer frequency and interfacial area leads to higher mass transfer coefficients on a laboratory scale, while on a large scale, stronger dispersion of “dead bubbles” (i.e., those with CO2 saturation) is useless.

[0021] In the case of oxygen mass transfer, further experiments have shown that equilibrium cannot be reached even on an industrial scale. Therefore, the higher the volumetric power input, the larger the interfacial area, and consequently the lower the volumetric mass transfer coefficient k. L a O2 The higher the flow rate, the higher the volumetric mass transfer coefficient of carbon dioxide. Therefore, it can be concluded that the volumetric mass transfer coefficient of carbon dioxide only increases significantly with higher gas flow rates, but not significantly with higher volumetric power inputs.

[0022] On an industrial scale, the difficulty in removing carbon dioxide is primarily related to the fact that the gas phase is already saturated with carbon dioxide just a short distance above or near the bottom of the submerged gas supply in a bioreactor or fermenter. Therefore, increasing the mass transfer performance of carbon dioxide is clearly the most feasible option, which is to increase the gas flow rate. However, this also often leads to an undesirable increase in the oxygen mass transfer rate, and consequently, makes it impossible to independently manage O2- and CO2- concentrations within the bioreactor or fermenter.

[0023] In the prior art, reactors with two bubblers are known and commercially available. For example, EP 0 099 634A2 describes a reactor apparatus for multiphase contact between gas, solid, and liquid phases, comprising a cylindrical container, an inlet tube, a conical bottom, and a gas bubbler system. A gas bubbler 16 is located at the lower end of the container and in the gap between the inner wall and the periphery of the conical surface, for allowing at least one gas to enter the continuous liquid phase in the form of bubbles, in which particulate solid phase is suspended within the container. An auxiliary gas bubbler (in the form of an annular bubbler 34) surrounds the inlet tube and is configured to radially eject gas into the liquid phase in the form of bubbles. EP 0 099 634 A2 makes no mention of the distance between the two bubblers.

[0024] WO 2002 / 33048 A1 discloses a method for culturing microorganisms under aerobic conditions in a fermentation vessel, comprising injecting a first oxygen-containing gas into the lower part of the vessel in a non-uniform flow (causing turbulent movement of the liquid culture medium), and introducing a second oxygen-containing gas into the vessel, characterized in that the second oxygen-containing gas is introduced as: a non-uniform flow of bubbles moving in all possible directions within the vessel, independent of the flow direction of the liquid culture medium, resulting in turbulent conditions at the injection point; and as a group of bubbles with a non-uniform size and a wide size distribution. The distance between the two bubblers is not mentioned and is not critical, as there are no restrictions on the inlet location of the second oxygen-containing flow, as described on page 4, I.20-24 of the specification.

[0025] In Sen Xu et al., “A practical approach in bioreactor scale-up and process transfer using a combination of constant P / V and vvm as the criterion”, Biotechnology Progress, Vol. 33, No. 4, 2017, pp. 1146-1159, bioreactor scale-up was evaluated as a key step in the production of therapeutic proteins such as monoclonal antibodies (MAbs). For example, bubbler k from a series of bioreactor scales (3-2000 L) with different bubblers was examined. L a and k L a CO2 (CO2 volumetric mass transfer coefficient). This relationship describes single-buffer and dual-buffer systems without disclosing any information about their geometry. Generally, in a dual-buffer system, the two bubblers are located at approximately the same position and not at different heights.

[0026] Furthermore, US 5,994,567 from the field of organic chemistry relates to instructions for oxygen-injected bubble-cap reactors, i.e., liquid-phase oxidation processes, wherein a first oxygen-containing gas is injected into the lower part of a bubble-cap reactor vessel containing an oxidizable organic liquid. At one or more points, a second oxygen-containing gas is further injected into the reactor, wherein the liquid has substantially depleted its dissolved oxygen prior to this injection. Oxygen from the first and second oxygen-containing gases is used to oxidize the organic liquid, such as cumene or cyclohexane. Therefore, instead of describing a stirred-tank bioreactor or fermenter for culturing cells or microorganisms, a chemical reaction is described, whereby the stripping of CO2 produced by the cultured living cells is not important.

[0027] The disclosure of WO 2008 / 088371 A2 relates to systems for containing and manipulating fluids, including systems and methods involving supporting collapsible bags that can be used as reactors for performing chemical, biochemical, and / or biological reactions. On one hand, fluid contained in a container can be sprayed, for example, such that the fluid is guided into a container's containment, and in some cases, the spray can be controlled as needed by rapidly activating or changing the spray intensity. It is mentioned that in some cases, multiple bubblers can be used. However, the document does not mention the specific geometric arrangement of these. The different bubblers 47 or 301 described are based on... Figure 1 Located at the same height at the bottom of the reactor, without any specific teachings on its physicochemical effects.

[0028] The possible location, design, and dimensions of the sparger relative to the agitator were evaluated and discussed in the following literature: Sardeing et al., “Gas-liquid mass transfer”, Chemical Engineering Research and Design, Elsevier, Amsterdam, NL, Vol. 82, No. 9, 2004, pp. 1161-1168; Birch et al., “The Influence of Sparger Design and Location on Gas Dispersion in Stirred Vessels”, Chemical Engineering Research and Design, Elsevier, Amsterdam, NL, Vol. 75, No. 5, 1997, pp. 487-496; and Rewatkar VB et al., “Role of sparger design on gas dispersion in mechanically agitated gas-liquid contactors”, Canadian Journal of Chemical Engineering, 1993, Vol. 71, No. 2, pp. 278-291. For evaluation purposes, only a single sparger was used. The simultaneous presence of two bubblers in the reactor and the distance between them are irrelevant and not mentioned.

[0029] Therefore, one object of the present invention is to overcome the deficiencies of the prior art and to provide a modified bioreactor or fermenter that allows for the management of carbon dioxide concentration independently of oxygen concentration within an aerated stirred bioreactor or fermenter on an industrial scale.

[0030] In addition, another objective is to provide a method for controlling cell culture or fermentation processes by independently managing carbon dioxide and oxygen concentrations within an industrial-scale aerated stirred bioreactor or fermenter. Summary of the Invention

[0031] Surprisingly, it was found that known drawbacks from existing technologies can be overcome, especially when a second gas supply (or optionally more gas supplies) is arranged within the bioreactor at a predetermined distance from the first gas supply, enabling independent management of O2- and CO2- concentrations in industrial-scale aerated stirred bioreactors or fermenters.

[0032] Therefore, to overcome the aforementioned drawbacks, a modified and thus improved bioreactor or fermenter is provided for culturing suspended cells or microorganisms in a liquid medium on an industrial scale. The bioreactor or fermenter 100 according to this disclosure for culturing suspended cells or microorganisms in a liquid medium on an industrial scale includes...

[0033] Container 102, which contains a culture in a liquid medium having a defined fill height;

[0034] A stirrer 120 is disposed in a container to stir a liquid medium;

[0035] A first bubbler 150, disposed in the bottom portion 105 of container 102, is configured to continuously supply bubbles 10, 10.1, 10.2, and 10.3 to the liquid medium, the gases being selected from air and / or oxygen; and

[0036] A second bubbler 160, disposed within container 102 and positioned above the first bubbler 150, continuously supplies additional air bubbles and / or additional oxygen bubbles 20, 20.1, 20.2, 20.3 to the liquid medium.

[0037] thus

[0038] The second bubbler 160 is located at a distance η above the first bubbler 150 in the bioreactor or fermenter 100, whereby η is selected to be within the range of approximately 0.4 m above at least the first bubbler 150 to approximately 0.5 m below the filling height of the bioreactor or fermenter 100, or

[0039] The first bubbler is located approximately 0.4m above the filling height of the bioreactor or fermenter 100, which is approximately 2 / 3 of the total filling height.

[0040] The first bubbler is located approximately 0.4m above the filling height of the bioreactor or fermenter 100, or approximately 1 / 2 of the filling height.

[0041] The distance may be approximately 0.4 m above the first bubbler to approximately 3.0 m above the first bubbler, or approximately 0.4 m to approximately 2.5 m, or approximately 0.4 m to approximately 2.0 m, or approximately 0.4 m to approximately 1.5 m, or approximately 0.4 m to approximately 1.0 m, or approximately 0.45 m to approximately 0.90 m, or approximately 0.5 m to approximately 0.80 m, or approximately 0.55 m to approximately 0.70 m, or at approximately 0.6 m.

[0042] Therefore, in order to improve the mass transfer performance of CO2 while ensuring that the mass transfer performance of O2 is not adversely affected, according to the invention, an additional second gas bubbler is positioned at a distance η above the first bubbler within the bioreactor or fermenter to achieve a much shorter residence time for the supplied additional gas (compared to the gas supplied from the submerged bubbler or the first bubbler). Due to the shorter residence time of the gas injected by the second bubbler, less oxygen is transferred to the liquid phase, while more and more CO2 can be stripped away.

[0043] According to an implementation, the second bubbler can be a side bubbler, that is, a bubbler that provides additional bubbles near the sidewall. Attached Figure Description

[0044] Embodiments of the prior art and the present invention will be described by way of example with reference to the accompanying drawings, which are schematic and not intended to be drawn to scale, so that no assumptions can be made about the precise geometric values ​​of the original dimensions. The drawings of this disclosure are incorporated in and form part of the specification, and illustrate embodiments of the invention, but are not limited to the specific embodiments described. The drawings, together with the general description and detailed description, serve to explain the principles of this disclosure. In all the drawings, the same features are indicated by the same reference numerals. In the drawings:

[0045] Figure 1 A schematic illustration of the processes and reactions involving the distribution of O2 and CO2 occurring in bubbles within a bioreactor or fermenter is shown.

[0046] Figure 2 This shows the results on a laboratory scale for different apparent gas velocities w. 0 g The volumetric mass transfer coefficient k of carbon dioxide L a CO2 Dependence on the power input P / V of the volumetric mixer;

[0047] Figure 3 This shows the effects of different apparent gas velocities w on an industrial scale. 0 g The volumetric mass transfer coefficient k of carbon dioxide L a CO2 Dependence on the power input P / V of the volumetric mixer;

[0048] Figure 4 It shows the basis Figure 2 and Figure 3 The obtained value is the mass transfer coefficient k of carbon dioxide between laboratory and industrial scales. L a CO2 Comparison;

[0049] Figure 5 The illustration shows a schematic depiction of the processes and reactions involving the distribution of O2 and CO2 in bubbles 10 within a bioreactor or fermenter, according to an exemplary embodiment of the present disclosure, with and without additional gas injection (part A) and with additional gas injection (part B; bubble 20).

[0050] Figure 6a The positions of a first bubbler 150 and a second bubbler 160 in a bioreactor or fermenter according to an exemplary embodiment of the present disclosure are illustrated.

[0051] Figure 6b The positions of the first bubbler 150, the second bubbler 160, and the third bubbler 170 in a bioreactor or fermenter according to an exemplary embodiment of the present disclosure are illustrated respectively.

[0052] Figure 7 Types of bubblers 150, 160, and 170 according to exemplary embodiments of the present disclosure are shown;

[0053] Figure 8 The embodiments (Example 1) have been explained and illustrate exemplary implementations according to this disclosure, from saturation c CO2 =14% to c CO2 =6% of k L a CO2 Descriptive evaluation of the value;

[0054] Figure 9 The embodiments (Example 2) are explained and show technical drawings of a vertical cross-section schematic diagram of an industrial-scale bioreactor according to an exemplary embodiment of the present disclosure and the installation location of an additional second bubbler (160).

[0055] Figure 10 The embodiments (Examples 2 and 3) are explained and illustrate how, according to the prior art and exemplary implementations of this disclosure, the oxygen mass transfer coefficient (k) can be determined. L a O2 A comparison of four measurements performed in relation to the gas flow rate;

[0056] Figure 11The embodiments (Example 3) have been explained, and illustrate how, according to the prior art and exemplary implementations, the mass transfer coefficient (k) of carbon dioxide can be determined. L a CO2 A comparison of four measurements performed in relation to the gas flow rate;

[0057] Figure 12a The embodiments (Example 3) are explained and illustrate the mass transfer coefficient (k) for oxygen determined according to this disclosure. L a O2 Influence factor C O2 ;

[0058] Figure 12b The embodiments (Example 3) are explained and illustrate the mass transfer coefficient (k) for carbon dioxide determined according to this disclosure. L a CO2 Influence factor C CO2 ;

[0059] Figure 13a The embodiments (Example 4) explain and illustrate how the oxygen mass transfer coefficient (k) is determined using side-buffer type A or side-buffer type B, according to the prior art and exemplary embodiments of this disclosure. L a O2 A comparison of four measurements performed in relation to the gas flow rate;

[0060] Figure 13b The embodiments (Example 4) explain and illustrate how the mass transfer coefficient (k) of carbon dioxide is determined using side-buffer type A or side-buffer type B, according to the prior art and exemplary embodiments of this disclosure. L a CO2 A comparison of four measurements performed in relation to the gas flow rate;

[0061] Figure 14a The explanation is provided in the Examples section (Example 5), and the method for determining the mass transfer coefficient (k) of carbon dioxide is shown. L a CO2 Measurements performed based on the dependence of stirrer frequency and submerged gas flow rate;

[0062] Figure 14b The explanation is provided in the Examples section (Example 5), and the determination of the oxygen mass transfer coefficient (k) is shown. L a O2 Measurements performed based on the dependence of stirrer frequency and submerged gas flow rate;

[0063] Figure 15a The explanation is provided in the Examples section (Example 5), and the method for determining the mass transfer coefficient (k) of carbon dioxide is shown.L a CO2 Measurements performed based on the dependence of stirrer frequency and side gas flow rate;

[0064] Figure 15b The explanation is provided in the Examples section (Example 5), and the determination of the mass transfer coefficient (k) of oxygen is shown. L a O2 Measurements performed based on the dependence of stirrer frequency and side gas flow rate;

[0065] Figure 16a The step response method, particularly the system input, is explained in the embodiments section (Example 6).

[0066] Figure 16b The step response method, particularly the system output, is explained in the embodiments section (Example 6).

[0067] Figure 17 The embodiments (Example 6) are explained and illustrated by way of example a bubble trap (funnel) 180 used in step response measurement;

[0068] Figure 18 The examples are explained in the Examples section (Example 6), and typical input and output signals from a step response method applied to a 12kL aerated stirred tank reactor are shown.

[0069] Figure 19 The examples are explained in the Examples section (Example 6), and a comparison of the step response at bubble trap (funnel) 180 caused by 100% and 50% input steps is shown.

[0070] Figure 20 Explained in the Examples section (Example 6), the results of measurements of the gas phase residence time in a laboratory-scale bioreactor or fermenter (30L) compared to an industrial-scale bioreactor or fermenter (12000L) are shown; and

[0071] Figure 21 The results of culturing antibody derivatives for 11 days in a conventional feed-batch process in a commercially available 12,000 L bioreactor (therefore, the bioreactor consists of only one bubbler) are explained in the Examples section (Comparative Example 1) and are shown.

[0072] Detailed illustrations of some figures are provided at the end of this instruction manual. Detailed Implementation

[0073] Terms not specifically defined herein should be given the meaning that would be given to them by those skilled in the art in light of this disclosure and the context.

[0074] A "bioreactor" is an apparatus or instrument in which living organisms, particularly bacteria and eukaryotic cells, grow and / or synthesize useful substances, thereby consuming nutrients in a culture medium and, in the case of aerobic cells or microorganisms, consuming O2 provided by technical devices (such as bubblers). In this disclosure, the bioreactor is an industrial-scale bioreactor. A bioreactor may consist of or include a biocompatible container in which chemical or biochemical methods involving organisms and / or biochemically active substances derived from these organisms are performed. Bioreactors utilize additional equipment, such as stirrers, baffles, one or more bubblers (as is the subject of this invention), and / or ports, which in particular allow for the culture and propagation of cells. Generally, bioreactors are in the form of a cylindrical tube with two ends forming the top and bottom of the bioreactor. Bioreactor sizes range from liters to cubic meters and are often made of stainless steel. The bioreactors according to this disclosure are used in large-scale production.

[0075] Cultured cells, especially eukaryotic cells (such as Chinese hamster ovary (CHO) or yeast cells), are used, for example, to produce antibodies, such as monoclonal antibodies, and / or recombinant proteins, such as recombinant proteins for therapeutic purposes. Alternatively, cells may produce, for example, peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites, or any other useful substances.

[0076] A "fermenter" is a device or apparatus in which microorganisms synthesize useful substances, thereby maintaining suitable growth conditions for the microorganisms. The details of the bioreactor mentioned above are applied with necessary modifications. The fermenter disclosed herein is used in large-scale fermentation. Known commercial products of large-scale fermenters include, for example, antibiotics, antibodies, hormones, or enzymes synthesized by these cells or microorganisms.

[0077] The generated microorganisms can be used for various purposes, such as wastewater treatment, food production in the food industry, the manufacture of pharmaceuticals (such as antibiotics or insulin) in the biotechnology sector, pest control, or the biodegradation of waste, pollutants (such as oil).

[0078] In this disclosure, the terms "industrial scale" and "large-scale" are used interchangeably and synonymously, and refer to articles produced in large quantities, whereby cost advantages often exist, with the cost per unit output decreasing as scale increases. All other things being equal, large manufacturing units are expected to have a lower cost per unit output than smaller units. In the context of cell culture, industrial scale can be understood as having a bioreactor volume equal to or greater than about 2000 L. In the context of microbial culture, industrial scale can be understood as having a fermenter volume equal to or greater than about 1000 L. According to another embodiment, the volume of the bioreactor or fermenter used for industrial scale can be equal to or greater than 6000 L, 8000 L, 10000 L, 12000 L, 15000 L, or even more.

[0079] A "stirrer" is an object or mechanical device used for stirring, such as a magnetic stirrer. Any kind of stirrer commonly used for the culture of cells or microorganisms can be used. Possible stirrers include, for example, impellers, Rushton turbines, impellers, blade stirrers such as swashplate stirrers, etc.

[0080] A "bubbler" is a gas supply or feed device used in a bioreactor or fermenter that provides oxygen and / or air bubbles into the liquid phase and their presence in the liquid phase in which cells or microorganisms are cultured. Bioreactors or fermenters of the prior art typically have only one bubbler, which is often located on or near its bottom. In this disclosure, this bubbler is also designated as a "first bubbler" or a "submerged bubbler," and these two expressions are used interchangeably and synonymously.

[0081] According to this disclosure, it has been found that providing an additional bubbler (i.e., a second bubbler positioned at a predetermined distance η above the first bubbler and continuously supplying additional air bubbles and / or additional oxygen bubbles to the liquid medium) has a number of advantages for the culture process, which will be apparent to those skilled in the art from the foregoing and following description.

[0082] The processes and reactions that occur in the bioreactor or fermenter according to this disclosure are described in more detail below.

[0083] In a bioreactor or fermenter with only one gas supply device (often located at the bottom or lower part of the bioreactor or fermenter), the bubbles entering the liquid phase have the ability to absorb CO2 from the cell culture medium, but this ability is gradually lost as the bubbles rise. At the top or upper part of the bioreactor or fermenter, the absorption of dissolved CO2 from the liquid phase by bubbles no longer occurs, depending on the height explained above. Therefore, the CO2 content increases from the bottom to the top of the bioreactor or fermenter, resulting in a CO2 gradient within the liquid phase.

[0084] The CO2 gradient that is normally present in the liquid phase of a bioreactor or fermenter, and the disadvantages associated with this non-uniform distribution of CO2, can be overcome by this disclosure, which proposes that a second bubbler be positioned above the first bubbler in the liquid phase of a large-scale bioreactor or fermenter, spaced apart from the first bubbler by a distance η. The second bubbler is configured to counteract CO2 saturation caused by the formation of bubbles that can no longer absorb CO2. The second bubbler adds new bubbles into the liquid phase, allowing the process of crossing O2 and CO2 to be additionally repeated above the first bubbler.

[0085] The processes and reactions discussed herein occur in the liquid phase of a large-scale bioreactor or fermenter, as illustrated in the illustrative manner. Figure 5 It is shown schematically in the middle. Figure 5 Part A on the left illustrates the distribution of dissolved CO2 in a schematic bioreactor or fermenter (with only one gas supply device or bubbler, near the stirrer (not shown)). The bubbler is located at or near the bottom of the bioreactor or fermenter. Standard aeration with only one bubbler clearly results in a CO2 gradient. After reaching a certain height, the bubbles 10.2 are saturated with CO2. Therefore, in Figure 5 In the lower part (part A), CO2 stripping performance is good, while in the upper part, CO2 stripping is poor and unacceptable.

[0086] Figure 5 Part B on the right illustrates the distribution of dissolved CO2 in a schematic bioreactor or fermenter on a large scale, as a result of an additional second gas supply device or second bubbler positioned above the first bubbler. As can be seen from... Figure 5 Derived from part B, the second bubbler provides bubble 20.1, which begins its rise to the surface in the liquid medium. As shown in bubble 20.2, O2 gas crosses from bubble 20.2 into the liquid phase (reaction (1)), while CO2 gas crosses from the liquid phase into bubble 20.2 (reaction (2)). Thus, due to the different Henry's constants, the rate of reaction (2) is much faster than that of reaction (1) (its rate in... Figure 5 (This is illustrated by arrows of varying thicknesses). Therefore, the stripping of CO2 from the middle and upper parts of the liquid phase in part B is similar to that in part A, as... Figure 5 The left side shows CO2 stripping in the lower part of the liquid phase. This avoids a CO2 gradient throughout the liquid medium. Cells or microorganisms present in the bioreactor or fermenter will experience a more consistent environment and will be subject to less fluctuation in the liquid medium (which can be reflected in the metabolic reactions of the cells or microorganisms).

[0087] The second bubbler is positioned above the first bubbler to shorten the absolute rise height of the bubbles, thus preventing them from becoming saturated with CO2 during their ascent through the liquid phase. Furthermore, the reduced residence time of bubbles originating from the second bubbler in the liquid phase allows them to rise to the liquid surface relatively quickly because they are not dispersed by a stirrer. Therefore, CO2 stripping performance is also good in the upper part of a bioreactor or fermenter.

[0088] According to another embodiment, a third and additional bubblers may also exist above the second bubbler, such that three, four, or more bubblers are present simultaneously in the bioreactor or fermenter. The above explanation of the second bubbler applies accordingly to the third, fourth, fifth, and additional bubblers.

[0089] Therefore, large-scale bioreactors or fermenters are subdivided into different sub-compartments, each containing a bubbler to directly influence the CO2 distribution in the liquid phase. This "separation" of large-scale bioreactors or fermenters into smaller units improves comparability and, consequently, the predictability of industrial-scale cultivation relative to laboratory scale, and vice versa, advantageously with increased metabolic rates, activity, and / or productivity.

[0090] Despite the use of large volumes, the inclusion of a second or optional third bubbler and additional bubblers within bioreactors or fermenters operating at large production scales can affect the coordination of the physicochemical environmental conditions of the cells or microorganisms being cultured. Therefore, this concept is a method for adapting industrial-scale processes to laboratory-scale processes, where CO2 distribution is more uniform throughout the liquid phase.

[0091] Furthermore, the presence of a second or optional third and additional bubbler can reduce the dissolved CO2 concentration in the liquid phase to a suitable level. It should be remembered that CO2 concentration affects cell culture performance, especially at higher concentrations; thus, high CO2 concentrations inhibit the growth of aerobic cells (see David R. Gray et al., CO2 in large-scale and high-density CHO cell perfusion culture, Cytotechnology 1996, 22, 65-78). High CO2 concentrations in the cell culture medium itself must be avoided. Second and additional third or additional bubblers help prevent this undesirable high CO2 content in the liquid phase.

[0092] In addition, the addition of a second, third, and other bubblers (which continuously supply additional air bubbles and / or additional oxygen bubbles to the liquid medium) also results in a more uniform distribution of O2 throughout the liquid phase.

[0093] Therefore, according to another embodiment, there may be one or more additional bubblers disposed above the first bubbler, the second bubbler and the third bubbler.

[0094] It has been found that, taking into account the management of O2- and CO2- concentrations in the liquid phase, the location of the second bubbler and additional bubblers can have a positive impact on the performance and efficiency of the culture.

[0095] Assuming the first bubbler is located at or near the bottom or lower part of the bioreactor or fermenter, the second bubbler is always located above the first bubbler, for example, in the middle or upper part of the bioreactor or fermenter. To further verify the location of the second bubbler, there are essentially two main directions in which its position can be altered within a large-scale bioreactor or fermenter. One main direction is the vertical direction, i.e., altering the bubbler's position from the bottom to the top of the bioreactor or fermenter. That is, the second bubbler can be positioned, for example, closer to the bottom of the bioreactor or fermenter, or closer to the top of the bioreactor or fermenter, or at any distance in between. In this respect, the fill height of the bioreactor or fermenter must be considered rather than its absolute volume, since the bubbles will be supplied within the existing liquid phase.

[0096] According to embodiments of the present invention, the bioreactor or fermenter includes a filling height in the following ranges: from about 8m to about 20m, or about 9m to about 15m, or about 9.5m to about 12m or about 10m.

[0097] The second primary direction to consider is the horizontal direction, i.e., the location of the bubbler between the sidewalls and the central axis of the bioreactor or fermenter. The central axis is a dashed line within the bioreactor or fermenter, presumably having a cylindrical geometry and whose distances to the surrounding sidewalls are everywhere equal or nearly everywhere equal. That is, the second bubbler can be located, for example, closer to the sidewalls or closer to the central axis of the bioreactor or fermenter, or at any distance in between.

[0098] According to another embodiment, the continuous bubblers can be precisely positioned one on top of the other in the vertical direction. According to another embodiment, the continuous bubblers can also be positioned laterally relative to each other with respect to the vertical direction.

[0099] Therefore, according to the present invention, the second bubbler is located inside the container of the bioreactor or fermenter at a distance η from the first bubbler. The distance η should be understood as a vertical distance, for example, along the sidewall of the bioreactor or fermenter, such that the second bubbler is located above the first bubbler at a distance η. The distance η is selected to be within the range of approximately 0.4 m above at least the first bubbler to approximately 0.5 m below the filling height of at most the bioreactor or fermenter, or...

[0100] The first bubbler is located approximately 0.4m above the top of the reactor to about 2 / 3 of the filling height of the bioreactor or fermenter.

[0101] The height of the first bubbler is approximately 0.4m above the top of the bioreactor or fermenter, up to approximately half the filling height.

[0102] Approximately 0.4m to approximately 3.0m, or approximately 0.4m to approximately 2.5m, or approximately 0.4m to approximately 2.0m, or approximately 0.4m to approximately 1.5m, or

[0103] At approximately 0.4m to approximately 1.0m above the first bubbler, or approximately 0.45m to approximately 0.90m, or approximately 0.5m to approximately 0.80m, or approximately 0.55m to approximately 0.70m, or at approximately 0.6m.

[0104] Therefore, the distance η between the first and second bubblers has a lower limit of approximately 0.4 m above the first bubbler and an upper limit of approximately 0.5 m below the fill height of the bioreactor or fermenter. The term "fill height" (used synonymously with "liquid height") should be understood to mean the level of liquid present in the bioreactor or fermenter at the start of the cultivation process, further defined by the surface of the liquid or the nominal volume of the liquid present. Therefore, 0.5 m below the fill height of the bioreactor or fermenter is synonymous with 0.5 m below the surface of the liquid present in the bioreactor or fermenter at the start of the cultivation process.

[0105] If, for example, the total fill height is 10m, then 0.5m below the fill height is 9.5m. Then, a distance η is selected within the range of approximately 0.4m to approximately 9.5m. The lower and upper limits of this range are considered the critical values ​​of the present invention.

[0106] The presence of two bubblers in a bioreactor inherently increases the area in the liquid medium where CO2 stripping will occur. If the second bubbler is placed near the surface of the liquid, for example, about 0.5 m below the fill height of the bioreactor or fermenter, it can strip the downstream area, while the first bubbler, placed near the bottom of the bioreactor, can strip the upstream area of ​​the liquid medium. In summary, the entire fill height of the bioreactor or fermenter is subject to CO2 stripping.

[0107] The distance η can be selected within the range of approximately 0.4 m above the first bubbler to approximately 2 / 3 of the filling height of the bioreactor or fermenter. The statement "approximately 2 / 3 of the filling height of the bioreactor or fermenter" should be understood to mean that the second bubbler is located at approximately 2 / 3 of the total filling height. For example, if the total filling height is 12 m, then 2 / 3 of the total filling height is 8.0 m. The distance η is then selected within the range of approximately 0.4 m to approximately 8.0 m.

[0108] The distance η can also be selected as a range from approximately 0.4 m above the first bubbler to approximately half the filling height of the bioreactor or fermenter. The statement "approximately half the filling height of the bioreactor or fermenter" should be understood to mean that the second bubbler is located at approximately half the total filling height or approximately 0.5 times the liquid volume. For example, if the total filling height is 11 m, then half the total filling height is 5.5 m. Then, the distance η is selected within the range of approximately 0.4 m to approximately 5.5 m.

[0109] The distance η can also be selected to be within the following ranges: from about 0.4m to about 3.0m, or from about 0.4m to about 2.5m, or from about 0.4m to about 2.0m, or from about 0.4m to about 1.5m, or from about 0.4m to about 1.0m, or from about 0.45m to about 0.90m, or from about 0.5m to about 0.80m, or from about 0.55m to about 0.70m, or at about 0.6m. Therefore, the distance η can be selected as approximately 3.0m, 2.9m, 2.8m, 2.7m, 2.6m, 2.5m, 2.4m, 2.3m, 2.2m, 2.1m, 2.0m, 1.9m, 1.8m, 1.7m, 1.6m, 1.5m, 1.4m, 1.3m, 1.2m, 1.1m, 1.0m, 0.95m, 0.90m, 0.85m, 0.80m, 0.75m, 0.70m, 0.65m, 0.6m, 0.55m, 0.45m, and 0.4m above the first bubbler, respectively.

[0110] In another embodiment, the distance η can be selected to be within the range of approximately 0.6 m above at least the first bubbler to approximately 0.5 m below the filling height of at most the bioreactor or fermenter, or

[0111] The first bubbler is located approximately 0.6m above the filling height of the bioreactor or fermenter, or approximately 2 / 3 of the filling height.

[0112] The first bubbler is located approximately 0.6m above the filling height of the bioreactor or fermenter, or approximately 1 / 2 of the filling height.

[0113] The distance above the first bubbler is approximately 0.6m to approximately 3.0m, or approximately 0.6m to approximately 2.5m, or approximately 0.6m to approximately 2.0m, or approximately 0.6m to approximately 1.5m.

[0114] The distance is approximately 0.6m to approximately 1.0m above the first bubbler, or approximately 0.6m to approximately 0.90m above the first bubbler, or approximately 0.6m to approximately 0.80m above the first bubbler, or approximately 0.6m to approximately 0.70m above the first bubbler, or approximately 0.6m above the first bubbler.

[0115] The term “about” followed by a value should be understood to mean: the value ±5%, or the value ±4%, or the value ±3%, or the value ±2%, or the value ±1%.

[0116] To determine the distance η, the location of the gas outlet opening of the bubbler (i.e., the opening through which the bubbles enter the liquid phase) is, of course, a key criterion. If there are several openings in a bubbler, the appropriate distance can be determined using the average value.

[0117] The range and value of the distance η mentioned above are the result of various experiments, calculations and evaluations performed. Based on these experiments, calculations and evaluations, the second bubbler is set in the bioreactor or fermenter at a distance from the first bubbler or at a height above the first bubbler, at which the bubbles have reached or will soon reach the CO2 gas phase saturation concentration.

[0118] The CO2 gas phase saturation concentration at the height of an industrial-scale aerated stirred bioreactor or fermenter has been evaluated based on the following considerations:

[0119] Based on our experiments (see...) Figures 2 to 4 The volumetric mass transfer coefficient k of carbon dioxide in a 12000L industrial-scale aerated stirred bioreactor or fermenter. L a CO2 The volumetric mass transfer coefficient k of carbon dioxide in laboratory-scale (2L) L a CO2 Approximately ten times lower. Conversely, the volumetric oxygen mass transfer coefficient k of both systems... L a O2 The quantities are comparable.

[0120] As is known and in contrast to oxygen mass transfer, the mass transfer of carbon dioxide from the continuous liquid phase to the gas phase is completed after a short period. If the gas phase residence time in the system is longer than this period, until bubble saturation occurs, these bubbles are no longer available for CO2 mass transfer. Therefore, this effect only occurs in industrial-scale reactors, not in laboratory-scale reactors.

[0121] To evaluate the specific spatial boundary interface and CO2 mass transfer coefficient k during the evaluation period (after which, in an industrial-scale system (e.g., 12000L) during operation, bubbles are saturated with CO2, and the bubbles no longer contribute to CO2 stripping), the following parameters were considered. L a CO2 It must be taken into account.

[0122] Therefore, the residence time distribution of the gas phase is first assessed as follows:

[0123] The gas phase residence time was determined using a step response method (“Sprungantwortmethode”), as explained in detail in the Experimental Section. Detailed results are given in the Experimental Section. The step response method is based on the use of two different gases: one gas (such as oxygen) is used to saturate the liquid (such as water), and another gas (such as carbon dioxide or nitrogen) is used to replace it and expel it from the liquid phase. The gas is added from the bottom of the bioreactor or fermenter, and the expelled gas is measured near the top of the reactor within the liquid phase. This method for measuring gas phase residence time is familiar to those skilled in the art.

[0124] Therefore, using the step function response method, the gas phase residence time of a laboratory-scale (30L) bioreactor or fermenter has been determined to be 5s, and the gas phase residence time of an industrial-scale (12000L) bioreactor or fermenter has been found to be 21s.

[0125] Then, based on further experimental measurements and evaluations described in the experimental section, the CO2 mass transfer coefficient was confirmed to be 4 ± 0.68 h⁻¹ in a laboratory-scale bioreactor or fermenter (2 L). -1 .

[0126] Under the assumption of a monodisperse bubble size distribution of d = 5 mm in a 12000 L system, the theoretical carbon dioxide profile in a single bubble can be calculated. This is based on the carbon dioxide mass transfer coefficient k... L a CO2 =4±0.68h -1 Also applicable under the assumption of industrial scale, the concentration profile of CO2 in bubbles can be calculated as follows:

[0127]

[0128] From the experiment shown in Example 6 (by Figure 20 (Note) It can be inferred that approximately 95% bubble saturation can be observed after about 3.5 seconds.

[0129] The average gas phase residence time, measured in industrial-scale bioreactors or fermenters according to the same embodiment, is approximately 21 s. Figure 20The total distance traveled by the bubbles was determined to be 3.6 m, and the average bubble rising velocity (velocity = distance / time) was determined to be 0.17 m / s. Therefore, after a height of approximately h = 0.6 m (3.5 s × 0.17 m / s), the gas phase was saturated with CO2, and thus CO2 stripping could no longer be observed.

[0130] Because the above assessments, measurements, and calculations include some evaluations and estimates, the obtained result of 0.6m is only an approximation of the distance η, which is better represented by the range of approximately 0.4m above at least the first bubbler to approximately 0.5m below the filling height of at most the bioreactor or fermenter.

[0131] Furthermore, experiments have shown that the presence of a second bubbler positioned at a distance η has particular advantages for culturing suspended cells or microorganisms in liquid media on an industrial scale. It is anticipated that the presence of the second bubbler (located above the first bubbler at a distance η, where η is selected within the aforementioned range) will have a positive effect on CO2 stripping. The presence of the second bubbler can be assumed to result in a reduction in the partial pressure of CO2 in the culture (liquid medium) of the bioreactor or fermenter. That is, when comparing two bioreactors / fermenters, thus two operating under the same conditions with the same liquid medium and respectively culturing the same cells or microorganisms, the only difference between the two bioreactors / fermenters is the use of one bubbler in one bioreactor / fermenter (and thus reflecting the current state of the technology), and the use of two bubblers positioned at a distance η in another bioreactor / fermenter according to the invention. It is then anticipated that, compared to a bioreactor / fermenter with one bubbler, the partial pressure of CO2 in a bioreactor / fermenter with two bubblers will be reduced by at least about 0.5%, or at least about 1%, or at least about 2% up to about 20%. The degree of decrease in the partial pressure of CO2 can be based on experiments performed using a bubbler (see Control Example 1 and...). Figure 21 The O2 and CO2 content in the lower, middle, and upper parts of the bioreactor or fermenter was estimated by combining evaluative measurements (in which two bubblers were used (see Examples 2 to 6), which allowed conclusions to be drawn about the content of O2 and CO2 present in the lower, middle, and upper parts of the bioreactor or fermenter).

[0132] Furthermore, if a second bubbler is present (as according to the invention), higher product titers and higher product yields can be expected compared to a bioreactor or fermenter using only one bubbler. The produced product titers and yields can be estimated to be at least about 1%, or at least about 5%, or at least about 10% up to about 30% higher than in the same bioreactor or fermenter operating under the same conditions using only one bubbler. The extent of the product titer or yield can be based on experiments performed using one bubbler (see Comparative Example 1 and...). Figure 21This is combined with evaluative measurements (where two bubblers have been used (see Examples 2 to 6), which allows conclusions to be drawn regarding the content of O2 and CO2 present in the lower, middle, and upper parts of the bioreactor or fermenter) to estimate. It should be noted that even a small improvement in a method used commercially on a large scale represents a technical problem worth solving. Assuming a representative large-scale bioreactor has a total volume of 10,000 L or more, with millions of, for example, protein-producing cells per ml, even a small improvement in yield or other industrial characteristics implies a highly relevant improvement in large-scale production and must be considered important.

[0133] Therefore, according to the present invention, it has been found that selecting a distance η within one or more ranges has significant advantages and technical effects or benefits, especially the reduction of CO2 partial pressure in the culture (liquid medium) of the bioreactor and the increase of product titer will occur respectively.

[0134] The presence of two bubblers in the bioreactor or fermenter according to the invention increases the total area in the liquid medium where CO2 stripping will occur. The second bubbler can be placed at a distance η, whereby η can be selected such that the areas of CO2 stripping performed by the first and second bubblers overlap to a certain extent. On the one hand, it is anticipated that increasing the degree of overlap of the bubbler areas will significantly enhance the beneficial effects. On the other hand, if the two bubblers are too close, such as a distance η less than 0.4 m or less than 0.3 m or even smaller, the second bubbler will most likely provide increasingly smaller additional effects on the first bubbler.

[0135] If the distance η is chosen to be within the range of approximately 0.4 m above the first bubbler to approximately 2 / 3 of the filling height of the bioreactor or fermenter, the advantageous technical effects are expected to become more pronounced.

[0136] If the distance η is chosen to be within the range of approximately 0.4 m above the first bubbler to approximately half the filling height of the bioreactor or fermenter, the advantageous technical effects are expected to become more pronounced.

[0137] If the distance η is selected to be within the following range: from about 0.4m to about 3.0m above the first bubbler, or more preferably from about 0.4m to about 2.5m above the first bubbler, or from about 0.4m to about 2.0m above the first bubbler, or from about 0.4m to about 1.5m above the first bubbler, or most preferably from about 0.4m to about 1.0m above the first bubbler, or from about 0.45m to about 0.90m above the first bubbler, or from about 0.5m to about 0.80m above the first bubbler, or from about 0.55m to about 0.70m above the first bubbler, or at about 0.6m above the first bubbler, it is expected that the advantageous technical effect will become particularly strong.

[0138] Therefore, the distances η between the first bubbler, the second bubbler, and all other bubblers (if present) are chosen to be within one or more ranges as described above.

[0139] Therefore, if several bubblers exist, in one embodiment, the second bubbler can be located at a distance η from the first bubbler, ranging from 0.4m to 10m. 1,2 The third bubbler can be located at a distance η from 0.4m to 10m above the second bubbler. 2,3 In order to distinguish between different distances, the distance η between the first bubbler and the second bubbler is specified as η. 1,2 The distance between the second and third bubblers is specified as η. 2,3 wait.

[0140] Therefore, the distance between two consecutive bubblers, i.e., one arranged above the other, can be chosen as η.

[0141] The distance η can be the same or different among all the bubblers that exist, but it is always selected from the publicly disclosed range.

[0142] To better understand, the following example is provided:

[0143] The bioreactor or fermenter comprises a container with a filling height of 10 m. A first bubbler is located near the bottom of the bioreactor or fermenter. A second bubbler may be located within the bioreactor of the fermenter. The distance η between the first and second bubblers (also specified as η) is... 1,2 At point ), therefore, the distance from η 1,2 The third bubbler is selected to be spaced approximately 0.6 m from the first bubbler (defined as the distance between the openings of the two bubblers, where bubbles enter the liquid phase). The distance between the second and third bubblers is η. 2,3 At the location. In this example, η 2,3 This is equivalent to 0.6m. That is, the second bubbler is located 0.6m above the first bubbler, and the third bubbler is located 0.6m above the second bubbler. Therefore, the third bubbler is located 2×η above the first bubbler. Therefore, η between the bubblers... 1,2 and η 2,3 Equally large. Other bubblers may exist that can have η. 3,4 η 4,5 η 5,6 The distance between , ...

[0144] However, it is not required that all distances have the same value, but they can be chosen independently from the range published above. In other words, η 1,2 η2,3 η 3,4 η 4,5 η 5,6 ... can be chosen independently of each other and can be the same or different.

[0145] According to the second main direction, the second bubbler may be located closer to the side wall of the bioreactor or fermenter or closer to the central axis of the bioreactor or fermenter, so that air bubbles and / or oxygen bubbles provided from the second bubbler enter the liquid phase, closer to the side wall or closer to the central axis of the bioreactor or fermenter.

[0146] According to another embodiment, the second bubbler may also be located at a position where the bubbler has the same distance to both the sidewall and the central axis of the bioreactor or fermenter.

[0147] Therefore, according to one embodiment, one or more bubblers may be central bubblers or side bubblers.

[0148] In this disclosure, "central bubbler" must be understood as meaning that, in a sense, the central bubbler is designed in such a way that air bubbles and / or oxygen bubbles supplied from the bubbler enter the liquid phase closer to the central axis in the horizontal direction, rather than closer to the sidewall of the bioreactor or fermenter.

[0149] In this disclosure, "side bubbler" must be understood as a bubbler designed in a manner that allows air bubbles and / or oxygen bubbles to be provided more closely to the sidewalls in the horizontal direction, rather than closer to the central axis of the bioreactor or fermenter.

[0150] According to one embodiment, the first bubbler can be a central bubbler or a side bubbler.

[0151] According to another embodiment, the first bubbler can be a central bubbler, and the second bubbler can also be a central bubbler.

[0152] According to another embodiment, the first bubbler can be a central bubbler, and the second bubbler can be a side bubbler.

[0153] According to another embodiment, the first bubbler and the second bubbler may be side bubblers, respectively.

[0154] According to one embodiment, the optional third bubbler can be a central bubbler or a side bubbler.

[0155] According to another embodiment, the first bubbler can be a central bubbler, the second bubbler can be a central bubbler, and an optional third bubbler can be a central bubbler.

[0156] According to another embodiment, the first bubbler can be a central bubbler, the second bubbler can be a side bubbler, and the third bubbler can be a side bubbler.

[0157] According to another embodiment, the first bubbler can be a central bubbler, and all other bubblers can be side bubblers.

[0158] According to another embodiment, the first bubbler can be a side bubbler, and all other bubblers can be side bubblers.

[0159] In one embodiment, it has been found that if the second bubbler is a side bubbler, the advantageous technical effects disclosed herein can be significantly increased.

[0160] According to another embodiment, the second bubbler can be constructed in such a way that its opening points downward, i.e., to provide bubbles toward the bottom of the bioreactor or fermenter.

[0161] The total number of bubblers in a bioreactor or fermenter can be selected as needed and depends on the existing fill height. Bubblers may be present throughout the entire fill height of the bioreactor or fermenter or only on a portion thereof. The number of bubblers used depends on the type of cells or microorganisms selected, the size of the bioreactor or fermenter, the culture conditions, etc. Based on the explanations and statements disclosed herein, those skilled in the art can readily select an appropriate number of bubblers for any culture system used.

[0162] According to another embodiment, it has been found advantageous that the position of the agitator in the bioreactor or fermenter relative to the first and / or second bubbler and optionally additional bubblers is taken into account. Although the agitator used is not limited in any way, any agitator selected has an agitator radius r. s The agitator radius is positioned around a central axis A passing through the bioreactor or fermenter. Numerous experiments have shown that when the first bubbler is positioned at a distance from the central axis A of the bioreactor or fermenter, this distance results in bubbles produced at a radius equal to or less than the agitator radius r. s It is advantageous to introduce the liquid medium at a distance from the agitator. In this respect, it is irrelevant whether the first bubbler is a central bubbler or a side bubbler. In this case, the bubbler provides bubbles near the agitator, thus providing maximum turbulence at the agitator blade tips. This type of high-energy mixing of liquid and gas is considered advantageous in the culture of cells or microorganisms.

[0163] Further experiments have shown that, preferably, a second bubbler and optionally an additional bubbler are installed at a distance r from the center of the stirrer. sA greater distance ensures that the provided bubbles are on the outer side, or significantly outside the direction of the stirrer's movement. That is, the second bubbler and optional additional bubblers are arranged at a distance A from the central axis, a distance such that the provided bubbles are at a distance greater than the stirrer radius r. s The gas enters the liquid phase at a distance from the agitator. In this respect, it is irrelevant whether the second bubbler (and other bubblers) is a central bubbler or a side bubbler. In the current case, the second bubbler provides bubbles spaced apart from the agitator, thus avoiding turbulence that could deform or damage the provided bubbles. Therefore, high-energy mixing of the liquid and gas is considered detrimental to the effectiveness of the second bubbler and optional other bubblers, as beneficial technical effects (such as CO2 stripping performance and product yield) can be negatively affected.

[0164] exist Figure 6a The location of a first bubbler 150 and a second bubbler 160 within the vessel 102 of a bioreactor or fermenter 100 is illustrated, by way of example. The bioreactor or fermenter 100 has a bottom 105 and sidewalls 110, and a diameter D. A gas supply pipe 140 or several gas supply pipes (not shown) are arranged near the sidewalls 110, designed and installed in a manner to form the first bubbler 150 and the second bubbler 160. The first bubbler 150 is located at or near the bottom 105 of the lower portion of the bioreactor or fermenter 100. In the illustrated embodiment, the first bubbler 150 is a central bubbler, meaning that bubbles enter the liquid phase closer to the central axis A in the horizontal direction than closer to the sidewalls 110. However, it is also possible that the first bubbler 150 could be a side bubbler as already explained.

[0165] The second bubbler 160 is disposed above the first bubbler 150. In the illustrated embodiment, the second bubbler 160 is a side bubbler, meaning that the bubbles entering the liquid phase are located closer to the sidewall 110 of the bioreactor or fermenter 100 in the horizontal direction compared to their distance from the central axis A. However, it is also possible that the second bubbler 160 can be a central bubbler as already explained.

[0166] Figure 7 A schematic diagram of an exemplary type of bubbler is shown, which can be used as a side bubbler 150, 160, 170. Of course, other useful bubbler types are also commercially available and usable.

[0167] exist Figure 6a In the bioreactor or fermenter 100, the second bubbler 160 is located above the first bubbler 150 at a height or distance η. 1,2 Location. Distance η 1,2Selected to be within the following range: at least about 0.4 m above the first bubbler 150 to about 0.5 m below the filling height of at most the bioreactor or fermenter 100, or

[0168] The first bubbler is located approximately 0.4m above the filling height of the bioreactor or fermenter 100, which is approximately 2 / 3 of the total filling height.

[0169] The first bubbler is located approximately 0.4m above the filling height of the bioreactor or fermenter 100, or approximately 1 / 2 of the filling height.

[0170] Approximately 0.4m to approximately 3.0m above the first bubbler, or approximately 0.4m to approximately 2.5m above the first bubbler, or approximately 0.4m to approximately 2.0m above the first bubbler, or approximately 0.4m to approximately 1.5m above the first bubbler, or approximately 0.4m to approximately 1.0m above the first bubbler, or approximately 0.45m to approximately 0.90m above the first bubbler, or approximately 0.5m to approximately 0.80m above the first bubbler, or approximately 0.55m to approximately 0.70m above the first bubbler, or approximately 0.6m above the first bubbler. Figure 6a In this configuration, the second bubbler 160 is located at approximately half the filling height of the bioreactor or fermenter 100. For example... Figure 6a As shown, the second bubbler 160 can acquire "new" bubbles that are capable of absorbing CO2 from the liquid phase. Therefore, better removal of dissolved CO2 is possible. In the liquid phase, the overall CO2 gradient is largely avoided. The environmental conditions in industrial-scale bioreactors or fermenters are thus adjusted to be more similar to those in small-scale bioreactors or fermenters, where bubbles do not reach CO2 saturation concentration during their upward path. Therefore, the removal of growth-inhibiting dissolved CO2 from the culture suspension is performed more effectively. Therefore, in Figure 6a On the right side, the two arrows (6) from the width to the arrowhead symbolize the absorption capacity of CO2 from the liquid phase to the gas phase, which is decreasing. Figure 6a The arrow (7) in the image symbolizes that from the tip to the width of the arrow, the bubbles become increasingly saturated with CO2, thus allowing CO2 to pass from the liquid phase into the gas phase much faster than the delivery of O2. The presence of additional bubblers further homogenizes the environment of the cultured cells or microorganisms.

[0171] In addition, refer to Figure 6a And the exemplary embodiment shown provides a stirrer with a radius r sThe stirrer 120 is configured such that its stirring axis or rotation axis corresponds to the central axis A. The stirrer 120 consists of three stirrers R1, R2, and R3. The first stirrer R1 is located above the first bubbler 150 (located at the bottom 105 or lower part of the bioreactor or fermenter 100). In addition to the first stirrer R1, two additional stirrers R2 and R3 are provided, located above and below the second bubbler 160. According to another embodiment, only the first stirrer R1 may be provided, or both the first stirrer R1 and the second stirrer R2 may be provided simultaneously. Embodiments with more than three stirrers are also possible.

[0172] exist Figure 6a In the embodiment shown, the stirrer 120 has a stirrer radius r s The radius of the agitator is symmetrically positioned around a central axis A passing through the bioreactor or fermenter 100, resulting in an agitator diameter d. s The first bubbler 150 is positioned at a distance A from the central axis (i.e., the opening of the first bubbler is positioned at a distance), said distance, such that the provided bubbles are equal to or less than the diameter d of the stirrer. s Or the radius of the mixer r s At a distance (such as at) Figure 6a (Diagrams d1 and d2 are used to schematically illustrate) the entry into the liquid phase. This finding is based on Klaas Van't Riet, Review of Measuring Methods and Results in Mass Transfer in Stirred Vessels Nonviscous Gas-Liquid, Ind. Eng. Chem. Process Des. Dev., Vol. 18, No. 3, 1979, pp. 357-364, which states that the bubbler may not be installed at a distance from the center greater than the radius of the stirrer. If the first bubbler is installed at a distance from the central axis equal to or less than the radius r of the stirrer... s Providing maximum turbulence at the blade tip of the agitator at a distance of [distance missing] is considered advantageous. Furthermore, high-energy mixing of liquids and gases is considered advantageous in the cultivation of cells or microorganisms. Therefore, this implementation is considered advantageous.

[0173] exist Figure 6a In this configuration, all agitators R1, R2, and R3 have the same dimensions, such that the agitator radius r s and diameter d s The three agitators R1, R2, and R3 are identical throughout the entire volume of the bioreactor or fermenter 100. Other implementations are possible.

[0174] In the exemplary embodiment shown, the stirrer has a stirrer radius r. s And therefore the diameter d of the stirrer s The second bubbler 160 is positioned symmetrically around a central axis A passing through the bioreactor or fermenter 100, such that the bubbler provides bubbles at a distance greater than the agitator radius r. s Or the diameter of the stirrer d s The gas enters the liquid phase at a distance from the agitator. If a second bubbler and optional (one or more) additional bubblers provide bubbles spaced apart from the agitator, turbulence that could deform or damage the provided bubbles can be avoided. Therefore, high-energy mixing of the liquid and gas is considered less advantageous to the effectiveness of the second bubbler and optional additional bubblers, as beneficial technical effects (such as CO2 stripping performance and product yield) can be negatively impacted.

[0175] When several agitators are present, the agitator radius or diameter is often related to the agitator located next to the bubbler in the discussion.

[0176] refer to Figure 6b In addition to the first bubbler 150 and the second bubbler 160, a third bubbler 170 is also provided, wherein the third bubbler 170 is located at a height or distance η above the second bubbler 160 in the bioreactor or fermenter 100. 2,3 Location. Distance η 2,3 The range above the second bubbler 160, as defined herein, can be selected. Figure 6b In the process, the second bubbler 160 is located at approximately 1 / 3 of the filling height, and the third bubbler 170 is located at approximately 2 / 3 of the filling height of the bioreactor or fermenter 100.

[0177] Compared to a bioreactor / fermenter with only one bubbler (reflecting the current state of the technology), the presence of the second bubbler according to the invention is expected to significantly reduce the partial pressure of CO2 by at least about 0.5%, or at least about 1%, or at least about 2% up to about 20%. Furthermore, compared to a bioreactor / fermenter using only one bubbler, it is expected to achieve an increase in product titer and product yield of at least about 1%, or at least about 5%, or at least about 10% up to about 30%. As stated above, even small improvements in methods used on an industrial scale as described in this invention represent significant improvements in aspects such as the possible duration of the entire process (extending the feed-batch process by several hours, up to 1 day, 2 days, or even 3 days), viable cell density (cell activity), or the titer of the produced product.

[0178] Furthermore, it has been found that in bioreactors or fermenters operating on a large scale, the supply of a second bubbler and optionally additional bubblers, in addition to a first bubbler, has provided the possibility of establishing independent management of O2- and CO2- concentrations within the bioreactor or fermenter.

[0179] Therefore, a method is provided for controlling and adjusting the dissolved CO2 and dissolved O2 content in a liquid medium in a bioreactor or fermenter 100 for culturing suspended cells or microorganisms on an industrial scale, said bioreactor or fermenter comprising a container 102 containing a culture in a liquid medium, the method comprising:

[0180] Stirring liquid media;

[0181] Bubbles 10, 10.1, 10.2, and 10.3 are continuously supplied to the liquid medium from a first bubbler 150 arranged in the bottom portion 105 of the container 102, the gas being selected from air and / or oxygen;

[0182] Bubbles 20, 20.1, 20.2, and 20.3 are continuously supplied to the liquid medium from a second bubbler 160 arranged in container 102. The gas is selected from air and / or oxygen. Thus, the second bubbler 160 is arranged above the first jet 150, and the second bubbler 160 is a side bubbler.

[0183] Based on the gas flow rate q of the submerged bubbler or the first bubbler 150 sub The gas flow rate q of the side bubbler or the second bubbler 160 side Select and adjust the corrected gas flow rate q mod (O2) and selection and adjustment of the corrected gas flow rate q mod (CO2), both are applicable to the culture process, thus the following equation is applied:

[0184] q mod (O2)=q sub +C O2 ×q side [1a]

[0185] as well as

[0186] q mod (CO2)=q sub +C CO2 ×q side [1b]

[0187] in

[0188] q sub This indicates the gas flow rate of the submerged bubbler or the first bubbler 150;

[0189] qside This indicates the gas flow rate of the side bubbler or the second bubbler 160;

[0190] C O2 This represents the influence factor C of volumetric oxygen mass transfer, hence C O2 =0.15; and

[0191] C CO2 The mass transfer factor C represents the volumetric carbon dioxide mass transfer influence factor, hence C CO2 =0.6.

[0192] The methods for controlling and adjusting the dissolved CO2 and dissolved O2 content in the liquid medium of bioreactors or fermenters used for industrial-scale cultivation of suspended cells or microorganisms will be explained in detail below:

[0193] It has been recognized that more accurate predictions of the oxygen and carbon dioxide mass transfer performance of industrial-scale aerated stirred bioreactors or fermenters can only be made based on detailed studies. Therefore, numerous experiments have been performed with only a single bubbler. Alternatively, the effect of an additional second bubbler has been investigated. It has been found that in the case of only one bubbler, the oxygen mass transfer coefficient k... L a O2 It is directly proportional to the gas flow rate. That is, when the gas flow rate doubles, the mass transfer coefficient almost doubles as well. In other words, a bubbler may already provide sufficient oxygen mass transfer (characterized by k). L a O2 This is to meet the oxygen requirements of the culture.

[0194] Furthermore, it has been surprisingly found that the presence of an additional side bubbler and therefore additional side inflation affects the oxygen mass transfer coefficient k. L a O2 There is almost no effect or only a very small effect. Therefore, the mass transfer coefficient k of oxygen... L a O2 Only the gas flow rate of the submerged bubbler or the first bubbler in the bioreactor or fermenter is important. Therefore, the effect of submersion aeration can be considered dominant for oxygen mass transfer.

[0195] Furthermore, it has been observed that side injection results in a slight increase in oxygen mass transfer performance, while significantly increasing carbon dioxide mass transfer performance. This finding, confirming another aspect of the invention, was also entirely unexpected. In fact, the side bubbler has a very strong influence on the carbon dioxide mass transfer coefficient, and the increase in the carbon dioxide mass transfer coefficient is even stronger with increasing side injection. Therefore, the effect of side injection can be considered dominant on carbon dioxide mass transfer.

[0196] Because side injection of gas in industrial-scale aerated stirred bioreactors or fermenters has different effects on the mass transfer coefficients of oxygen and carbon dioxide, independent management of oxygen and carbon dioxide concentrations is possible. Independent management can be based on two principles: if the total gas flow rate is constant, then CO2 mass transfer is almost constant; and side injection has different effects on CO2 and O2 mass transfer.

[0197] In many evaluation measurements, it has been found that the gas flow rate of the side-ejected gas cannot be simply added to the submerged gas flow rate. In fact, a "corrected" gas flow rate must be assumed. Therefore, the corrected carbon dioxide gas flow rate (q) mod (CO2)) and the corrected oxygen gas flow rate (q) mod (O2) must be taken into account, and the corrected gas flow rate is considered to be related to the mass transfer coefficient (k). L a O2 or k L a CO2 The gas flow rate is proportional to the CO2 and O2 mass transfer during the cultivation process. Therefore, the gas flow rate can be used to directly influence the CO2 and O2 mass transfer during the cultivation process, respectively. (Experiment by...) Figure 10 and Figure 11 (Note) As shown, the “corrected” gas flow rate can be expressed by the following equation:

[0198] For oxygen:

[0199] q mod (O2)=q sub +C O2 ×q side [1a]

[0200] Among them, C O2 =0.15.

[0201] For carbon dioxide:

[0202] q mod (CO2)=q sub +C CO2 ×q side [1b]

[0203] Among them, C CO2 =0.6.

[0204] The corrected gas flow rate includes immersion filling q sub and side inflation q side (It is corrected and weighted using an influence factor C). The higher the factor C, the greater the impact of side-filling on mass transfer performance. The factor C can be calculated under the assumption that mass transfer is linearly proportional to the submerged gas flow rate. The detailed calculation of the influence factor C is explained and demonstrated in the Examples section (Example 3).

[0205] The results show that the side injection of gas causes the oxygen mass transfer coefficient to decrease only slightly due to the influence factor C. O2 The mass transfer coefficient of carbon dioxide increases by 0.15, while the mass transfer coefficient of carbon dioxide can be affected by the factor C. CO2 The increase was 0.6. In other words, a slight increase in the mass transfer performance of oxygen was observed, while the mass transfer performance of carbon dioxide increased significantly.

[0206] This method enables independent management of carbon dioxide and oxygen in industrial-scale aerated stirred bioreactors or fermenters. In other words, compared to submerged aeration, the influence factor (k) of carbon dioxide in side-injection gas is significantly reduced. L a CO2 Approximately four times higher. Therefore, the actual or modified gas flow rates of oxygen and carbon dioxide can be selected and adjusted to achieve optimal conditions in the culture system.

[0207] The following exemplary cases should illustrate the above findings, as follows:

[0208] The gas flow rate q of the submerged bubbler or the first bubbler sub Selected to have the following values: q sub =120L / min.

[0209] The gas flow rate q of the second bubbler or side bubbler side Selected to have the following values: q side =60L / min.

[0210] Then, the corrected oxygen mass transfer gas flow rate q mod (O2) can be calculated according to equation [1a] as follows:

[0211] q mod (O2)=120L / min+0.15×60L / min=129L / min.

[0212] Furthermore, the corrected gas flow rate q for carbon dioxide mass transfer mod (CO2) can be calculated according to equation [1b] as follows:

[0213] q mod (CO2)=120L / min+0.6×60L / min=156L / min.

[0214] Because q mod (O2) indicates that it is considered to be related to the mass transfer coefficient k. L a O2 A proportional correction to the total gas flow rate allows those skilled in the art to have a direct measure of the impact on oxygen mass transfer. Furthermore, q mod (CO2) represents the mass transfer coefficient k. L a CO2A proportional correction to the total gas flow rate, and a direct measure of the impact on the mass transfer of carbon dioxide, is also available to those skilled in the art. Therefore, the equations [1a] and [1b] mentioned above allow for the control and adjustment of O2 and CO2 contents based on the selected gas flow rate. Those skilled in the art can readily select and adjust suitable and desired q. mod (O2) and q mod (CO2), which are optimal for individual culture processes and have a direct impact on cell or microbial culture performance.

[0215] Using this method, if a second bubbler in the form of a side-buffered reactor is provided, independent management of carbon dioxide and oxygen is thus possible in an industrial-scale aerated stirred bioreactor or fermenter. Furthermore, those skilled in the art can adjust and select the gas flow rates of the first and second bubblers such that k L a O2 and k L a CO2 Any specific method that can be controlled and adjusted for the cultivation of cells or microorganisms.

[0216] Although according to one embodiment of the present invention, the gas flow rate q of the first bubbler sub The gas flow rate q can be selected to be greater than that of the second bubbler. side Alternatively, you can choose to adjust q. sub For greater than q side (q side >q sub ).

[0217] According to one embodiment and as already explained, the distance η between the first submerged bubbler and the second side bubbler 1,2 It can be selected within the range defined herein. Thus, the second bubbler can be positioned at a height above the first bubbler, at which the bubbles have reached or will soon reach the CO2 gas phase saturation concentration.

[0218] In another embodiment, a third bubbler 170 and optional (one or more) additional bubblers are disposed above the first bubbler 150 and the second bubbler 160 in the bioreactor or fermenter 100, two consecutive bubblers, i.e., one arranged above the other, and the distance between the bubblers 150, 160 and / or 160, 170 is selected as η.

[0219] In addition to the first submerged bubbler and the second side bubbler, other bubblers may be present in a bioreactor or fermenter as already described. The distance η between the bubblers... 1,2 η 2,3 η 3,4The selection can be made within the range already disclosed above. Additional bubblers can be central bubblers or side bubblers, determined individually for each bubbler. Based on the above findings and explanations regarding the independent management of CO2 and O2, according to one embodiment, the additional bubblers are all side bubblers.

[0220] Therefore, according to another embodiment, the first bubbler is a central bubbler or a side bubbler, and the second bubbler and optionally a third bubbler and additional bubblers are side bubblers. According to another embodiment, the first bubbler is a central bubbler, and the second bubbler and optionally a third bubbler and additional bubblers are side bubblers.

[0221] Besides changes in the gas flow rate of the bubbler used, there are other parameters and conditions that can help improve the culture performance of cells or microorganisms, such as temperature, pH or concentration of specific nutrients, type of stirrer, stirrer speed, type of bubbler, size of bubbler, geometry of bubbler, etc. These parameters and how to modify them are familiar to those skilled in the art.

[0222] Those skilled in the art can select any known bubbler from the prior art for use in cell or microbial culture processes. However, the type of bubbler used can affect the mass transfer performance of oxygen and carbon dioxide. The number and size of the openings present, as well as the geometry and size of the selected bubbler, can play a role. It is presumed that larger bubbles, due to larger openings, higher bubble rise velocities, and therefore shorter contact times between the gas and the medium, will allow for more effective removal of carbon dioxide even with only a weak oxygen supply. Therefore, those skilled in the art can readily select a suitable bubbler based on their average skill in the field, and the selected bubbler is advantageous for each culture process.

[0223] According to one embodiment, the first bubbler, the second bubbler, and optionally another bubbler are static bubblers selected from the following: bubblers having a tubular geometry, such as tubular bubblers, open-tube bubblers, sintered plates, perforated plates, annular bubblers, spider bubblers, disc bubblers, plate bubblers, cup bubblers, and bushing bubblers.

[0224] In another embodiment, the first bubbler, the second bubbler, and optional additional bubblers are the same or different bubblers.

[0225] According to another embodiment, the first bubbler, the second bubbler, and optional additional bubblers are bubblers having a tube geometry, such as a tubular bubbler or an open tube bubbler.

[0226] In another embodiment, the first bubbler, the second bubbler, and an optional additional bubbler are crescent-shaped bubblers.

[0227] According to another embodiment, the first bubbler, the second bubbler, and an optional additional bubbler are crescent-shaped open tube bubblers.

[0228] Tubular aerators, especially open-tube aerators (such as crescent open-tube aerators), have the advantage of good cleaning ability, as well as the advantage of easier cleaning in place (CIP) and sterilization in place (SIP).

[0229] Furthermore, according to this disclosure, the bioreactor or fermenter is not limited. Any known aerated and stirred bioreactor or fermenter can be used. Bubble column trickle bed reactors, loop reactors, etc., can also be used.

[0230] Furthermore, the cells used are not limited according to this disclosure. In embodiments of this disclosure, the cells can be eukaryotic cells, such as mammalian cells, especially yeast (Saccharomyces cerevisiae, Pichia pastoris), Chinese hamster ovary (CHO) cells, human cells (e.g., HEK 293), or insect cells. Other cells may also be used.

[0231] According to this disclosure, microorganisms are not limited. In embodiments of this disclosure, microorganisms can be prokaryotic cells, such as Escherichia coli or Bacillus subtilis.

[0232] This disclosure also relates to a method for culturing cells or microorganisms in a bioreactor or fermenter as described herein, wherein a second bubbler is disposed in the bioreactor or fermenter at a defined distance η to promote the growth, activity, productivity and / or any other metabolic conditions of the cells or microorganisms to be cultured.

[0233] This disclosure also relates to a method for culturing cells or microorganisms in a bioreactor or fermenter as described herein, wherein, in addition to a first and a second bubbler, one or more additional bubblers are respectively disposed in the bioreactor or fermenter at a defined distance η to promote the growth, activity, productivity and / or any other metabolic conditions of the cells or microorganisms to be cultured.

[0234] This disclosure also relates to a second bubbler disposed in a bioreactor or fermenter for culturing cells or microorganisms as already described, wherein the second bubbler is disposed in the bioreactor or fermenter to promote the growth, activity, productivity and / or any other metabolic conditions of the cells or microorganisms to be cultured, wherein the second bubbler is located at a distance η above the first bubbler in the bioreactor or fermenter, wherein η is selected to be at least about 0.4 m above the first bubbler to at most about 0.5 m below the filling height of the bioreactor or fermenter, or

[0235] The first bubbler is located approximately 0.4m above the filling height of the bioreactor or fermenter, or approximately 2 / 3 of the filling height.

[0236] The first bubbler is located approximately 0.4m above the filling height of the bioreactor or fermenter, or approximately 1 / 2 of the filling height.

[0237] At approximately 0.4m to approximately 3.0m above the first bubbler, or approximately 0.4m to approximately 2.5m, or approximately 0.4m to approximately 2.0m, or approximately 0.4m to approximately 1.5m, or approximately 0.4m to approximately 1.0m, or approximately 0.45m to approximately 0.90m, or approximately 0.5m to approximately 0.80m, or approximately 0.55m to approximately 0.70m, or at approximately 0.6m.

[0238] This disclosure also relates to a second bubbler and one or more additional bubblers disposed in a bioreactor or fermenter for culturing cells or microorganisms as already described, wherein the second bubbler and one or more additional bubblers are disposed in the bioreactor or fermenter to promote the growth, activity, productivity and / or any other metabolic conditions of the cells or microorganisms to be cultured, wherein the second bubbler is located at a distance η above the first bubbler in the bioreactor or fermenter, wherein η is selected to be at least about 0.4 m above the first bubbler to at most about 0.5 m below the filling height of the bioreactor or fermenter, or

[0239] The first bubbler is located approximately 0.4m above the filling height of the bioreactor or fermenter, or approximately 2 / 3 of the filling height.

[0240] The first bubbler is located approximately 0.4m above the filling height of the bioreactor or fermenter, or approximately 1 / 2 of the filling height.

[0241] At approximately 0.4m to approximately 3.0m above the first bubbler, or approximately 0.4m to approximately 2.5m, or approximately 0.4m to approximately 2.0m, or approximately 0.4m to approximately 1.5m, or approximately 0.4m to approximately 1.0m, or approximately 0.45m to approximately 0.90m, or approximately 0.5m to approximately 0.80m, or approximately 0.55m to approximately 0.70m, or at approximately 0.6m.

[0242] Therefore, additional gas injection at higher positions, such as additional side injections, with large bubbles, high bubble rise rates, and thus short contact time between the gas and the medium, leads to improved performance in the cell culture process.

[0243] Furthermore, according to the present invention, the volumetric carbon dioxide mass transfer coefficient k of large-scale systems can now be improved. L a CO2 without significantly affecting the oxygen mass transfer coefficient k L a O2 Therefore, in the simplified form, the first bubbler is used to control and adjust O2 mass transfer, while the second bubbler (in the form of a side bubbler) and optional (one or more) additional bubblers can be used to control and adjust CO2 mass transfer. Specifically, the first bubbler can be primarily used to provide more O2 to the culture process, while the second bubbler and optional (one or more) additional bubblers can be primarily used to reduce CO2 levels.

[0244] Example

[0245] Example 1: k L a CO2 The determination

[0246] To determine the volumetric mass transfer coefficient k L a CO2 The dynamic method was used. In the method used, carbon dioxide was introduced into the bioreactor until 15% saturation was reached. Subsequently, the desired stirrer frequency n and the desired aeration rate q were set, and the decrease in carbon dioxide concentration was recorded. The relationship between the recorded carbon dioxide level and the corresponding time t is shown in the following equation [2], and can be described as follows.

[0247]

[0248] Wherein, the volumetric mass transfer coefficient k L a CO2 And the saturation concentration c*. This equation can be transformed into the following logarithmic expression:

[0249]

[0250] Wherein, the concentration c' at time t' CO2 and the concentration c” at time t” CO2 Equation [3] provides k L a CO2 The value of is used as the slope of the logarithmic function of the carbon dioxide plot. An exemplary evaluation can be performed... Figure 8 Found it. (Reference) Figure 8 k L a CO2 The evaluation of the value is based on the concentration c.CO2 =14% to c CO2 =6% was carried out. Therefore, k L a CO2 It is shown as a negative value because a decrease in CO2 occurred. Therefore, k L The value of 'a' is derived from the slope represented by the logarithm of the defined curve.

[0251] Example 2: First Evaluation Measurement

[0252] To examine the oxygen mass transfer performance of an industrial-scale aerated stirred bioreactor or fermenter, a second bubbler (in the form of a side bubbler) was designed and installed in the reactor. Measurements were performed in a 15kL bioreactor filled with approximately 12kL (see Table 1 below) of 0.9% (w / v) NaCl / H2O. Technical drawings of the 15kL bioreactor used and the installation location of the additional side bubbler are shown in [Table 1]. Figure 9 As shown in the figure. In these measurements, the additional side bubbler 160 is installed at a position where the filling height represents half of the reactor volume. V Fill This indicates the filling volume of the container of bioreactor 100. That is, the side bubbler 160 is located at 0.5 times (1 / 2V) of the filling volume. Fill At the location of ), in the current case, it is about 1 / 2 of the filling height of the bioreactor or fermenter, because the bioreactor or fermenter is cylindrical in shape.

[0253] The immersion bubbler or first bubbler (not shown) used in these measurements is a side bubbler, which is in the form of a tubular bubbler with 31 × 2 mm boreholes, i.e., 31 openings, each with a diameter of 2 mm.

[0254] The second bubbler used in these measurements was also a side bubbler, which was in the form of a tubular bubbler with 15×3mm holes, i.e., 15 openings, each with a diameter of 3mm.

[0255] The gas used in the immersion bubbler (first bubbler) and the side bubbler (second bubbler) is air.

[0256] The first evaluation measurement was performed to measure the dependence of oxygen mass transfer rate on gas flow rate, with a stirrer frequency of n = 60 rpm. A detailed overview of the operating conditions and ventilation strategies studied is given in Table 1.

[0257] Table 1: Dependence of oxygen mass transfer rate on gas flow rate

[0258]

[0259] Therefore, in measurements 1 through 4, the gas flow rate has changed, while the stirrer rate has remained constant.

[0260] In Measurement 1, the gas flow rate of the submerged bubbler or the first bubbler was set to 120 L / min. The side bubbler or the second bubbler did not supply air to the liquid medium; that is, the gas flow rate of the second bubbler was 0 L / min. The overall or total gas flow rate (q) in Measurement 1... total Therefore, it is:

[0261] q sub +q side =120mL / min+0mL / min=120mL / min.

[0262] In measurement 2, the gas flow rate of the submerged bubbler or the first bubbler was set to 100 L / min; the gas flow rate of the side bubbler or the second bubbler was set to 20 L / min, so the total gas flow rate was 120 L / min.

[0263] In measurement 3, the gas flow rate of the submerged bubbler or the first bubbler was set to 120 L / min; the gas flow rate of the side bubbler or the second bubbler was set to 20 L / min, so the total gas flow rate was 140 L / min.

[0264] In measurement 4, the gas flow rate of the submerged bubbler or the first bubbler was set to 120 L / min; the gas flow rate of the side bubbler or the second bubbler was set to 60 L / min, so the total gas flow rate was 180 L / min.

[0265] Measurement 1 (in which no side ventilation occurred) has been used as the standard, and the volumetric mass transfer coefficient k of measurements 2 through 4 has been determined relative to this standard. L a O2 Volumetric mass transfer coefficient k L a O2 It represents a direct measure of the rate of oxygen mass transfer.

[0266] In additional measurements, the mass transfer coefficient k has been found to be... L a O2 The mass transfer coefficient is directly proportional to the gas flow rate. When the gas flow rate is doubled, the mass transfer coefficient almost doubles as well. This finding was confirmed in Measurement 2, where the gas flow rate of the submerged bubbler was reduced from 120 L / min to 100 L / min. Although the total gas flow rate remained constant at 120 L / min in Measurements 1 and 2, the mass transfer coefficient k increased significantly compared to Measurement 1. L a O2 In measurement 2, the value decreased to -18%. The side-buffered motor with a gas flow rate of 20 L / min effectively affected the mass transfer coefficient k. L a O2There is no effect. Therefore, the gas flow rate that only submerges the bubbler (first bubbler) has no effect on k. L a O2 It works. Therefore, the effect of immersion aeration is considered to be dominant for oxygen mass transfer.

[0267] In fact, in measurements 1 to 4, the mass transfer coefficient was not significantly affected by the additional side injection of gas. This confirms that side injection has a significant impact on the oxygen mass transfer coefficient k. L a O2 It has little or no impact.

[0268] For illustrative purposes, the results obtained in Table 1 are... Figure 10 It was described in the text. Figure 10 The comparison of the four measurements performed is shown. From Figure 10 As can be seen, compared to Measurement 1, a 16% reduction in immersion aeration in Measurement 2 significantly affected the O2 mass transfer coefficient, even though the total aeration rate was constant in both Measurements 1 and 2. However, compared to Measurement 1, a 16% increase in side injection in Measurement 3 showed that the oxygen mass transfer coefficient k in Measurement 3 was significantly lower. L a O2 An increase of only 2%, and a 50% increase in side injection in measurement 4, show that the oxygen mass transfer coefficient k L a O2 Only an 8% increase.

[0269] The first evaluation measurement thus confirmed that the side injection of air has only a small effect on oxygen mass transfer performance.

[0270] Example 3: Second Evaluation Measurement

[0271] The same measurement as in the first evaluation measurement has been performed, but the volumetric mass transfer coefficient k of carbon dioxide is... L a CO2 It has been determined (see Figure 11 ).from Figure 11 The mass transfer coefficient with oxygen (8%) can be derived (see...). Figure 10 In contrast, the mass transfer coefficient of carbon dioxide increases much more strongly with the increase of side injection (up to 23% in measurement 4).

[0272] First and second evaluation measurements confirmed that side injection has different effects on the mass transfer coefficients of oxygen and carbon dioxide, and therefore cannot be simply added to the submerged gas flow rate. Therefore, side injection of gases in industrial-scale aerated stirred bioreactors or fermenters allows for independent management of oxygen and carbon dioxide concentrations.

[0273] The above evaluation leads to the conclusion that a "corrected" gas flow rate exists. To better describe this "corrected" gas flow rate, it can be expressed by the following equation:

[0274] q mod =q sub +C×q side [1]

[0275] In other words, a modified gas flow rate can be introduced, which includes immersion filling q. sub and side inflation q side (Weighted by an influence factor C). The higher the factor C, the greater the impact of side-filling on mass transfer performance. The factor C can be calculated under the assumption that mass transfer is linearly proportional to the submerged gas flow rate. This finding is based on... Figure 12a and Figure 12b This was explained in the text. Figure 12a and 12b The volumetric mass transfer coefficient k for oxygen can be derived. L a O2 The side injection factor can be calculated as C. O2 =0.15, while for carbon dioxide (k L a CO2 The factor is four times higher, which is C. CO2 =0.6.

[0276] Therefore, the additional side injection results in a slight increase in oxygen mass transfer performance, while the mass transfer performance of carbon dioxide increases significantly. In fact, the side injection of the gas causes the oxygen mass transfer coefficient to increase by a factor of 0.15, while the carbon dioxide mass transfer coefficient can increase by a factor of 0.6.

[0277] Example 4: Third Evaluation Measurement

[0278] The same measurements as in the first and second evaluation measurements have been performed, but different side bubblers (second bubblers) have been used. Side bubbler type A has a 10×3 mm borehole, and side bubbler type B has a 32×5 mm borehole. Compared to type A, side bubbler type B has more openings, and each opening has a larger diameter.

[0279] The results obtained are Figure 13a and Figure 13b This is explained in the text. (See reference.) Figure 13a and Figure 13b It was confirmed that if the total gas flow rate is constant, the CO2 mass transfer is also almost constant. Furthermore, side-filling has a greater impact on CO2 mass transfer compared to O2 mass transfer. Specifically, the volumetric mass transfer coefficient k of CO2 in side-buffereder type B is compared to the second evaluation measurement of the CO2 mass transfer coefficient. L a CO2 The increase is much stronger with the addition of side injection (see...) Figure 13b (Up to 30% in measurement 4). That is to say, CO2 mass transfer can be further increased by selecting bubbler type B.

[0280] Therefore, it is speculated that the additional side injection of gas with more and larger bubbles at higher positions, i.e., higher bubble rising speed and thus shorter contact time between the gas and the medium, leads to improved performance in the cell culture process.

[0281] Example 5: Fourth Evaluation Measurement

[0282] To verify the mass transfer coefficient k of carbon dioxide L a CO2 Mass transfer coefficient k of oxides L a O2 Further measurements were performed to investigate the dependence of stirrer frequency and gas flow rate. Results were obtained in... Figure 14a and Figure 14b as well as Figure 15a and Figure 15b As shown in the image.

[0283] exist Figure 14a , 14b In the legends for 15a and 15b (provided at the end of the instruction manual), the results are summarized using a plus sign ("+"). The plus sign in the legends has the following meanings:

[0284] +................minor impact

[0285] +++............significant impact

[0286] exist Figure 14a and Figure 14b In this process, only one aerator is used, which is a submersible aerator located at the bottom of the container.

[0287] According to other experiments, in Figure 14a The mass transfer coefficient k of carbon dioxide relative to the power input has been found. L a CO2 It has a small effect (indicated by a plus sign (+)), while the apparent gas flow rate has a significant impact on the mass transfer coefficient k of carbon dioxide. L a CO2 It has a significant impact (+++).

[0288] refer to Figure 14b The mass transfer coefficient k of oxides L a O2 It increases with increasing specific power input and increasing apparent gas flow rate.

[0289] exist Figure 15a and Figure 15b In this experiment, the second bubbler used was a side bubbler. According to other experiments, in... Figure 15a The mass transfer coefficient k of carbon dioxide relative to the power input has been found. L aCO2 It has a small effect (indicated by a plus sign (+)), while the apparent gas flow rate has a significant impact on the mass transfer coefficient k of carbon dioxide. L a CO2 It has a significant impact (+++).

[0290] refer to Figure 15b The mass transfer coefficient k of oxides L a O2 It increases with increasing specific power input. However, increasing the apparent gas flow rate has less effect on the mass transfer coefficient k of the oxide. L a O2 It has only a minor impact.

[0291] Based on this method, independent management of carbon dioxide and oxygen is possible in industrial-scale aerated stirred bioreactors or fermenters. Independent management can be based on two principles: that CO2 mass transfer is nearly constant if the total gas flow rate is constant, and that side aeration has different effects on CO2 and O2 mass transfer.

[0292] Example 6: Evaluation of distance η

[0293] The distance η between the first bubbler and the second bubbler (also designated as η) 1,2 The CO2 saturation concentration of the bubbles and the time it takes for the bubbles to reach CO2 saturation in an industrial-scale bioreactor or fermenter are used to determine the gas phase residence time. Therefore, the gas phase residence time at the reactor height has been determined using the step response method (“Sprungantwortmethode”), the fundamental problem of which will be explained below.

[0294] 6.1. Measuring the gas phase residence time in an aerated stirred tank reactor using the step response method.

[0295] Only a very limited number of publications address the determination of gas-phase residence time (Wachi S. and Nojima Y., Gas-Phase Dispersion in Bubble Columns, Chemical Engineering Science, Vol.45, No.4, pp. 901-905, 1990; Yianatos JB and Bergh LG, International Journal of Mineral Processing, 36(1992), pp.81-91). The gas-phase residence time distribution in two-phase flows can be determined using pulse or step response methods. However, the described pulse response method is difficult to adapt due to the use of either radioactive or toxic tracer gases. Furthermore, no studies on the gas-phase residence time distribution in aerated stirred tank reactors have been published to date. Therefore, this paper uses a measurement technique based on a correction to the step response method to determine the gas-phase residence time.

[0296] According to control theory, the behavior of a system can be determined using either impulse or step response methods. The difference between the two methods lies in the information obtained about the system. The residence time distribution can be determined using the impulse response method, while the residence time itself can be determined using the step response method. For example, the output of a system responding to a step input... Figure 16a and Figure 16b As shown in the image. Figure 16a and Figure 16b Explanation of the step response method: Based on Leigh, JR (2004). Control Theory 2nd ed., IET control engineering series, London, the input to the system is shown ( Figure 16a ) and system output ( Figure 16b ).

[0297] As the measurement signal at the system output, radiation from a radioactive gas tracer (Yianatos JB, Bergh, LG, Duran, OU, Diaz, FJ, Heresi, NM (1994), Measurement of Residence Time Distribution of the Gas Phase in Flotation Columns, Minerals Engineering Vol. 7, pp. 333-344) or dichlorodifluoromethane (Wachi S. and Nojima Y., in the above citations) is used. For practical reasons, it is often impossible to use a radioactive gas tracer to achieve the input pulse. Furthermore, in the current case, information only about the residence time (and not about the distribution) is sufficient.

[0298] Therefore, for our application in a 12kL acrylic glass stirred tank reactor, we only changed the gas type during aeration to introduce a step signal into the system.

[0299] For the application of the step response method, the aerated stirred tank reactor should be operated under steady-state process conditions, for example, being aerated until dissolved oxygen concentration equilibrium is reached. During steady-state operation, the subsequent aeration is converted to pure nitrogen. Oxygen concentration is continuously measured at the reactor inlet and outlet. To minimize the effects of gas mixing in the reactor headspace, a funnel is installed on the water surface as a "bubble trap." This bubble trap (funnel) 180 (which minimizes the effects of the reactor headspace) in... Figure 17 The example is shown below. The bubble trap (funnel) 180 is designed to absorb bubbles across its cross-section and guide the collected gas to a gas sensor. As the gas sensor at the reactor inlet and outlet, an optical oxygen sensor (PreSens Precision Sensing GmbH) has been used in this case, with a very low response time of t. response <2s. In Figure 17 The bubble trap (funnel) 180 described herein shows the PreSens port 185 and the exhaust gas 189.

[0300] Typical input signals and corresponding output signals of oxygen concentrations located at the submerged bubbler and funnel, respectively, from a step response method applied to a 12kL aerated stirred tank reactor. Figure 18 As shown in the image.

[0301] Assuming the residence time scale is significantly smaller than the mass transfer time scale, the gas-phase residence time is defined as the time between a step input and the point in time when the oxygen concentration in the exhaust gas has decreased by more than 1%. This assumption can be demonstrated by comparing the system response resulting from a 100% and a 50% step input. Figure 19 The diagram shows a comparison of the step responses at the funnel caused by 100% and 50% input step changes, respectively. Regarding... Figure 19 No difference in response signal was detected regarding the time of the first drop in the output oxygen signal.

[0302] The step response method is associated with some drawbacks, which are minor in the current case for the following reasons:

[0303] - The signal begins to drop once the first bubble reaches the funnel. Depending on the bubble size distribution, this can occur very early (through large bubbles), while the largest number of small bubbles may remain in the system longer. However, due to the use of PBS and Prönkel as solvents (phosphate-buffered saline + 1 g / L Prönkel), the bubble size distribution is very narrow, and this method should have acceptable accuracy.

[0304] - Under heterogeneous flow conditions, it is possible that the main bubble plume will not be captured by the funnel. In this case, the residence time will be overestimated.

[0305] - An exchange of dissolved oxygen and nitrogen may occur between oxygen and nitrogen bubbles, as well as additional coalescence and splitting of cocoa. This effect is assumed to be negligible.

[0306] In summary, the step response method for determining gas phase residence time is an easy-to-apply method with acceptable accuracy for the described system and conditions.

[0307] The solvents used in the step response method are PBS (phosphate-buffered saline) and 1.0 g / L Prönnicke.

[0308] The results of the step response method are in Figure 20 As shown in [the image]. Figure 20 In the figure, oxygen concentration (in [%)) is plotted against sampling time t (in [s]). A laboratory-scale bioreactor or fermenter with a capacity of 30 L has been compared with an industrial-scale bioreactor or fermenter with a capacity of 12000 L. From laboratory to industrial scale, stirrer frequency n (in [rpm]) and gas flow rate (in [L h]) are also compared. -1 The agitator frequency has been modified to provide comparable power input for each system. Therefore, the agitator frequency is 300 rpm for industrial-scale systems and 80 rpm for laboratory-scale systems. The gas flow rate for the laboratory system is 1 L / min. -1 And for industrial-scale gas flow rates of 60 L / min-1 .

[0309] exist Figure 20 Two curves are shown for laboratory scale (30L) and two curves for industrial scale (12000L). Curves 1 and 2 show the measurements at the industrial scale, and curves 3 and 4 show the measurements at the laboratory scale. Gas concentrations were measured at the gas inlet at the bottom (feed) and top (top) of the bioreactor or fermenter. As can be derived from the provided curves, the residence time t r as follows:

[0310] t r,30L =5s

[0311] t r,12kL =21s.

[0312] Therefore, the gas phase residence time for laboratory-scale bioreactors or fermenters has been determined to be 5 s, and the gas phase residence time for industrial-scale bioreactors or fermenters has been determined to be 21 s.

[0313] 6.2. CO2 mass transfer coefficient k L a CO2 Assessment

[0314] CO2 mass transfer coefficient k L a CO2 The assessment has been performed as follows:

[0315] The specific gas boundary interface and volumetric CO2 mass transfer coefficient k in a 2L bioreactor or fermenter have been studied. L a CO2 Measurements were performed. The results are summarized in Table 2 below.

[0316] Table 2: Measurement results of specific gas interface and volumetric CO2- mass transfer coefficient in a 2L bioreactor or fermenter

[0317]

[0318] Based on the above measurements, the CO2 mass transfer coefficient has been confirmed to be 4 ± 0.68 h⁻¹ in a 2L bioreactor or fermenter. -1 .

[0319] 6.3. Estimation of average bubble rising velocity

[0320] Under the following assumptions: the bubble size in the 12000L system is monodisperse with d = 5 mm, and the carbon dioxide mass transfer coefficient k L a CO2 =4±0.68h -1(As mentioned above) This also applies to industrial scale; the theoretical carbon dioxide profile in a single bubble at 37°C can be calculated as follows:

[0321]

[0322] C CO2 carbon dioxide concentration

[0323] C * CO2 saturation concentration of carbon dioxide

[0324] k L Mass transfer coefficient

[0325] 'a' is the area of ​​the interface, therefore a = A / V.

[0326] A bubble Surface area of ​​bubbles

[0327] V bubble Bubble volume

[0328] t time

[0329] Therefore, it can be inferred that the bubble saturation reaches 95% after about 3.5 seconds.

[0330] Based on an average gas phase residence time of approximately 21 s measured in industrial-scale bioreactors or fermenters and a total measured distance of 3.6 m for bubble travel, the average bubble rise velocity can be calculated as follows:

[0331] Velocity u = Distance / Time: u = 0.17 m / s.

[0332] Therefore, at a height of approximately h = 0.6 m (3.6 m × 0.17 m / s), the gas phase becomes saturated with CO2, and thus CO2 stripping can no longer be observed.

[0333] Because the above assessment, measurement and calculation include some evaluation and estimation, the obtained result of 0.6m is only an approximation of the distance η, which is better represented by the range as required.

[0334] Comparative Example 1: Bioreactor containing only one bubbler

[0335] Proprietary BI HEX ( B oehringer- I ngelheim H igh EThe xpression CHO-DG44-derived cell line (expressing antibody-like proteins) was cultured for 11 days in a commercially available 12000L bioreactor using a standard fed-batch process. The bioreactor included Rushton and slant paddle stirrers with a 2:1 H / D (height / diameter ratio). The distance between the lower and upper impellers was 1.8 m. The liquid level (fill height) within the bioreactor at nominal volume was 4.2 m. A bubbler was located below the lowest stirrer. Proprietary technology was used for this experiment. The platform's growth, production, and feed media. Culture began at 9000L and ended at approximately 1100L with feed addition. Throughout the process, the culture temperature was controlled at 36.5±0.5℃, the pH maintained within the range of 7±0.6, and the glucose concentration within the range of 0 to 10 g / L. Oxygen was supplied via jet air and oxygen injection. The dissolved oxygen concentration was maintained at 30%.

[0336] The results are shown in Tables A through D below, and in Figure 21 The following diagrams (A through D) illustrate the process. Cells grow exponentially until day 5, after which the cell number remains roughly constant. During the 11 days of culture, cell viability steadily declines, eventually falling slightly below 80%. Product titers are measured starting on day 3 and increase significantly until day 11. The pCO2 profile of the culture begins at around 10% and declines sharply until day 4, after which pCO2 increases again until it reaches its initial value on day 11.

[0337] Table A: Live-cell growth curves for this run. Data are given as a percentage of the maximum cell density achieved using this run.

[0338] Runtime [d] Viable cell density [%) 0 3 1 6 2 14 3 32 4 56 5 79 6 85 7 87 8 88 9 86 10 100 11 77

[0339] Table B: Cell Viability

[0340] Runtime [d] Cell viability [%) 0 98 1 98 2 98 3 98 4 97 5 97 6 94 7 89 8 83 9 79 10 76 11 77

[0341] Table C: Concentration curves of antibody derivatives produced by cells. Values ​​are given as a percentage of the maximum product concentration achieved during operation.

[0342] Runtime [d] Titration [%] 0 0 1 0 2 0 3 6 4 12 5 21 6 31 7 45 8 58 9 73 10 87 11 100

[0343] Table D: Partial pressure of CO2 in the bioreactor

[0344] Runtime [d] pCO2 [%] 0 9.9 1 8.5 2 6.6 3 6.1 4 5.5 5 6.9 6 7.9 7 8.5 8 8.7 9 9.0 10 9.2 11 9.9

[0345] The results from Tables A to D above are as follows: Figure 21The following is an explanation. The figure shows culture data from an exemplary 12000L manufacturing run according to Comparative Example 1. In (A), the viable cell growth curve for 11 days of culture is given. The data are given as a percentage of the maximum cell density achieved in this run. In (B), the cell viability used for culture is depicted. (C) shows the concentration curve of the antibody derivative produced by the cells. This value is given as a percentage of the maximum product concentration achieved in the run. In (D), the partial pressure of CO2 within the bioreactor is given.

[0346] Example 7: A bioreactor including a first bubbler and a second bubbler

[0347] The experiment according to Comparative Example 1 can be performed, but two bubblers can be used instead of just one, as per the invention. The first bubbler can be located below the lowest agitator and can be a central bubbler or a side bubbler. The second bubbler can be located above the first bubbler at a distance η in the bioreactor or fermenter, whereby η can be selected to be in the range of at least about 0.4 m to at most about 0.5 m below the filling height of the bioreactor or fermenter. The second bubbler is either a central bubbler or a side bubbler.

[0348] The presence of two bubblers in a bioreactor increases the area in the liquid medium where CO2 stripping will occur. If the second bubbler is placed near the surface of the liquid, for example, about 0.5 m below the fill height of the bioreactor or fermenter, it can strip the downstream area, while the first bubbler, placed near the bottom of the bioreactor, can strip the upstream area of ​​the liquid medium. In summary, the entire fill height of the bioreactor or fermenter undergoes CO2 stripping.

[0349] It is anticipated that the presence of a second bubbler located at a distance η (selected within the range described above) above the first bubbler will have a significant impact on CO2 stripping. That is, compared to the embodiment of Comparative Example 1, it is presumed that the partial pressure of CO2 in the culture (liquid medium) of the bioreactor will be reduced by at least about 0.5%, or at least about 1%, or at least about 2% up to about 20%.

[0350] Furthermore, higher product titers and higher product yields are expected compared to Control Example 1. Compared to the implementation of Control Example 1, the concentration of antibodies or antibody derivatives produced by cells can be presumed to increase by at least about 1%, or at least about 5%, or at least about 10% up to about 30%.

[0351] In the current context, even small improvements in methods used at large scale (such as at least about 0.5% or at least about 1%) represent a meaningful contribution. Even small improvements in methods, such as stripping performance and yield, are highly relevant improvements in large-scale production and must be considered important.

[0352] With the second bubbler selected as a side bubbler, it is expected that the advantageous technical effects disclosed herein, particularly the reduction in CO2 partial pressure and the increase in product titer in the culture (liquid medium) of the bioreactor, will be more pronounced.

[0353] Example 8: Change in the distance η between the first and second bubblers

[0354] An experiment according to Example 7 can be performed, wherein the distance η between the first bubbler and the second bubbler will be changed. The first bubbler may be located below the lowest stirrer; the second bubbler may be located above the first bubbler at a distance η in the bioreactor or fermenter. The second bubbler is located above the first bubbler at a distance η, where η is approximately 2 / 3 of the filling height of the bioreactor or fermenter, approximately 1 / 2 of the filling height of the bioreactor or fermenter, approximately 3.0 m, approximately 2.9 m, approximately 2.8 m, approximately 2.7 m, approximately 2.6 m, approximately 2.5 m, approximately 2.4 m, approximately 2.3 m, approximately 2.2 m, approximately 2.1 m, approximately 2.0 m, approximately 1.9 m, approximately 1.8 m, approximately 1.7 m, approximately 1.6 m, approximately 1.5 m, approximately 1.4 m, approximately 1.3 m, approximately 1.2 m, approximately 1.1 m, approximately 1.0 m, approximately 0.95 m, approximately 0.90 m, approximately 0.85 m, approximately 0.80 m, approximately 0.75 m, approximately 0.70 m, approximately 0.65 m, approximately 0.6 m, approximately 0.55 m, approximately 0.45 m, and approximately 0.4 m.

[0355] The presence of two bubblers in the bioreactor increases the area in the liquid medium where CO2-stripping will occur. The second bubbler can be placed at a distance η, thereby allowing η to be chosen such that the areas where CO2-stripping is performed by the first and second bubblers overlap to a certain extent.

[0356] Therefore, it is anticipated that the presence of a second bubbler located at a distance η (having one of the above values) above the first bubbler will have a significant impact on CO2 stripping. That is, compared to the embodiment of Comparative Example 1, it is presumed that the partial pressure of CO2 in the culture (liquid medium) of the bioreactor will be reduced by at least about 0.5%, or at least about 1%, or at least about 2% up to about 20%.

[0357] Furthermore, higher product titers and higher product yields are expected compared to Control Example 1. Compared to the implementation of Control Example 1, the concentration of the antibody derivative produced by cells can be presumed to increase by at least about 1%, or at least about 5%, or at least about 10% up to about 30%.

[0358] Furthermore, even small improvements in methods used on a large scale (such as at least about 0.5% or at least about 1%), as is the case now, represent a meaningful contribution. Even small improvements in the method, such as stripping performance and yield, are highly relevant improvements in large-scale production and must be considered significant.

[0359] With the second bubbler selected as a side bubbler, it is expected that the advantageous technical effects disclosed herein, particularly the reduction in CO2 partial pressure and the increase in product titer in the culture (liquid medium) of the bioreactor, will be more pronounced.

[0360] Comparative Example 2: The bioreactor includes two bubblers, but the distance η is outside the scope of the claims.

[0361] The experiment according to Example 7 can be performed, wherein the distance η between the first and second bubblers is outside the scope of the claims. Specifically, the distance η is less than 0.4 m, such as 0.35 m, 0.3 m, 0.2 m, or 0.1 m. It is anticipated that the technical effects will not be achieved due to the presence of the second bubbler, i.e., the advantages resulting from the reduction in the partial pressure of CO2 in the culture (liquid medium) of the bioreactor, as well as the higher product titers and higher product yields, will not be obtained. The positive effects of having two bubblers simultaneously in the bioreactor will not occur. In fact, the performance of the bioreactor will be close to that of a bioreactor with only one bubbler as described in Comparative Example 1. Therefore, the lower value of 0.4 m can be considered a critical value.

[0362] Reference Symbol List

[0363] 10, 10.1, 10.2, 10.3 Bubbles from the first bubbler

[0364] 20, 20.1, 20.2, 20.3 Bubbles from the second bubbler

[0365] 100 Bioreactors or Fermenters

[0366] 102 Containers

[0367] 105 Bottom section

[0368] 110 sidewall

[0369] 120 mixer

[0370] 140 Gas Supply Pipe

[0371] 150 First Bubbler

[0372] 160 Second Bubbler

[0373] 170 Third Bubbler

[0374] 180 Bubble Collector (Function Box)

[0375] 185 PreSens port

[0376] 189 Exhaust Gas

[0377] A Central Axis

[0378] r s Mixer radius

[0379] d s stirrer diameter

[0380] d1 and d2 are determined by the radius r of the stirrer. s Limited distance

[0381] D. Diameter of the bioreactor or fermenter

[0382] R1, R2, R3, R4 mixers

[0383] η is the distance between the two bubblers.

[0384] η 1,2 The distance between the first bubbler and the second bubbler

[0385] η 2,3 The distance between the second and third bubblers

[0386] η 3,4 The distance between the third and fourth bubblers

[0387] Legends for some of the figures:

[0388] Figure 2 :

[0389] ◆w 0 g =0.96mm s -1

[0390] ●w 0 g =0.75mm s -1

[0391] w 0 g =0.53mm s -1

[0392] □w 0 g =0.32mm s -1

[0393] Mass transfer measurement:

[0394] System: 0.9% NaCl - Water / Air

[0395] Mixer: Rushton / Slant Paddle

[0396] Volume: 2L

[0397] Temperature: 37℃

[0398] Figure 3 :

[0399] ◆w 0 g =0.95mm s -1

[0400] ●w 0 g =0.64mm s -1

[0401] w 0 g =0.32mm s -1

[0402] Mass transfer measurement:

[0403] System: 0.9% NaCl - Water / Air

[0404] Mixer: Rushton / Slant Paddle

[0405] Volume: 12000L

[0406] Temperature: 37℃

[0407] Figure 4 :

[0408] Mass transfer measurement:

[0409] System: 0.9% NaCl - Water / Air

[0410] Mixer: Rushton / Slant Paddle

[0411] Apparent gas velocity: 0.96 mm / s -1

[0412] Volume: 12000L and 2L

[0413] Temperature: 37℃

[0414] Figure 14a :

[0415]

[0416] Figure 14b :

[0417]

[0418] Figure 15a :

[0419]

[0420] Figure 15b :

[0421]

[0422] Figure 18 :

[0423] Dwell time measurement:

[0424] Filling volume: 12m³ 3

[0425] Mixer: Rushton / Slant Paddle

[0426] Mixer frequency: 60 rpm

[0427] Gas flow rate: 60 L / min

[0428] Medium: DI-water

[0429] Temperature: T = 37℃

[0430] Figure 19 :

[0431] Dwell time measurement:

[0432] Filling volume: 12m³ 3

[0433] Mixer: Rushton / Slant Paddle

[0434] Mixer frequency: 60 rpm

[0435] Gas flow rate: 60 L / min

[0436] Medium: DI-water

[0437] Temperature: T = 37℃

[0438] Figure 20 :

[0439] -1- n=300rpm / q=1l min -1 Feeding

[0440] -2- n=300rpm / q=1l min -1 top

[0441] -3- n=80rpm / q=60l min -1Feeding

[0442] -4- n=80rpm / q=60l min -1 top

Claims

1. A bioreactor or fermenter (100) for culturing suspended cells or microorganisms in a liquid medium on an industrial scale, comprising: Container (102) containing a culture in a liquid medium having a defined filling height; A stirrer (120) is provided in a container to stir a liquid medium; The first bubbler (150), which is arranged in the bottom portion (105) of the container (102), is configured to continuously supply bubbles (10, 10.1, 10.2, 10.3) to the liquid medium, the gas being selected from air and / or oxygen; as well as A second bubbler (160) is arranged in the container (102) and positioned above the first bubbler (150) to continuously supply additional air bubbles and / or additional oxygen bubbles (20, 20.1, 20.2, 20.3) to the liquid medium. thus The second bubbler (160) is located above the first bubbler (150) in the bioreactor or fermenter (100) at a distance of At this location, It is selected to be within the range of at least 0.4 m above the first bubbler (150) to at most 0.5 m below the filling height of the bioreactor or fermenter (100).

2. Bioreactor or fermenter (100) according to claim 1, characterized in that is chosen to be in the range: 0.4 m above the first sparger to 2 / 3 of the fill height of the bioreactor or fermenter (100).

3. The bioreactor or fermenter (100) according to claim 1, characterized in that selected to be in the range of 0.4 m above the first sparger to 1 / 2 of the fill height of the bioreactor or fermenter (100).

4. The bioreactor or fermenter (100) according to claim 1, characterized in that was chosen to be in the range: 0.4 m to 3.0 m above the first bubbler.

5. The bioreactor or fermenter (100) according to claim 1, characterized in that, It was selected to be within the range of 0.4 m to 2.5 m above the first bubbler.

6. The bioreactor or fermenter (100) according to claim 1, characterized in that was chosen to be in the range: 0.4 m to 2.0 m above the first bubbler.

7. The bioreactor or fermenter (100) according to claim 1, characterized in that, was chosen to be in the range: 0.4 m to 1.5 m above the first bubbler.

8. The bioreactor or fermenter (100) according to claim 1, characterized in that, was chosen to be in the range: 0.4 m to 1.0 m above the first bubbler.

9. The bioreactor or fermenter (100) according to claim 1, characterized in that, was chosen to be in the range: 0.45 m to 0.90 m above the first bubbler.

10. The bioreactor or fermenter (100) according to claim 1, characterized in that, It was selected to be within the range of 0.5 m to 0.80 m above the first bubbler.

11. The bioreactor or fermenter (100) according to claim 1, characterized in that, was chosen to be in the range: 0.55 m to 0.70 m above the first bubbler.

12. The bioreactor or fermenter (100) according to claim 1, characterized in that, was chosen to be in the range: 0.6 m above the first bubbler.

13. The bioreactor or fermenter (100) according to claim 1. Its features The bioreactor or fermenter (100) includes a filling height ranging from 8 m to 20 m.

14. The bioreactor or fermenter (100) according to claim 1. Its features The bioreactor or fermenter (100) includes a filling height ranging from 9 m to 15 m.

15. The bioreactor or fermenter (100) according to claim 1. Its features The bioreactor or fermenter (100) includes a filling height ranging from 9.5 m to 12 m.

16. The bioreactor or fermenter (100) according to claim 1. Its features The bioreactor or fermenter (100) includes a filling height of 10 m.

17. The bioreactor or fermenter (100) according to any one of claims 1-16. Its features The second bubbler (160) is a side bubbler.

18. A bioreactor or fermenter (100) according to any one of claims 1 to 17. Its features A third bubbler (170) and one or more optional additional bubblers are disposed above the first bubbler (150) and the second bubbler (160) in the bioreactor or fermenter (100), two consecutively arranged, i.e., one above the other, the distance between the bubblers being selected as follows: .

19. The bioreactor or fermenter (100) according to any one of claims 1 to 18. Its features The stirrer (120) has a stirrer radius r s positioned around a central axis A through the bioreactor or fermenter (100), Thus, the first bubbler (150) is positioned at a distance A from the central axis of the bioreactor or fermenter (100), such that the provided bubbles (10, 10.1, 10.2, 10.3) are at or below the agitator radius r. s It enters the liquid medium at a distance; and / or Thus, the second bubbler (160) and optional additional bubblers are arranged at a distance A from the central axis, such that the provided bubbles (20, 20.1, 20.2, 20.3) are greater than the stirrer radius r. s It enters the liquid phase at a distance of [distance].

20. The bioreactor or fermenter (100) according to any one of claims 1 to 19. Its features In addition to the aforementioned agitators (120, R1), one or more additional agitators (R2, R3, R4) are provided, which are located above and / or below the second bubbler (160).

21. The bioreactor or fermenter (100) according to any one of claims 1 to 20. Its features The first bubbler (150), the second bubbler (160), and optional additional bubblers are static bubblers selected from the following: tubular bubblers, sintered plates, perforated plates, annular bubblers, spider bubblers, disc bubblers, plate bubblers, cup bubblers, and bushing bubblers.

22. The bioreactor or fermenter (100) according to claim 21, characterized in that The tubular bubbler includes an open-tube bubbler.

23. The bioreactor or fermenter (100) according to any one of claims 1 to 21. Its features The first bubbler (150) is a central bubbler or a side bubbler, and the second bubbler (160) and optional third bubbler (170) and the additional bubbler is a side bubbler.

24. A method for controlling and adjusting the content of dissolved CO2 and dissolved O2 in a liquid medium in a bioreactor or fermenter (100) for industrial-scale cultivation of suspended cells or microorganisms, said bioreactor or fermenter comprising a container (102) containing a culture in a liquid medium; said method comprising: Stirring liquid media; Bubbles (10, 10.1, 10.2, 10.3) are continuously supplied to the liquid medium from a first bubbler (150) arranged in the bottom portion (105) of the container (102), the gas being selected from air and / or oxygen; Bubbles (20, 20.1, 20.2, 20.3) are continuously supplied to the liquid medium from a second bubbler (160) arranged in a container (102), the gas being selected from air and / or oxygen, whereby the second bubbler (160) is arranged above the first bubbler (150), and the second bubbler (160) is a side bubbler; gas flow rate q based on the submerged sparger or first sparger (150) sub and gas flow rate q of the side sparger or second sparger (160) side , the modified gas flow rate q mod (O2) and the modified gas flow rate q mod (CO2), both applicable to the method, whereby the following equations are applied: q mod (O2) = q sub +C O2 × q side [1a] as well as q mod (CO2) = q sub +C CO2 x q side [1b] in q sub gas flow rate to the submerged bubbler or first bubbler (150); q side gas flow rate indicative of the side bubbler or second bubbler (160); C O2 C represents the influence factor of volumetric oxygen mass transfer, whereby C O2 = 0.15; and C CO2 represents the influence factor of the volume carbon dioxide mass transfer, whereby C CO2 = 0.

6.

25. The method according to claim 24, Its features q sub adjusted to be greater than q side .

26. The method according to any one of claims 24 or 25, Its features The second bubbler (160) is positioned in the bioreactor or fermenter (100) at a distance above the first bubbler (150). Place, from here It is selected to be within the range of at least 0.4 m above the first bubbler (150) to at most 0.5 m below the filling height of the bioreactor or fermenter (100).

27. The method according to claim 26, characterized in that, is chosen to be in the range: 0.4 m above the first sparger to 2 / 3 of the fill height of the bioreactor or fermenter (100).

28. The method according to claim 26, characterized in that, selected to be in the range of 0.4 m above the first sparger to 1 / 2 of the fill height of the bioreactor or fermenter (100).

29. The method according to claim 26, characterized in that, was chosen to be in the range: 0.4 m to 3.0 m above the first bubbler.

30. The method according to claim 26, characterized in that, was chosen to be in the range: 0.4 m to 2.5 m above the first bubbler.

31. The method according to claim 26, characterized in that, was chosen to be in the range: 0.4 m to 2.0 m above the first bubbler.

32. The method according to claim 26, characterized in that, was chosen to be in the range: 0.4 m to 1.5 m above the first bubbler.

33. The method according to claim 26, characterized in that, It was selected to be within the range of 0.4 m to 1.0 m above the first bubbler.

34. The method according to claim 26, characterized in that, was chosen to be in the range: 0.45 m to 0.90 m above the first bubbler.

35. The method according to claim 26, characterized in that, was chosen to be in the range of 0.5 m to 0.80 m above the first bubbler.

36. The method according to claim 26, characterized in that, was chosen to be in the range: 0.55 m to 0.70 m above the first bubbler.

37. The method according to claim 26, characterized in that, was chosen to be in the range: 0.6 m above the first bubbler.

38. The method according to any one of claims 24 to 26, Its features A third sparger (170) and optionally one or more additional spargers are disposed in the bioreactor or fermenter (100) above the first sparger (150) and the second sparger (160), the distance between the two consecutive, i.e. one arranged above the other, spargers being selected to be .

39. The method according to any one of claims 24 to 38, Its features The stirrer (120) has a stirrer radius r s positioned around a central axis A through the bioreactor or fermenter (100), Thus, the first bubbler (150) is positioned at a distance A from the central axis of the bioreactor or fermenter (100), such that the provided bubbles (10, 10.1, 10.2, 10.3) are at or below the agitator radius r. s It enters the liquid medium at a distance; and / or Thereby, the second sparger (160) is arranged at a distance from the central axis A which is such that the provided gas bubbles (20, 20.1, 20.2, 20.3) enter the liquid phase at a distance larger than the stirrer radius r s .

40. The method according to any one of claims 24 to 39, Its features In addition to the first agitator (120, R1), one or more additional agitators (R2, R3, R4) are provided, which are located above and / or below the second bubbler (160).

41. The method according to any one of claims 24 to 40, Its features The first bubbler (150) is a central bubbler or a side bubbler, and the second bubbler (160) and optional third bubbler (170) and optional additional bubbler are side bubblers.

42. The method according to any one of claims 24 to 41, Its features The first bubbler (150), the second bubbler (160), and optional additional bubblers are static bubblers selected from the following: bubblers with tubular geometry, sintered plates, perforated plates, annular bubblers, spider-type bubblers, disc-type bubblers, plate-type bubblers, cup-type bubblers, and bushing-type bubblers.

43. The method of claim 42, wherein, The bubbler with tubular geometry includes a tubular bubbler.

44. The method of claim 43, wherein, The tubular bubbler includes an open-tube bubbler.

45. Method for cultivating cells or microorganisms in a bioreactor or fermenter (100) according to any of the preceding claims 1 to 23, wherein, A second bubbler (160) and an optional third bubbler (170) and one or more optional additional bubblers are provided in the bioreactor or fermenter (100) to promote the growth, activity, productivity and / or any other metabolic conditions of the cells or microorganisms to be cultured.

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