阅读说明：本技术 用于细胞培养缩放的系统和方法 (System and method for cell culture scaling ) 是由 C·布劳 N·琼斯 M·史密斯 于 2019-01-03 设计创作，主要内容包括：本发明实施例集合涉及一种用于产生可缩放生物反应器系统的生物生产系统、方法和装置。具体来说,本发明实施例集合使得能够通过小测试规模的生物反应的操作参数与商业规模的生物反应的操作参数之间的匹配来确定商业规模的生物反应性能特性。所述系统和方法不仅仅依赖于使跨规模的生物反应器装置在尺寸上相同,因为这并没有考虑非常小与非常大的体积之间在流体动力学属性上的差异,而是需要彼此结合来调试各种系统(混合总成、鼓泡器系统和顶部空间气流系统)以实现预期结果。(The present set of embodiments relates to a bioproduction system, method, and apparatus for producing a scalable bioreactor system. In particular, the present set of embodiments enables commercial scale bioreaction performance characteristics to be determined by a match between operating parameters of small test scale bioreactions and operating parameters of commercial scale bioreactions. The systems and methods do not rely solely on making the cross-scale bioreactor apparatus the same in size, as this does not account for differences in hydrodynamic properties between very small and very large volumes, but rather requires the tuning of various systems (mixing assemblies, bubbler systems, and headspace gas flow systems) in conjunction with one another to achieve the desired results.)

1. A scalable bioreactor system for transitioning from testing to commercial production, comprising:

a first bioreactor, comprising:

a first bioprocessing container having a first end, a second end, and a sidewall;

a first configurable mixing assembly suspended between the first end and the second end of the first bioprocessing container; and

a first impeller having a first diameter, the first impeller attached to the first configurable mixing assembly at a first location, wherein the first diameter and the first location are selected to achieve a set of operational parameters;

a second bioreactor comprising

A second bioprocessing container having a first end, a second end, and a sidewall, wherein the second bioprocessing container is a different volume than the first bioprocessing container;

a second configurable mixing assembly suspended between the first end and the second end of the second bioprocessing container; and

a second impeller having a second diameter different from the first diameter, the second impeller attached to the second configurable mixing assembly at a second location, wherein the second diameter and the second location are selected to match the set of operating parameters and the operating parameters include power per volume and impeller tip speed.

2. The scalable bioreactor system of claim 1, wherein the first bioprocessing container comprises a first bubbler attached to the first end of the first bioprocessing container and having a first number of holes and each hole having a first diameter, and the second bioprocessing container comprises a second bubbler attached to the first end of the second bioprocessing container and having a second number of holes and each hole having a second diameter.

3. The scalable bioreactor system of claim 2, wherein the first number of pores is different from the second number of pores, the first diameter is different from the second diameter, and the number of pores and pore size are selected such that the first and second bioreactors achieve the same kLa.

4. The scalable bioreactor system of claim 2, wherein the first and second positions are selected to re-entrain bubbles rising from the first and second bubblers.

5. The scalable bioreactor system of claim 1, wherein the first bioreactor comprises a first headspace gas flow means and the second bioreactor comprises a second headspace gas flow means, and each cross-flow sparger operates to provide a different gas flow rate across the headspace to match the CO2 removal rate of the liquid phase to within five percent between the first bioreactor and the second bioreactor.

6. The scalable bioreactor system of claim 1, wherein the second bioreactor comprises a third impeller having a third diameter different from the first diameter, wherein the third impeller is attached to the second configurable mixing assembly, and the third diameter and third attachment position and the second diameter and the second position are selected to match the set of operating parameters.

7. The scalable bioreactor system of claim 1, wherein a ratio of the first impeller diameter to the first bioprocessing vessel width is different than a ratio of the second impeller diameter to the second bioprocessing vessel width.

8. The scalable bioreactor system of claim 1, wherein the set of operating parameters further comprises total fluid flow and T95 mixing time.

9. The scalable bioreactor system of claim 1, wherein the set of operating parameters is selected based on optimal growth conditions for cells.

10. The scalable bioreactor system of claim 9, wherein the cells are eukaryotic and sensitive to shear forces that increase as the impeller tip speed increases.

11. The scalable bioreactor system of claim 1, wherein the first bioprocessing vessel is a bench-scale volume between 0.1 liters and 50 liters and the second bioprocessing vessel is a commercial volume between 50 liters and 10,000 liters.

12. The scalable bioreactor system of claim 1, wherein the first and second bioprocessing containers are rectangular in shape and the first and second configurable mixing assemblies are offset from a central axis to increase total fluid flow.

13. The scalable bioreactor system of claim 1, wherein the aspect ratio of the first and second bioprocessing containers is greater than 1.5.

14. The scalable bioreactor system of claim 1, wherein the first bioprocessing container has an aspect ratio of between 1.5 and 2 and the second bioprocessing container has an aspect ratio of between 1.75 and 4.

15. A method of matching fluid mixing characteristics between bioreactors having different volumes, comprising:

selecting a first bioreactor having an operational parameter set, the first bioreactor comprising:

a first bioprocessing container having a first end, a second end, and a sidewall; and

a first configurable mixing assembly suspended between the first end and the second end of the first bioprocessing container;

selecting a first impeller having a first diameter;

attaching the first impeller to the configurable mixing assembly, wherein the first diameter and the attachment location are selected to conform to the operating parameters;

selecting a second bioreactor comprising:

a second bioprocessing container having a first end, a second end, and a sidewall, wherein the second bioprocessing container is a different volume than the first bioprocessing container; and

a second configurable mixing assembly suspended between the first end and the second end of the second bioprocessing container;

selecting a second impeller having a second diameter, the second diameter being different from the first diameter;

attaching the second impeller to the second configurable mixing assembly, wherein the second diameter and the second attachment position are selected to match the set of operating parameters, wherein the set of operating parameters includes power per volume and impeller tip speed.

16. The method of claim 15, further comprising the step of: selecting a third impeller having the third diameter different from the first diameter and attaching the third impeller to the second configurable mixing assembly, wherein the third diameter and the third attachment location and the second diameter and the second attachment location are selected to match the set of operational parameters.

17. The method of claim 16, wherein the step of adding the third impeller reduces second and third impeller tip speeds required to maintain power per volume and impeller tip speed in the first and second bioreactors.

18. The method of claim 15, wherein a ratio of the first impeller diameter to the first bioprocessing container width is different than a ratio of the second impeller diameter to the second bioprocessing container width.

19. The method of claim 15, wherein the set of operational parameters further includes total fluid flow and T95 mixing time.

20. The method of claim 19, wherein the set of operational parameters is selected based on optimal growth conditions for the cell.

21. The method of claim 20, wherein the cell is eukaryotic and sensitive to shear forces that increase as the impeller tip speed increases.

22. The method of claim 15, wherein the first bioprocessing container is a bench-scale volume of between 0.1 liters and 50 liters and the second bioprocessing container is a commercial volume of between 50 liters and 10,000 liters.

23. The method of claim 22, wherein the first and second biological treatment vessels are rectangular in shape and the first and second configurable mixing assemblies are offset from a central axis to increase overall fluid flow.

24. The method of claim 23 wherein the first and second bioprocessing containers have an aspect ratio greater than 1.5.

25. A method of matching fluid mixing characteristics between bioreactors having different volumes, comprising:

selecting a first bioreactor having operating parameters, the first bioreactor comprising:

a first bioprocessing container having a first end, a second end, and a sidewall; and

a first configurable mixing assembly suspended between the first end and the second end of the first bioprocessing container;

selecting a first sparger having a first number of holes, wherein the holes have a first diameter, wherein the first number and first diameter are selected to conform to the operating parameters, wherein the first sparger is attached to the first end;

selecting a second bioreactor comprising:

a second bioprocessing container having a first end, a second end, and a sidewall, wherein an aspect ratio of the second bioprocessing container is different from an aspect ratio of the first bioprocessing container; and

a second configurable mixing assembly suspended between the first end and the second end of the second bioprocessing container;

selecting a second bubbler having a second number of holes, wherein the holes have a second diameter, and

the second sparger is attached to the first end of the second biological treatment vessel,

wherein the second number of holes is different from the first number of holes and the second diameter is different from the first diameter, wherein the second number of holes and the second diameter are selected to match the operating parameter to within five percent, wherein the operating parameter is kLa.

26. The method of claim 25, wherein the first bioreactor comprises a first headspace gas flow means and the second bioreactor comprises a second headspace gas flow means, and each headspace gas flow means is operated to provide a different gas flow rate across the headspace to match the operating parameter.

27. The method of claim 25, further comprising the step of attaching a second impeller to the second mixing assembly, wherein the second impeller is configured to re-entrain bubbles rising from the second bubbler, wherein the locations of the bubblers and second impeller and the second number and second aperture are selected to match the operating parameters.

28. The method of claim 25 wherein the aspect ratio of the first bioprocessing container is between 1.5 and 2 and the aspect ratio of the second bioprocessing container is between 1.75 and 4.

29. The method of claim 25, wherein the first bioprocessing container is a bench top volume of between 0.1 liters and 50 liters and the second bioprocessing container is a commercial volume of between 50 liters and 10,000 liters.

30. The method of claim 25, wherein the first and second bioprocessing containers are rectangular in shape and the first and second mixing assemblies are offset from a central axis to achieve a desired kLa.

31. The method of claim 25, wherein kLa refers to O2.

32. The method of claim 25, wherein kLa refers to CO 2.

Background

The biopharmaceutical industry uses a wide range of mixing systems for a variety of processes, such as in the preparation of media and buffers and in the growth, mixing, and suspension of cells and microorganisms. Some conventional mixing systems, including bioreactors and fermentors, include a flexible bag disposed within a rigid support housing. An impeller is disposed within the flexible bag and is coupled with the drive shaft. Rotation of the drive shaft and impeller promotes mixing and/or suspension of the fluid contained within the flexible bag.

Scientists and engineers have focused on creating stirred tank reactors that can not only provide a sterile and well controlled environment for cell culture growth, but also provide robust scale-up solutions from research to manufacturing scale. The traditionally good engineering principles have been applied to design reactors in this way based on linear geometry scaling methods (i.e., vessels will have similar height/diameter ratios and similar impeller/trough diameter ratios). This approach works well in many cases, but the operator must often make many complex decisions as to how best to select the operational settings to obtain repeatable results through scaling up. Since it is a biphasic and metabolism-based system, most of the operating parameters interact dynamically and respond generally non-linearly; optimal parameter selection and process results are often too unpredictable, expensive, and time consuming to properly resolve at the operator level.

Those skilled in the art of biopharmaceutical manufacturing recognize the importance of having good scale-up and scale-down models that are capable of simulating or ideally replicating the physiological growth conditions found in large-scale bioreactors. The reason for the importance of scaling down models is often related to the need for rapid screening of multiple parameters (optimizing cell cloning, culture media, key operating parameters or product quality) and it would be beneficial to be able to perform a wide range of experiments under well controlled conditions at lower cost and with less labor. A good scale-down model will also remove the risk of a desired process in which the likelihood of problems occurring on a large scale will be extremely low, which were not previously identified when occurring on a small scale.

Because larger bioreactors are two-phase systems (gas and liquid based systems), it is difficult to scale down the system in a predictable manner, largely due to solubility differences of the gas types and because the fluidic mechanisms and/or physics of the system are very dynamic (non-linear) due to viscosity, shear forces, density, and surface interactions that are affected across scale. While the overall power dissipation rates using conventional bioreactor designs may be similar, the resulting total flows and measured mixing times are rarely similar on all scales. Thus, problems that are not considered problematic in small scale systems typically occur on a large scale because the same pH, nutrient, gas concentration, or shear gradient found on a large scale cannot be easily replicated in a single operating state produced in a small scale bioreactor.

The main design method currently employed is the geometric scaling of the system (slot height to diameter ratio and impeller to diameter ratio); this operates in conjunction with 1) near constant impeller tip speed or 2) near constant P/V (power input/volume). These methods are quite successful because they allow the operator to compensate for some of the dynamics and non-linearities of mass transfer in rotary agitator-based systems when charging air. However, shear force is conventionally referenced based on the tip velocity of the impeller (where tip velocity is a function of the square of the impeller diameter). Power input/volume is more commonly employed because it is advantageous to meet the practical mechanical design scaling up constraints (meaning that power requirements are within reasonable magnitudes across scale), because power transfer is a non-linear function of speed and diameter (and part of the impeller geometry).

Once the general design of the reactor is known, it is common practice to focus on one of two key process parameters. Layer 1 target constants were chosen-cultures sensitive to shear force typically use impeller tip speed, while cultures more resistant to shear force typically use input power/volume (P/V). Mixing efficiency is very important regardless of the scale parameters chosen, and it is optimal to verify that similar or at least reasonable blending times can be achieved across scale (most desirably T90 mixing times <30 seconds, but are generally not achievable across scale due to the overall fluid mixing dissipation issues inherent with water-based fluids). Subsequently, a second layer of targets is usually associated with pH control and dissolved gases — mass transfer kLa for oxygen (primary) and kLa for carbon dioxide (secondary). Some systems are further optimized by using Computational Fluid Dynamics (CFD). This is a good method for determining the impeller power number (Np) and for modeling the fluid flow pattern in the system. The power transfer can be estimated and used to predict the amount of (hybrid) power transferred into the system. Computational or hybrid models (RPT) can also predict flow field direction, velocity and local shear conditions.

Familiar operators often achieve acceptable results with great success if the system is designed correctly, but the overall process is still often plagued by unknown events and time-consuming troubleshooting efforts, especially when it is necessary to facilitate the transfer of the process between different systems as part of a scale-up or to another location.

The problem that is often ignored is the total fluid flow of the system and how it varies across scales. The fluid is displacement and velocity is not linear across scale, so the mixing curve for liquid movement will vary based not only on volume, tip velocity, and diameter. The mixing curve also depends on the spatial distribution of the power within the reactor. In practice, the design should detune the local mixing performance of the smaller volume system to match the overall mixing and mass transfer performance of the large scale system. Since some randomness in mixing or container inversion is beneficial, good design should take into account the effects of obstructions or spoilers in the design.

Disclosure of Invention

In one aspect, a scalable bioreactor system for transitioning from testing to commercial production is disclosed. The system may include: a first bioreactor comprising a first bioprocessing vessel having a first end, a second end, and a sidewall, a first configurable mixing assembly suspended between the first end and the second end of the first bioprocessing vessel, the first impeller having a first diameter, and a first impeller attached to the first configurable mixing assembly at a first location, wherein the first diameter and the first location are selected to achieve a set of operational parameters; and a second bioreactor comprising a second bioprocessing container having a first end, a second end, and a sidewall, and a second configurable mixing assembly suspended between the first end and the second end of the second bioprocessing container, wherein the second bioprocessing container is a different volume than the first bioprocessing container, and a second impeller having a second diameter different from the first diameter, the second impeller attached to the second configurable mixing assembly at a second location, wherein the second diameter and the second location are selected to match a set of operating parameters, and the operating parameters include power per volume and impeller tip speed. In some embodiments, the first bioprocessing container includes a first sparger attached to a first end of the first bioprocessing container and having a first number of holes, and each hole having a first diameter, and the second bioprocessing container includes a second sparger attached to a first end of the second bioprocessing container and having a second number of holes, and each hole having a second diameter. In some embodiments, the first number of wells is different from the second number of wells, the first diameter is different from the second diameter, and the number of wells and the size of wells are selected such that the first and second bioreactors achieve the same kLa. In some embodiments, the first and second positions are selected to re-entrain bubbles rising from the first and second bubblers. In some embodiments, the first bioreactor comprises a first headspace gas flow means and the second bioreactor comprises a second headspace gas flow means, and each cross-flow sparger operates to provide a different gas flow rate across the headspace to match the rate of CO2 removal of the liquid phase to within five percent between the first bioreactor and the second bioreactor. In some embodiments, the second bioreactor comprises a third impeller having a third diameter different from the first diameter, wherein the third impeller is attached to the second configurable mixing assembly, and the third diameter and third attachment location and the second diameter and second location are selected to match the set of operating parameters. In some embodiments, the ratio of the first impeller diameter to the first bioprocessing container width is different than the ratio of the second impeller diameter to the second bioprocessing container width. In some embodiments, the set of operational parameters further includes total fluid flow and T95 mixing time. In some embodiments, the set of operating parameters is selected based on optimal growth conditions for the cell. In some embodiments, the cell is eukaryotic and sensitive to shear forces that increase with increasing impeller tip speed. In some embodiments, the first bioprocessing container is a bench-scale volume between 0.1 liters and 50 liters and the second bioprocessing container is a commercial volume between 50 liters and 10,000 liters. In some embodiments, the first and second biological treatment vessels are rectangular in shape and the first and second configurable mixing assemblies are offset from the central axis to increase overall fluid flow. In some embodiments, the first and second bioprocessing containers have an aspect ratio greater than 1.5. In some embodiments, the first bioprocessing container has an aspect ratio of between 1.5 and 2 and the second bioprocessing container has an aspect ratio of between 1.75 and 4.

In one aspect, a method of matching fluid mixing characteristics between bioreactors having different volumes is disclosed. The method may include selecting a first bioreactor having a set of operating parameters, the first bioreactor comprising: a first bioprocessing container having a first end, a second end, and a sidewall; and a first configurable mixing assembly suspended between the first end and the second end of the first bioprocessing container. The method may include selecting a first impeller having a first diameter and attaching the first impeller to the configurable mixing assembly, wherein the first diameter and attachment location are selected to conform to operating parameters. The method may comprise selecting a second bioreactor comprising: a second bioprocessing container having a first end, a second end, and a sidewall, wherein the second bioprocessing container is a different volume than the first bioprocessing container; and a second configurable mixing assembly suspended between the first end and the second end of the second biological treatment vessel. The method may include selecting a second impeller having a second diameter different from the first diameter, and attaching the second impeller to a second configurable mixing assembly, wherein the second diameter and the second attachment location are selected to match a set of operational parameters, wherein the set of operational parameters includes power per volume and impeller tip speed. The method may include the step of selecting a third impeller having a third diameter different from the first diameter and attaching the third impeller to the second configurable mixing assembly, wherein the third diameter and third attachment location and the second diameter and second attachment location are selected to match the set of operating parameters. In some embodiments, the step of adding a third impeller reduces the second and third impeller tip speeds required to maintain power per volume and impeller tip speed in the first and second bioreactors. In some embodiments, the ratio of the first impeller diameter to the first bioprocessing container width is different than the ratio of the second impeller diameter to the second bioprocessing container width. In some embodiments, the set of operational parameters further includes total fluid flow and T95 mixing time. In some embodiments, the set of operating parameters is selected based on optimal growth conditions for the cell. In some embodiments, the cell is eukaryotic and sensitive to shear forces that increase with increasing impeller tip velocity. In some embodiments, the first bioprocessing container is a bench-scale volume between 0.1 liters and 50 liters and the second bioprocessing container is a commercial volume between 50 liters and 10,000 liters. In some embodiments, the first and second biological treatment vessels are rectangular in shape and the first and second configurable mixing assemblies are offset from the central axis to increase overall fluid flow. In some embodiments, the first and second bioprocessing containers have an aspect ratio greater than 1.5.

In one aspect, a method of matching fluid mixing characteristics between bioreactors having different volumes is disclosed. The method may include selecting a first bioreactor having operating parameters, the first bioreactor including a first bioprocessing vessel having a first end, a second end, and a sidewall; and a first configurable mixing assembly suspended between the first end and the second end of the first bioprocessing container. The method may comprise the step of selecting a first sparger having a first number of holes, wherein the holes have a first diameter, wherein the first number and the first diameter are selected to meet operating parameters, wherein the first sparger is attached to the first end. The method may comprise the step of selecting a second bioreactor comprising: a second bioprocessing container having a first end, a second end, and a sidewall, wherein an aspect ratio of the second bioprocessing container is different from an aspect ratio of the first bioprocessing container; and a second configurable mixing assembly suspended between the first end and the second end of the second biological treatment vessel. The method may comprise the step of selecting a second bubbler having a second number of wells, wherein the wells have a second diameter and attaching the second bubbler to the first end of the second biological treatment vessel, wherein the second number of wells is different from the first number of wells and the second diameter is different from the first diameter, wherein the second number of wells and the second diameter are selected to match an operating parameter to within five percent, wherein the operating parameter is kLa. In some embodiments, the first bioreactor comprises a first headspace gas flow means and the second bioreactor comprises a second headspace gas flow means, and each headspace gas flow means is operative to provide a different gas flow rate across the headspace to match the operating parameter. The method may include the step of attaching a second impeller to the second mixing assembly, wherein the second impeller is configured to re-entrain bubbles rising from the second bubbler, wherein the locations of the bubblers and the second impeller and the second number and second aperture are selected to match the operating parameters. In some embodiments, the first bioprocessing container has an aspect ratio of between 1.5 and 2 and the second bioprocessing container has an aspect ratio of between 1.75 and 4. In some embodiments, the first bioprocessing container is a bench volume between 0.1 liters and 50 liters and the second bioprocessing container is a commercial volume between 50 liters and 10,000 liters. In some embodiments, the first and second bioprocessing containers are rectangular in shape and the first and second mixing assemblies are offset from the central axis to achieve the desired kLa.

Drawings

To readily identify the discussion of any particular element or act, one or more of the most significant digits in a reference number refer to the figure number in which the element is first introduced.

FIG. 1 illustrates a hybrid system 100 according to one embodiment.

FIG. 2 illustrates a hybrid system 200 according to one embodiment.

Fig. 3 shows a flexible compartment 300 according to an embodiment.

FIG. 4 illustrates a mixing system 400 including a flexible compartment 402 and a spiral assembly 426 according to one embodiment.

FIG. 5 illustrates an exploded view of a screw assembly 500 according to one embodiment.

Fig. 6 illustrates a mixing system 600 including a flexible container 604 and a bubbler, according to one embodiment.

Fig. 7 illustrates a bubbler 700 design according to one embodiment.

Fig. 8 shows a bubbler 800 according to one embodiment.

FIG. 9 illustrates a mixing system 900 including a flexible compartment 902 and an offset drive shaft 910 according to one embodiment.

FIG. 10 shows a mixing system 1000 according to one embodiment comprising a flexible compartment 902 with an offset and angled drive shaft 910.

FIG. 11 shows a hybrid system 1100 incorporating a gas delivery system according to one embodiment.

Fig. 12 shows a gas delivery system 1200 according to an embodiment.

Fig. 13 shows a mixing system 1300 that exhibits bubble trajectories according to a single impeller embodiment.

Fig. 14 shows a hybrid system 1400 exhibiting a bubble trajectory with a sub-optimally placed bubbler.

Fig. 15 shows a hybrid system 1500 exhibiting a bubble trajectory with a second impeller placed suboptimally.

Fig. 16 shows a mixing system 1600 exhibiting bubble trajectories with optimally placed bubbler and impeller sets.

Fig. 17 shows a bubbler layout 1700 according to one embodiment.

Fig. 18A to F show gas distribution patterns using different bubbler locations according to one embodiment.

Fig. 19 shows bubble residence times 1900 from gas generated at various bubbler locations according to one embodiment.

Fig. 20 shows a kLa trend 2000 using various bubbler locations, according to one embodiment.

Fig. 21 shows bubbler performance 2100 according to various embodiments.

Fig. 22 shows mixing consistency 2200 across a volume scale.

Fig. 23 illustrates a matching operational parameter graph 2300, according to one embodiment.

Fig. 24 illustrates a method of matching fluid mixing characteristics between bioreactors having different volumes.

Fig. 25 illustrates a method of matching fluid mixing characteristics between bioreactors having different volumes.

FIG. 26 shows a comparison of Kscore between conventional bioreactors with different volumes.

Fig. 27 shows kLa comparison data across scale.

FIG. 28 shows predicted Kscores between scales in the system described herein.

Detailed Description

Description of the invention

Embodiments of systems, methods, and apparatuses for bioreactor scaling are described in the accompanying description and drawings. In the drawings, numerous specific details are set forth in order to provide a thorough understanding of certain embodiments. The skilled artisan will appreciate that the scalable bioreactor systems and methods described herein can be used for a variety of applications, including, but not limited to, the introduction of cell culture products from laboratory or bench scale into commercial scale production. In addition, the skilled person will understand that certain embodiments may be practiced without these specific details. Moreover, those of skill in the art will readily appreciate that the specific order in which the methods are presented and performed is illustrative and it is contemplated that the order may be varied and still remain within the spirit and scope of certain embodiments.

While the present teachings are described in conjunction with various embodiments, there is no intent to limit the present teachings to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

Moreover, in the various embodiments described, the specification may have presented the method and/or process as a particular sequence of steps. However, to the extent that a method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. Other orders of steps may be possible, as will be appreciated by one of ordinary skill in the art. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. Additionally, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art will readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.

While preferred embodiments of the present invention have been shown and described herein, it should be obvious to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will now recognize numerous variations, changes, and substitutions without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

The invention described herein is a scalable set of bioreactor systems and methods that enable a manufacturer in a bioproduction space to predict the previously discussed physiological growth conditions by altering hardware between scales to achieve similar mixing curves and growth conditions between two or more scales. For example, a scaled bioreactor system may include two or more different sized bioreactors of similar total fluid, similar shear forces at the impeller tips, and similar power input per volume ratios by modifying the geometry of the tank, the size and number of impellers, the ratio of the diameters (widths) of the impellers to the tank, the sparger design, and the CO2 blanket removal system between two or more scales. In other words, two or more bioreactors of the present invention may have distinct physical characteristics while still achieving similar mixing profiles and growth conditions in order to predict commercial scale bioproduction systems at small scale (including bench scale).

The magnitude of the local shear force, the mixed power dissipation ratio and the total fluid flow should be more fully addressed in the scaled-up design. This can be done by optimizing the power distribution of the impellers (increasing the number of impellers in a larger vessel). The overall fluid mixing properties are optimized by limiting how far apart the impellers (increasing in number with increasing volume) are spaced or using CFD or RPT models, and also modifying the size or shape of the impellers (not necessarily shape, but also swept area, blade geometry, surface texture properties or diameter to match the hydrodynamic (vortex) profile created by the fluid movement of the agitator). The main goal is to achieve nearly the same maximum shear level across at least 3 volume-scale values while delivering similar P/V, total flow, T95 mixing time and doing so using reasonable power input values for cell culture and within practical limits imposed by cell culture sensitivity, which may occur in extremely large working volumes (> 1000L). The invention described herein will make the scaling up of the reactor more predictable across scale and therefore easier/logistically debugged because the shear force profiles are similar and the overall fluid movement is nearly the same. The object of the present invention is to solve the now unmet need due to the wide variety of cell culture process sensitivity differences that are inherent in altering cell lines, fluid types, cell densities or media formulations.

The methods herein are designed to achieve the desired result that designing and characterizing reactor scaling-up will be more predictable across scale and therefore easier/logically tuned because the shear force profiles are more similar and the overall fluid movement is nearly the same. The objective here is to address the now unmet need for a wide variety of cell culture process sensitivity differences that are inherently affected when cell lines, fluid types, cell densities or media formulations are altered.

With respect to non-circular containers, it is beneficial to use a rectangular container with a centrally driven agitator that is offset from the centerline for a variety of reasons. An unbalanced design of these two geometries (non-square and non-centered) may produce improved blocking effects and desired mixing inversion (total flow) within the system. Our data supports the idea of improved mass transfer and mixing performance, showing by our performance measurements > 2X improvement in kLa and mixing compared to a circular design at similar working volumes. Scaling the impeller to match tip speed and P/V will produce a proportionally smaller impeller as the vessel's rated working volume decreases. This helps compensate for the inherent changes in fluid mechanics that occur at smaller scales (a proportionally larger impeller-to-vessel wall gap will excite and induce a total flow that is more representative than that of a large scale system). These mixing design features are then combined with optimal sparging locations and this will result in a large increase in bioreactor efficiency at large scale. Two-phase mixing efficiency gains will increase the residence time of the bubbles within the liquid column, and the overall improved bubble distribution from multiple impellers is a highly desirable attribute known to significantly improve mass transfer performance.

Fig. 1 illustrates a fluid mixing system 100, in accordance with various embodiments. The hybrid system 100 generally includes a rigid housing 102; an engine 104 mounted to the rigid housing 102; a first bearing assembly 106 in rotational communication with the motor 104 via a drive shaft 120 and providing rotational movement to the interior of the flexible compartment 118; a hinge 108 to secure the door 110 to the rigid housing 102 and provide a cover for the flexible compartment 118; a rigid housing support 112 for mounting the rigid housing 102 thereto; and support wheels 114 attached to the rigid housing support 112 and providing mobility for mixing. The rigid housing 102 may have a rigid housing opening 122 cut into the rigid housing floor 124 for holding one port 228 or more from the flexible compartment 218 and the second bearing assembly 222. In some embodiments, the rigid housing may be fixed in place and support wheels 114 are not required. In such embodiments, the rigid housing support 112 may be bolted to a screw, or held in place solely by the weight of the rigid housing 102.

Fig. 2 illustrates a cross-sectional view of a fluid mixing system 200, in accordance with various embodiments. The hybrid system 200 includes an engine 202 mounted to a rigid housing 208 having a drive shaft 120 in sterile rotational communication with the interior of a flexible compartment 218 through a first bearing assembly 204. Hybrid system 200 also includes a screw assembly 214 comprised of a yoke 230 and a yoke/impeller 232 to suspend drive train 206 between a first end 234 and a second end 236 of flexible compartment 218. A yoke/impeller 232 may be mounted to the second bearing assembly 222 to provide rotational movement to the screw assembly 214 on the opposite end of the flexible compartment 218. One impeller 216 or more may be mounted to the screw assembly 214 to provide mixing to the fluid within the flexible compartment 218. To facilitate installation of the flexible compartment 218 into the rigid housing 208, a pull handle 220 may be mounted to the second end 236 of the flexible compartment 218, and in some embodiments, may be mounted to the second bearing assembly 222. The rigid housing 208 may be mounted to the rigid housing support 224 and the support wheels 226 may be attached to the rigid housing support 224 to provide mobility to the hybrid system 200. In various embodiments, the flexible compartment 218 further includes at least one port 228 that may protrude through the rigid housing floor 124, 238.

In various embodiments, a user may open the door 110 of the rigid housing 102, 208 for easy installation of the flexible compartment 118, 218. As seen in fig. 1, the top surface 126 of the rigid housing 102, 208 may be fully open on the front when the door 110 is moved to the open position. The top surface 126 may be in the shape of a "U" shaped perimeter that includes a rear portion and two side portions that extend toward the door. When the door 110 is in the open configuration, the flexible compartment 118, 218 may be moved into the chamber of the rigid housing 102, 208. The first bearing assembly 106, 204 located on the first end 116, 234 of the flexible compartment 118, 218 may then be inserted onto the drive shaft 120, 240. Additional disclosure relating to mounting the flexible compartments 118, 218 to the drive shaft 120 may be found in US2017-0183617 filed 2016, 12, 28, specifically incorporated herein by reference in its entirety. Hooks (not shown) attached to the rigid housing 102, 208 may be hooked over loops (not shown) on the flexible compartment 118, 218 to further secure the flexible compartment 118, 218 to the top surface 126 of the rigid housing 102, 208. Once the first end 116 of the flexible compartment 118, 218 is secured to the top surface 126 of the rigid housing 102, 208, the second end 236 may be slid into the rigid housing floor 124, 238. In various embodiments, the flexible compartment 118, 218 will include one port 228 or more and the second bearing assembly 222 projecting from the exterior of the second end 236 of the flexible compartment 118, 218. The rigid housing opening 122 in the rigid housing floor 124, 238 may be configured to receive one port 228 or more and the second bearing assembly 222, thereby securing the second end 236 of the flexible compartment 118, 218 to the rigid housing floor 124, 238 of the rigid housing 102, 208. In some embodiments, a cover (not shown) may cover the rigid housing opening 122 to further secure one port 228 or more and the second bearing assembly 222 to the rigid housing floor 124, 238 of the rigid housing 102, 208. In various embodiments, a user may grasp the pull handle 220 at the second end 236 of the flexible compartment 118, 218 to pull the flexible compartment 118, 218 into place within the rigid housing 102, 208.

In various embodiments, once installation is achieved, fluids may be fed into the sterile flexible compartment 118, 218, which may require mixing. The engine 104, 202 may be started using a controller (not shown) that may then rotate the drive shaft 120, 240 previously inserted into the first bearing assembly 106. In some embodiments, there may be a single drive shaft 120, 240 projecting from the engine 104, 202 and protruding into the sterile flexible compartment 118, 218, and in other embodiments, the first bearing assembly 106 will be enclosed and have a second drive shaft portion 242 extending from the first bearing assembly 106. In various embodiments, the drive shaft 120 or the second drive shaft portion 242 will be mounted to a magnetic yoke 230 that serves to space the first line 210 from the second line 212 of the drive train 206. On the second end 236 of the flexible compartment 118, 218, there may be a second bearing assembly 222 comprising a yoke/impeller 232 that operates to suspend the other ends of the first and second wires 210, 212 and provide mixing as they rotate. The second bearing assembly 222 may be designed to provide rotational movement such that rotation allows the screw assembly 214 to rotate freely as the motor 104, 202 drives the screw assembly 214 from the opposite end. In addition to the yoke/impeller 232, one impeller 216 or more may provide mixing.

An additional advantage of the yoke/impeller 232 in various embodiments is that it provides extremely low volumetric mixing. For example, the biological reaction may require a smaller volume at the beginning of the reaction, and the fluid volume may increase as the biological reaction is fully completed. Currently available bioreactors have limitations in scaling up, and this example reduces the limitations. One impeller 216 or more may also be attached at various locations on the screw assembly 214 when considering the optimal scaling up of a given bioreactor. In some embodiments, the yoke/impeller 232 can maintain uniform mixing in the fluid at very low volumes during the discharge process.

Fig. 3 illustrates a flexible compartment 300 according to various embodiments. The flexible compartment 300 includes a first end 302; an opposite second end 304; a sidewall 306 connecting the first end 302 and the second end 304; at least three panels 308 joining the first end 302 and the second end 304; a sidewall line 310; a centerline 312; and a corner line 314.

In various embodiments, the centerline 312 is an indicator of a vertical axis running from the center of the first end 302 to the center of the second end 304 of the flexible compartment 300. For example, the centerline 312 may be placed such that the lengths from the centerline 312 to the opposing panel 308 are equal. In various embodiments, the sidewall line 310 may be an indicator of a plane running from the first end 234 to the second end 304 of the flexible compartment 300 and extending from the centerline 312 to a midpoint of the panel 308. In various embodiments, the angle line 314 may be a planar indicator that extends from the first end 302 to the second end 304 of the flexible compartment 300 and extends from the centerline 312 to a position where the two panels 308 are joined to form an angle. In various embodiments, the indicators listed above may be used to determine where the spiral assembly 214 will reside in the flexible compartment 300 when reducing blind zones and increasing overall fluid and thereby improving overall mixing efficiency within the mixing systems 100, 200.

Fig. 4 illustrates a hybrid system 400 in accordance with various embodiments. The mixing system 400 includes a flexible compartment 402 having a first end 404 and an opposing second end 406 joined together by a sidewall 408 having at least three panels 410 and a sidewall angle 412 where the panels intersect. The flexible compartment 402 may further include one or more inlets 414, one or more outlets 416, one or more bubblers 418, one or more sensor ports 422, optionally containing a sensor 420 and a vent 424. In various embodiments, the screw assembly 426 may be suspended between a first end 434 and a second end 436 of the flexible compartment 402 and have one or more impellers 428 positioned thereon. In various embodiments, the drive shaft 430 may protrude into the first bearing assembly 432 and the first bearing assembly 432 may provide a sterile connection between the drive shaft 430 on the exterior of the flexible compartment 402 to the magnetic yoke on the interior of the flexible compartment 402. In various embodiments, second bearing assembly 440 may be positioned on second end 406 of flexible compartment 402 and may include a pull 444 protruding onto an exterior portion and an opposing/interior portion of flexible compartment 402, second bearing assembly 440 may be connected to yoke/impeller 442. In various embodiments, the spiral assembly 426 may be comprised of a first line 446 and a second line 448 each having a first end 434 connected to the yoke 438 and a second end 436 connected to the yoke/impeller 442, and during operation, may impart rotational movement to mix the fluid 450 within the flexible compartment 402. In various embodiments, the flexible compartment 402 may include an attachment ring 452 attached to or molded as part of the second bearing assembly 222 used, which may slide into a retaining feature on the rigid housing 102, 208 during installation. In some embodiments, the design may include a snap ring that fits onto the pin and can slide into the bottom port of the flexible compartment 402.

In various embodiments, the flexible compartment 402 may include one or more inlets 414 and outlets 416. The inlet 414 may be used during the installation process to add gas into the flexible compartment 402 in order to expand the flexible compartment 402 to its working volume. In addition, inlet 414 may be used to introduce dry media, buffers, liquid nutrients, or anything else that requires mixing. After the mixing process is complete or the bioreaction is achieved to a desired state, the outlet 416 may be used to collect the contents of the flexible compartment 402. Additionally, the vent 424 may be used to empty the flexible compartment 402 of waste. There are various ways known in the art for attaching the inlet 414, outlet 416, and exhaust 424. A common technique is to weld the components to the flexible compartment 402. For example, the components may comprise a polymer that is weldable to the polymer comprising the flexible compartment 402. US 2017-.

In various embodiments, the sensor 422 may be used to monitor environmental conditions within the flexible compartment 402. There are a variety of sensors and sensor ports 420 available on the market, including those described in US 2008-. Various techniques are described in the above-cited references which disclose the manner in which the sensor port 420 is bonded to the flexible compartment 402 using a welding and adhesion method.

In various embodiments, the mixing system 400 described herein can be used to culture cells and then harvest the cells in their entirety or harvest cell byproducts, such as proteins or enzymes. Such biological reactions typically require the introduction of a gas, which is typically accomplished in the bioproduction art using a bubbler 418. Various bubbler 418 designs and methods of attachment thereof are described in US 2013-.

In various embodiments, the first and second bearing assemblies 432, 440 may include first and second annular sealing flanges 454, 456 that may be sealed to the opening on the flexible compartment 402 by welding or bonding around the perimeter. This allows rotational movement of the hub while the housing remains fixed to the flexible compartment 402, allowing the helix assembly 426 to rotate freely within the flexible compartment 402 while remaining sterile to the outside, as disclosed in US 2017-.

In various embodiments, the attachment ring 452 may be engageable to a retaining feature on the rigid housing 102, 208. The retaining means may take the form of a bracket or some other physical structure capable of retaining and/or limiting the movement of the attachment ring 452. Typically, during the installation process, the user pulls the pull handle 444 into the rigid housing opening 122 to facilitate the attachment loop 452 and retaining means interaction to complete the installation of the flexible compartment 402.

In various embodiments, as depicted in fig. 4, the optimal position of the spiral assembly 426 relative to the flexible compartment 402 will be along the centerline 312.

Fig. 5 is an illustration of an exploded view of a portion of a screw assembly 500 according to various embodiments. The spiral assembly 500 may include a first line 502, a second line 504, one or more rungs 516, one or more stabilizers 524, and one or more impellers 536. Each line may include a first end 506, 510 and a second end 508, 512.

In various embodiments, the helical assembly may include one or more rungs 516 having a first protrusion 518 protruding through the opening 514 on the first line 502 and a second protrusion 520 protruding through the opening 514 on the second line 504. In some embodiments, the rail cover 522 can be snapped onto the projections 518, 520 to secure the rail 516 to the wires 502, 504.

In various embodiments, the screw assembly 500 may include a stabilizer 524 including a cross-beam 526 having a first end 530 protruding through the opening 514 on the first line 502 and a second end 532 protruding through the opening 514 on the second line 504. Stabilizer cap 534 may snap onto ends 530, 532 to secure stabilizer 524 to screw assembly 500. In some embodiments, the rod 528 may protrude from the center and perpendicular to the beam 526.

In various embodiments, the impeller 536 may include a fin 538 having a first attachment 542 protruding through the opening 514 on the first wire 502 and a second attachment 544 protruding through the opening 514 on the second wire 504. In some embodiments, the impeller cover 546 may be snapped onto the attachments 542, 544 to secure the impeller 536 to the screw assembly 500. In various embodiments, the receiver 540 may extend from the center and perpendicular to the impeller 536.

In various embodiments, receiver 540 may be tubular in nature and receive rod 528 from stabilizer 524. As the rate of rotation of the screw assembly 500 varies, the receiver 540 and the rod 528 can slide relative to each other.

FIG. 6 is an illustration of one embodiment of a hybrid system 600 incorporating features of the present invention. The mixing system 600 includes a substantially rigid support housing 602 in which a flexible container 604 is disposed. The rigid support housing 602 has an upper end 606, a lower end 608, and an inner surface 666 that engages the compartment 612. A bottom plate 614 is formed at the lower end 608. A surrounding sidewall 616 extends upwardly from the bottom panel 614 to the upper end 606. As will be discussed in more detail below, one or more openings 618 may extend through the floor 614 or side wall 616 of the rigid support housing 602 to communicate with the compartment 612. An example of a bubbler device and system that may be used in the disclosed invention is disclosed in U.S. patent No. 9,643,133 issued on 5/9/2017, hereby specifically incorporated by reference.

Upper end 606 terminates at a lip 620 that joins inlet opening 622 to compartment 612. If desired, a cover (not shown) may be mounted on the upper end 606 so as to cover the inlet opening 622. Likewise, access may be made at another location on the rigid support housing 602, such as through the side wall 616 or through the floor 614 at the second end. The access port is large enough that an operator can access the access port to assist in manipulating and positioning the flexible container 604. The access opening may be selectively closed by a door or cover.

It should be appreciated that the rigid support housing 602 may have a variety of different sizes, shapes, and configurations. For example, the floor 614 may be flat, frustoconical, or have other slopes. The sidewalls 616 may have a circular, polygonal cross-section or have other configurations. The rigid support housing 602 may be insulated and/or jacketed such that heated or cooled fluid may flow through the jacket to heat or cool the fluid contained in the flexible container 604. The flexible container 604 may be any desired volume, such as those discussed below.

As also depicted in fig. 6, the flexible container 604 is disposed at least partially within a compartment 612 that supports the rigid support housing 602. The flexible container 604 includes a container 624 having one or more ports 626 mounted thereon. In the depicted embodiment, the container 624 includes a flexible bag having an inner surface 610 that engages a chamber 628 adapted to receive a fluid 630 or other type of material. More specifically, the container 624 includes a sidewall 632 that may have a generally circular or polygonal cross-section extending between a first end 634 and an opposing second end 636 when the container 624 is expanded. The first end 634 terminates at a top end wall 638 and the second end 636 terminates at a bottom end wall 640.

The container 624 may be comprised of one or more flexible sheets of water impermeable material, such as low density polyethylene or other polymeric sheets having a thickness generally in the range of between about 0.1mm to about 5mm, with about 0.2mm to about 2mm being more common. Other thicknesses may also be used. The material may consist of a single layer of material or may comprise two or more layers that are sealed together or separated to form a double-walled container. Where the layers are sealed together, the material may comprise a laminated or extruded material. The laminate may comprise two or more separately formed layers which are subsequently secured together by an adhesive.

The extruded material may comprise a single unitary sheet comprising two or more layers of different materials each separated by a contact layer. All layers were co-extruded simultaneously. An example of an extrusion material that may be used in the present invention is HyQ CX3-9 film available from HyClone Laboratories, Inc. of Logan, Utah. The HyQ CX3-9 film is a three layer 9mil cast film produced in a cGMP facility. The outer layer is a polyester elastomer coextruded with an ultra low density polyethylene product contact layer. Another example of an extruded material that may be used in the present invention is HyQ CX5-14 cast film also available from HyClone Laboratories, Inc. The HyQ CX5-14 cast film includes an outer polyester elastomer layer, an ultra-low density polyethylene contact layer, and an EVOH barrier layer disposed therebetween. In yet another example, a multi-web film made from three separate blown film webs may be used. The two inner webs were each a 4mil monolayer polyethylene film (HyClone referred to as HyQ BM1 film) while the outer barrier web was a 5.5mil thick 6 layer coextruded film (HyClone referred to as HyQ BX6 film).

The material may be approved for direct contact with living cells and enables the solution to remain sterile. In this embodiment, the material may also be sterilized, such as by ionizing radiation. Examples of materials that may be used in different situations are disclosed in U.S. patent No. 6,083,587, issued on 7/4/2000 and U.S. patent publication No. US 2003/0077466 a1, issued on 24/4/2003, each of which is hereby specifically incorporated by reference.

In one embodiment, the receptacle 624 comprises a two-dimensional pillow pouch in which two sheets of material are placed in an overlapping relationship and the two sheets are joined together at their peripheries to form an interior chamber 628. Alternatively, a single sheet of material may be folded and sewn around the periphery to form the interior chamber 628. In another embodiment, the vessel 624 may be formed from a continuous tubular extrudate of polymeric material that is cut to length and seamed into a closed end.

In still other embodiments, the container 624 may comprise a three-dimensional bag having not only a circular sidewall but also a two-dimensional top end wall 638 and a two-dimensional bottom end wall 640. For example, the three-dimensional vessel 624 may include a sidewall 616 formed from a continuous tubular extrudate of polymeric material cut to length, as shown in fig. 7. Rounded top and bottom end walls 638, 640 may then be welded to the opposite ends of the side wall 616. In yet another embodiment, the three-dimensional container 624 may be comprised of a plurality of discrete panels (typically three or more, and more typically between four and six). Each panel may be substantially identical and include a portion of the side walls 632, top end walls 638, and bottom end walls 640 of the receptacle 624. The peripheral edges of the abutting panels are sewn together to form a receptacle 624. The gap is typically formed using methods known in the art, such as thermal energy, RF energy, sonic energy, or other sealing energy. In alternative embodiments, the panels may be formed in a variety of different patterns.

It should be appreciated that the container 624 may be manufactured to have virtually any desired size, shape, and configuration. For example, the container 32 may be formed as a chamber 628 having a size of 10 liters, 30 liters, 100 liters, 250 liters, 500 liters, 750 liters, 1,000 liters, 1,500 liters, 3,000 liters, 5,000 liters, 10,000 liters, or other desired volume. The chamber 628 may also have a volume in a range between about 10 liters to about 5,000 liters or about 30 liters to about 1,000 liters. Any other range selected from the above volumes may also be used. Although the receptacle 624 may be any shape, in one embodiment, the receptacle 624 is specifically configured to complement or substantially complement the compartment 612 of the rigid support housing 602.

However, in either embodiment, it is generally desirable that when the container 624 is received within the compartment 612, the container 624 is generally uniformly supported by the supporting rigid support housing 602. The at least substantially uniform support of the reservoir 624 by the rigid support housing 602 helps prevent failure of the reservoir 624 when filled with fluid caused by hydraulic forces applied to the reservoir 624.

Although in the above-discussed embodiments, the container 624 is in the form of a flexible container 604, in alternative embodiments, it should be appreciated that the container 624 may comprise any form of collapsible container, flexible container 604, or semi-rigid container. Further, the container 624 may include an open top liner as opposed to having a closed top end wall 638. The container 624 may also be transparent or opaque and may have an ultraviolet inhibitor incorporated therein.

Mounted on the top end wall 638 are a plurality of ports 626 that are in fluid communication with the chamber 628. Although two ports 626 are shown, it should be understood that there may be one or three or more ports 626, depending on the intended use of the container 624. As such, each port 626 may be used for a different purpose depending on the type of processing to be performed. For example, port 626 may be coupled with a tube 642 for dispensing a fluid or other component into chamber 628 or withdrawing a fluid from chamber 628. Additionally, port 626 may be used to provide various probes, such as a temperature probe, a pH probe, a dissolved oxygen probe, etc., to access chamber 628, such as when container 624 is used as a bioreactor for growing cells or microorganisms. It should be appreciated that the port 626 may have a variety of different configurations and may be placed at any number of different locations on the container 624, including the side wall 616 and the bottom end wall 640.

Although not required, in one embodiment, means for mixing the fluid 630 within the chamber 628 is provided. The means for mixing may be in the form of a mixing assembly. By way of example but not limitation, in one embodiment as shown in fig. 6, a drive shaft 646 projects into the chamber 628 and has an impeller 648 mounted on an end thereof. The dynamic seal 650 forms a seal between the drive shaft 646 and the reservoir 624. The external rotation of the drive shaft 646 facilitates rotation of an impeller 648 that mixes and/or suspends the fluid 630 within the chamber 628. Specific examples of how to incorporate a rotary mixing assembly into a flexible container are disclosed in U.S. patent No. 7,384,783 issued on 10.6.2008 and U.S. patent No. 7,682,067 issued on 23.3.2010, which are specifically incorporated herein by reference.

In yet another alternative embodiment of the means for mixing or mixing assembly, mixing may be achieved by vertically reciprocating a vertical mixer within the chamber 628. Additional disclosure regarding the assembly and operation of the vertical mixer is disclosed in U.S. patent publication No. 2006/0196501, published 2006, 9, 7, which is specifically incorporated herein by reference. In still other embodiments, it will be appreciated that mixing can be achieved by simply circulating the fluid through the chamber 628, such as by using a peristaltic pump to move the fluid into or out of the chamber 628 by rotating a magnetic impeller or stir bar within the vessel 624 and/or by injecting sufficient bubbles within the fluid to mix the fluid. Other conventional mixing techniques may also be used.

Continuing with fig. 6, bottom end wall 640 has a plurality of bubblers incorporated therein. Specifically, the bottom end wall 640 includes a first sheet 652 having a first side 654 and an opposing second side 656. The first sheet 652 overlies a second sheet 658, which likewise has a first side 660 and an opposing second side 662. The first and second sheets 652, 658 typically comprise flexible polymer sheets, such as those discussed above with respect to the container 624. As discussed above with respect to the bottom end wall 640, the first tab 652 may comprise a continuous tab that is welded to the side wall 632 about the peripheral edge 702, as depicted in fig. 7. Alternatively, the first sheet 652 may comprise an integral part of the side wall 616, or may comprise a plurality of separate sheets secured together, attached to the side wall 616 or an integral part of the side wall 616. The second sheet 658 can be welded, for example, to the second side 656 of the first sheet 652 and/or to the side walls 616 along the peripheral edge 704 of the second sheet 658. In other embodiments, the second tab 658 may be welded to the side wall 616 or comprise an integral portion of the side wall 616, as discussed above with respect to the first tab 652, while welding or otherwise securing the first tab 652 to the first side 660 of the second tab 658 and/or the side wall 616.

A top view of the first sheet 652 overlaying the second sheet 658 is depicted in fig. 8. In this embodiment, first sheet 652 and second sheet 658 are welded together by weld line 802. As with the other weld lines discussed herein, the weld line 802 may be formed using any conventional technique, such as laser welding, sonic welding, thermal welding, and the like. The weld line 802 is shown as welding the peripheral or outer edges of the first and second sheets 652, 658 together, but may be formed radially inward from one or both of the peripheral edges or at other locations. As also shown in fig. 8, four separate bubblers 804, 806, 808, 810 are formed by creating additional weld lines between first sheet 652 and second sheet 658.

For example, the bubblers 804, 806, 808, 810 are formed by forming weld lines 812, 814, 816, 818 starting at a first location 820 located at or adjacent to the peripheral edge of the first and/or second sheets 652, 658 and extending along the predetermined path of the bubbler 804 into the interior of the first and second sheets 652, 658 and then circulating back to a second location 822 adjacent to the first location 820 at or adjacent to the peripheral edge of the first and/or second sheets 652, 658. Weld line 812 joins the perimeter of bubbler path 824, which is the area joined between first sheet 652 and second sheet 658 and partially surrounded by weld line 812. In the depicted embodiment, bubbler path 824 includes a gas delivery path 826 extending from a first end 830 to an opposing second end 834. An opening 828 is formed at the first end 830 between the first position 820 and the second position 822 and between the first sheet 652 and the second sheet 658 through which gas can be fed into the gas delivery path 826. Bubbler path 824 also includes a bubbling region 832 formed at second end 834, which is in fluid communication with gas transfer path 826. In the depicted embodiment, the gas delivery path 826 is a narrow elongated path, while the sparging zone 832 forms an enlarged circular area. Other configurations may also be used.

A plurality of perforations 836 extend through the first sheet 652 of the sparging zone 832 such that gas can enter the sparging zone 832 along the gas delivery path 826 and then exit through the perforations 836 to form bubbles within the fluid 630 disposed within the chamber 628. The bubblers 806, 808, 810 are similarly formed, with like reference numerals being used to identify like elements. By using this technique, a plurality of discrete bubblers can be easily formed on the container 624. Each bubbler may be positioned at any desired location and may be of any desired size, shape or configuration. Likewise, although four bubblers are shown, it should be understood that any number of bubblers, such as 1, 2,3, 5 or more, may be formed from first sheet 652 and second sheet 658. The blister zones may be evenly distributed over the first sheet 652 and the second sheet 658, or may be located at defined locations of optimal blisters. For example, the sparger can be disposed directly below the means for mixing such that the mixing or movement of the fluid 630 produced by the mixer helps to entrain the gas bubbles within the fluid 630.

In some embodiments, each bubbler may have the same number of perforations 836, and all perforations 836 may be the same size and shape. In an alternative embodiment, the perforations 836 may be different between two or more different bubblers. For example, different bubblers may have different numbers, sizes, and/or shapes of perforations 836 to optimize performance under different conditions. Larger perforations 836 create larger bubbles that may be optimal for stripping C02 from fluid 630, while smaller perforations create smaller bubbles that may be preferred for oxygenating fluid 630. Likewise, increasing the number of perforations 836 may help mix the fluid with the bubbles and/or increase stripping or oxygenation. In other embodiments, it should be appreciated that one or more of the bubblers 804, 806, 808, 810 may have a combination of different perforations 836. For example, a single bubbler may have both smaller and larger perforations 836. In one embodiment, the smaller bubbles are formed by perforations 836 typically having a diameter of less than 0.8mm, 0.4mm, or 0.2mm, 0.1mm, while the larger bubbles are formed by perforations typically having a diameter of greater than 1.5mm, 0.8mm, 0.4mm, or 0.15 mm. Other diameters of perforations may also be used. The size of the perforations and resulting bubbles depends on the intended use and the size of the reservoir 624. For example, when processing a larger volume of fluid in a larger container, the larger bubbles are typically larger than when processing a relatively smaller volume of fluid in a smaller container. The variance or increment between the diameter of the perforations for small bubbles and the diameter of the perforations for large bubbles is typically at least 0.15mm, 0.3mm, 0.5mm or 1mm and is typically within ± 0.1mm or ± 0.5 of these values. Other variances may also be used.

As discussed in more detail below, the bubblers 804, 806, 808, 810 may be operated simultaneously, or alternatively, a manifold or other regulator may be used so that one or more of the bubblers may be operated without operating the other bubblers. Thus, by having different bubblers with different perforations 836, the selective bubbler may be used at different instances or times to optimize performance.

In some embodiments, it should be appreciated that the gas delivery path 826 of the bubbler 804 is not required. For example, perforations 836 may be formed through the first sheet 652 covering the gas delivery path 826 so as to transfer the gas delivery path 826 in a portion of the bubbling region 832. It should be appreciated that any conventional technique may be used to form the perforations 836. For example, the perforations 836 may be formed as part of the manufacturing process of the sheet, or may be subsequently created by a punch or other technique. In one embodiment, one or more lasers may be used to form the through-holes 836. The advantage of using a laser is that the perforations 836 can be formed at precise locations and with precise diameters so that bubbles of precise predetermined sizes can be formed. In addition, when a laser is used to form the perforations, the material melted by the laser collects around the peripheral edges of the perforations, thereby strengthening the perforations and helping to prevent sheet breakage.

In one embodiment of the invention, a manifold may be used to control the flow of gas to one or more of the bubblers 804, 806, 808, 810. For example, one embodiment of a manifold 664 incorporating features of the present invention is depicted in FIG. 8. The manifold 664 includes a body 838 having an air inlet 840 and a plurality of air outlets 842, 844, 846, 848. The air outlets 842, 844, 846, 848 communicate in parallel with the air inlet 840 through the forked flow path 850. A gas source (e.g., a compressor or a compressed gas tank) is fluidly coupled to the gas inlet 840. The gas may be air, oxygen, or any other gas or combination of gases. The gas lines 706 extend from the gas outlets 842, 844, 846, 848, respectively, to corresponding openings 828 at the first end 830 of each bubbler 804, 806, 808, 810, respectively. Gas lines 706 may be welded between the first 652 and second 658 sheets at the opening 828 to seal the closed opening 828. The gas line 706 may comprise a flexible or rigid tube, and may be integrally formed with or separately attached to the body 838.

Valves 852 are mounted on the body 838 and control the flow of gas to each of the gas lines 706 individually. In one embodiment, the valve 852 may be an electrically actuated valve, such as a solenoid valve, that may be used to open, close, or restrict gas flow to the bubblers 804, 806, 808, 810. In this embodiment, electrical wire 854 may be coupled to valve 852 for controlling the operation thereof. In other embodiments, valve 852 may comprise a manually, hydraulically, pneumatically, or otherwise operated valve. By using the manifold 664, different bubblers or different combinations of bubblers can be used at different times to optimize performance, as described above.

Fig. 9 illustrates a hybrid system 900 in accordance with various embodiments. The mixing system 900 can include a flexible compartment 902 having a first end 904, a second end 906, and a sidewall 908; an offset drive shaft 910 disposed within the flexible compartment 902 and having a first bearing assembly 912 and a second bearing assembly 914, wherein a first impeller 916 is attached to the first end 904 and the second end 906 of the flexible compartment 902, respectively, and the first impeller 916 and the second impeller 918 are attached to the drive shaft 910, wherein the flexible compartment 902 further comprises a bubbler 920 having at least one perforation 922 designed to release a bubble 924 into the fluid 926.

In various embodiments, sparger 920 releases a gas into fluid 926 to add dissolved oxygen into fluid 926. The location of the bubbler 920 on the second end 906 of the flexible compartment 902 may be selected such that the bubble 924 optimally interacts with the first impeller 916 and the second impeller 918. For example, if the biological reaction requires a specified amount of dissolved oxygen, the mixing system 900 may optimize the residence time of the bubbles 924 within the fluid by re-entraining the bubbles 924 with the impellers 916, 918. This may be accomplished by adjusting the position of the bubbler 920 such that all of the bubbles 924 are re-entrained, no bubbles 924 are re-entrained, or a certain percentage of the bubbles 924 are re-entrained. For example, bubbler 920 may be moved away from second bearing assembly 914, which would make bubbles 924 less likely to be re-entrained. In various embodiments, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the bubbles 924 are re-entrained once by the first impeller 916 and/or the second impeller 918, or in any combination. In various embodiments, the impeller rotation rate may be adjusted to affect the amount of re-entrainment that occurs in the mixing system 900. The selection of the combination of bubble 924 diameter and impeller properties may also affect the residence time of the bubbles 924.

In various embodiments, the diameter and number of perforations 922 may be selected based on the type of cells being grown to optimize the operating characteristics of the mixing system 900. For example, if the residence time in the mixing system 900 needs to be increased to achieve the desired dissolved oxygen content in the fluid 926, the perforations 922 may be smaller to create smaller bubbles 924. Residence time may also be increased if the location and nature of the sparger 920 is selected in conjunction with the location of the impeller on the drive shaft 910 to provide more or less re-entrainment as needed for the desired biological reaction.

In various embodiments, the scalable mixing system 900 may include two or more mixing systems 900 as shown in fig. 9, where the volume of the systems may vary. The volume may range between a test size of less than 50 liters and a commercial size of greater than 50 liters. The volume may be selected based on the scale up/down cost. For example, optimal conditions for production of a product using a given cell type can be determined in a smaller test volume size, such that results can be predicted in expensive commercial systems without having to expend resources on a larger volume reaction for optimization. In some embodiments, the impeller tip velocity (shear force) and power input per volume may remain constant while the number of impellers and their diameters may vary between two or more mixing systems 900 (see fig. 23). In some embodiments, the location of the bubblers 920 and the number and size of perforations 922 may vary between test sizes and commercial size mixing systems 900.

Fig. 10 illustrates a hybrid system 1000 in accordance with various embodiments. The mixing system 900 can include a flexible compartment 902 having a first end 904, a second end 906, and a sidewall 908; an offset drive shaft 910 disposed within the flexible compartment 902 and having a first bearing assembly 912 and a second bearing assembly 914, wherein a first impeller 916 is attached to the first end 904 and the second end 906 of the flexible compartment 902, respectively, and the first impeller 916 and the second impeller 918 are attached to the drive shaft 910, wherein the flexible compartment 902 further comprises a bubbler 920 having at least one perforation 922 designed to release a bubble 924 into the fluid 926.

In various embodiments, the drive shaft 910 may be angled and offset within the flexible compartment 902. Such a configuration would change the operating parameters of the biological reaction by altering the amount of re-entrainment of bubbles 924 that occurs. For example, if the bubbler 920 is positioned proximate to the second bearing assembly 914, the first impeller 916 may re-entrain the bubbles 924 while the second impeller 918 may not contact as many bubbles 924 for re-entrainment. Thus, the dissolved oxygen content within the fluid 926 may be altered depending on the angle of the drive shaft 910. Additionally, the blocking effect of the rectangular flexible compartment 902 may be increased, decreased, or the mixing pattern may be altered entirely, based on the amount of offset and angulation of the drive shaft 910 within the flexible compartment 902.

Fig. 11 illustrates a hybrid system 1100 incorporating systems and methods for gas stream mass transfer, in accordance with various embodiments. Although gas flow mass transfer is discussed herein primarily with respect to oxygenating a biological culture, the same methods and systems may also be used to oxygenate other types of liquids (such as those mentioned above). Additionally, as discussed in more detail below, the methods and systems of the present invention are not limited to oxygenating a fluid, but may be used with other gases to affect any type of mass transfer into and/or out of a liquid.

Gas stream mass transfer, particularly over conventional sparging techniques, has a number of processing advantages when it is used to oxygenate biological cultures within a reactor vessel. Where the reactor vessel is designed to handle a culture of cells or microorganisms with relatively large changes in fluid volume, the diameter of the vessel generally needs to be relatively large to maintain the geometry and height requirements. As the diameter of the vessel increases relative to the volume, the depth of the culture within the vessel decreases. Thus, for a very small volume of culture in a container, such as when transferring an initial volume of culture into the container, the residence time of the oxygenating bubbles that bubble from the floor of the container into the culture is typically insufficient to properly oxygenate the cultureOxygen. That is, as the depth of the culture is too shallow, the time of the oxygenated bubbles within the culture is insufficient to completely oxygenate the culture as the bubbles travel from the sparger to the top surface of the culture. Likewise for stripping CO₂The residence time of the larger bubbling bubbles is also insufficient to completely remove the unwanted CO from the culture₂. Due to CO₂Gas is heavier than air, so that CO₂Like a cover layer, on the top surface of the culture, further hindering oxygenation and CO removal of the culture₂Thus, this problem is further exacerbated.

Oxygenation or mass transfer of a gas stream by blowing air or other gas stream containing oxygen over the top surface of the culture becomes more efficient as the depth of the culture or other fluid being treated decreases, as compared to sparging which becomes more efficient as the depth of the culture increases. Thus, gas flow oxygenation is particularly useful for shallow depth cultures disposed within a reactor (including reactors that start at a smaller volume and grow to a larger volume). In addition, bubbling is known to produce unwanted foam on the top surface of the culture, especially when the bubblers used produce extremely small bubbles (sub-millimeter diameter). In contrast, gas stream mass transfer produces minimal foam and can help reduce vessel foam production by reducing the amount of conventional bubbling required. In addition, oxygenation of the gas stream prevents the formation of CO on the surface of the culture₂And (4) a covering layer. In this way, the gas on the surface of the culture is both well controlled and thoroughly mixed, thereby enabling CO to be present₂Dissipated from the culture, mixed into the headspace of the reactor, and exited via the system vent. The interaction of gas stream oxygenation with system liquid also helps to directly promote stripping of CO from the culture₂. Thus, for relatively shallow depth cultures, oxygenation of the gas stream can be used to oxygenate the culture and remove CO from the culture₂In some cases, the need for conventional bubbling in some forms of the present invention is eliminated.

Filling the culture at the bottom of the reactor by oxygenation of the gas stream as the depth of the culture in the reactor increasesThe efficiency of oxygen decreases. Thus, as the depth of the culture increases, dissolved O can be used₂A sensor or other parameter or mechanism to determine when bubbling or other oxygenation methods should be initiated. That is, as the depth of the culture increases, sparging can be initiated, such as by a stepwise incremental increase or by a continuous gradual increase, in order to ensure that the culture is always properly oxygenated. The applied oxygenation of the gas stream may decrease as sparging increases or may remain constant. Even if the gas stream does not completely oxygenate the culture, the gas stream will still balance the upper region of the culture and prevent CO₂This in turn aids in the conventional bubbling operation. Thus, even for relatively deep volumes of culture, gas flow oxygenation can continue to be used in conjunction with bubbling or other oxygenation methods. It should be appreciated that an electronic controller may be used to automatically initiate and/or regulate bubbling and gas flow based on sensor readings.

Examples of systems that may be used to perform gas stream oxygenation/mass transfer will now be discussed. Additional examples of headspace gas flow devices and systems that may be used in the disclosed invention are disclosed in U.S. patent No. 9,388,375 issued on 2016, 7, 12, which is hereby specifically incorporated by reference. One embodiment of a reactor system 10 incorporating features of the present invention is depicted in FIG. 11. Generally, the reactor system 10 includes a support housing 12 that engages a chamber 14; a container assembly 16 disposed within the chamber 14; and a mixing system 17 coupled to the container assembly 16. Support housing 12 typically includes a rigid slot, such as a metal slot. The tank may be jacketed for controlling the temperature of the culture within the vessel assembly 16. Support housing 12 may be any desired size, shape, or configuration that will properly support container assembly 16, as described below.

With continued reference to FIG. 11, the container assembly 16 includes a container 18 having sides 20 extending from an upper end 22 to an opposite lower end 24. The upper end 22 terminates at an upper end wall 33 and the lower end 24 terminates at a lower end wall 34. The container 18 also has an inner surface 26 that engages a compartment 28. The compartment 28 is configured to receive a fluid. The fluid may comprise a biological culture comprising cells or microorganisms, culture medium, and other nutrients and additives. Any other type of fluid requiring mass transfer with a gas may also be used. The fluid may be, for example, a chemical, biological, food, or other fluid. For the examples herein, the fluid will be discussed as biological culture 29. Culture 29 has a top surface 31. A headspace 37 is disposed within compartment 28 and is joined between the top surface 31 and the upper end wall 33 of culture 29.

In the depicted embodiment, container 18 comprises a flexible bag composed of a flexible, water-impermeable material, such as low density polyethylene or other polymeric sheet or film having a thickness in a range between about 0.1mm to about 5mm, with about 0.2mm to about 2mm being more common. Other thicknesses may also be used. The material may consist of a single layer of material, or may comprise two or more layers that are sealed together or separated to form a double-walled container. Where the layers are sealed together, the material may comprise a laminated or extruded material. The laminate comprises two or more separately formed layers which are subsequently secured together by an adhesive. Examples of extrusion materials that may be used in the present invention include HyQ CX3-9 and HyQ CX5-14 films available from HyClonelaboratories, Utah. The material may be approved for direct contact with living cells and enables the solution to remain sterile. In this embodiment, the material may also be sterilized, such as by ionizing radiation. Prior to use, container assembly 16 is typically sealed closed and sterilized such that compartment 28 is sterile prior to introduction of culture 29.

In one embodiment, container 18 may comprise a two-dimensional pillow pouch. In another embodiment, vessel 18 may be formed from a continuous tubular extrudate of polymeric material cut to length. The ends may be sewn closed, or panels may be sealed over the open ends to form a three-dimensional bag. The three-dimensional bag has not only an annular side wall, but also a two-dimensional top end wall and a two-dimensional bottom end wall. Three-dimensional containers may comprise a plurality of discrete panels, typically three or more, and more typically four or six. Each panel is substantially identical and comprises a portion of the side, top and bottom end walls of the container. The corresponding peripheral edges of each panel are sewn together. The gap is typically formed using methods known in the art, such as thermal energy, RF energy, sonic energy, or other sealing energy.

In alternative embodiments, the panels may be formed in a variety of different patterns. Additional disclosure regarding a method of manufacturing a three-dimensional bag is disclosed in U.S. patent publication No. US 2002-0131654a1, published on 9/19/2002, which is specifically incorporated herein by reference in its entirety.

It should be appreciated that the container 18 may be manufactured to have virtually any desired size, shape, and configuration. For example, the container 18 may be formed as a compartment having a size of 10 liters, 30 liters, 100 liters, 250 liters, 500 liters, 750 liters, 1,000 liters, 1,500 liters, 3,000 liters, 5,000 liters, 10,000 liters, or other desired volume. The size of the compartment may also be in a range between any two of the above volumes. Although receptacle 18 may be any shape, in one embodiment receptacle 18 is specifically configured to be substantially complementary to chamber 14 of support housing 12 that receives receptacle 18 such that receptacle 18 is properly supported within chamber 14.

Although in the embodiments discussed above, container 18 is depicted as a flexible bag, in alternative embodiments, it should be appreciated that container 18 may comprise any form of collapsible or semi-rigid container. In still other embodiments, container 18 may be rigid and support housing 12 may be omitted.

Continuing with FIG. 11, formed on container 18 is an example of a plurality of different ports that may be mounted thereon, wherein each port is in communication with a compartment 28. Specifically, mounted on upper end wall 33 are inlet ports 40 and 41 having wires 39A and B, respectively, coupled thereto. Access ports 40 and 41 can be used to deliver gas, media, culture, nutrients, and/or other components into vessel 18, and can be used to withdraw culture 29 or gas from within headspace 37. For example, in some forms of the invention, port 40 may serve as a gas inlet into headspace 37, while port 41 may serve as a gas outlet from headspace 37. Any desired number of access ports may be formed on container 18. Sensor port 42 is formed on side 20 of container 18. The sensor 50 is disposed within the sensor port 42 so as to communicate with the compartment 28 generally at a lower end thereof. It should be appreciated that any number of sensor ports 42 may be formed on container 18, each having a corresponding sensor 50 disposed therein. Examples of sensors 50 that may be used include temperature probes, pH probes, dissolved oxygen sensors, carbon dioxide sensors, cell quality sensors, nutrient sensors, and any other sensor that allows for testing or inspection of a culture or production. The sensors may also be in the form of optical sensors and other types of sensors.

The bubbling ports 43 and 44 are mounted on the lower end wall 34. A first sparger 52 is mounted to port 43 and is designed to deliver small bubbles of gas to culture 29 to oxygenate culture 29. Bubbler 52 may be integrally formed with or attached to port 43. A second sparger 54 is mounted to port 44 and is designed to deliver larger bubbles to culture 29 for stripping CO from culture 29₂. As such, the bubbles from the first bubbler 52 are smaller than the bubbles from the second bubbler 54. In some forms of the invention, second bubbler 54 may be an open tube or a tube having a porous frit with relatively large pores, while first bubbler 52 may be a tube having a porous frit with relatively small pores. The first bubbler 52 may further include a perforated or porous film installed on the end of the port 43 or the inner surface of the lower end wall 34 so as to extend over the port 43. It should be appreciated that bubblers are present in a variety of different configurations, and that any type of bubbler may be used as desired or needed for the intended culture volume, cells and conditions.

Note again that container 18 may be formed with any desired number of ports, and the ports may be formed at any desired location on container 18. The ports may be of the same configuration or of different configurations and may serve a variety of different purposes, such as, but not limited to, those listed above. Examples of ports and how various probes, sensors and wires may be coupled to the ports are disclosed in U.S. patent publication nos. 2006-0270036, 2006-11-30 and 2006-0240546, 2006-10-26, which are specifically incorporated herein by reference in their entirety. The ports may also be used to couple the vessel 18 to a secondary vessel, a condenser system, and other desired fittings.

A plurality of vertically spaced gas ports 45 through 47 are also disposed along the side 20 of the vessel 18. Each of the ports 45 to 47 forms part of a corresponding gas delivery system designed for delivering gas into the compartment 28 to produce gas stream oxygenation/mass transfer. An enlarged view of the gas delivery system 1200, 60A including the gas port 45 is depicted in fig. 12. Port 45 includes a flange 62 mounted to container 18 and a tubular stem 64 projecting outwardly therefrom. The rod 64 engages a passage 66 extending longitudinally therethrough to communicate with the compartment 28. An annular barb 68 is formed on the free end of the stem 64 and is coupled to a tube 70. In turn, tube 70 is coupled with sterile connector 72.

The aseptic connector 72 includes a first connector portion 74 that selectively mates with and fluidly couples a second connector portion 76. A tubular stem 75 projects from the first connector portion 74 and is fluidly coupled to the tube 70. Each of the connector portions 74 and 76 has a sealing layer 78A and B, respectively, which covers the openings of the connector portions 74 and 76. After coupling connector portions 74 and 76 together, sealing layers 78A and B are pulled out from between the connector portions to form a sterile fluid connection between connector portions 74 and 76. Sterile connectors are known in the art. One example of a sterile connector is manufactured by Pall CorporationA connector is provided. A PALL connector is described in detail in U.S. patent No. 6,655,655, the contents of which are incorporated herein by reference in their entirety. Other sterile connectors may also be used.

A tube 80 is fluidly coupled to the second connector portion 76 and extends to a gas supply 82. Gas supply 82 delivers gas through sterile connector 72, port 45, into compartment 28. The gas may be oxygen, or it may be an oxygen containing gas, such as air. Other gases may also be used depending on the desired application. The gas supply 82 may include a pressurized tank, compressor, or other gas supply. Along the tube 80 is disposed a gas filter 84 that sterilizes the gas as it passes therethrough. A valve 86 is also mounted along the tube 80. Valve 86 is used to selectively stop the flow of gas through delivery system 60A and prevent the egress of culture 29 within container 18 through delivery system 60. The valve 86 may have a variety of different configurations. For example, valve 86 may comprise a ball valve, a gate valve, a clamp that clamps pipe 80, or any other type of valve for the intended purpose. The valve 86 may be manually controlled, or may be electric, hydraulic, pneumatic, etc. It should be appreciated that valve 86 may be positioned anywhere along delivery system 60, but is typically located proximate to gas port 45. In one embodiment, valve 86 may be mounted on tube 70 adjacent port 45 or directly on port 45.

As previously discussed, the goal of the gas delivery system 60 is to deliver a flow of gas over the top surface 31 of the culture 29 or other applicable fluid at a sufficient velocity and direction such that the flow of gas creates turbulence on the top surface 31 sufficient to oxygenate the culture to grow cells or microorganisms therein. The term "above" is broadly intended to encompass gas traveling over the top surface 31 in any desired direction (e.g., horizontal, substantially horizontal, downwardly inclined, or upwardly inclined). The gas stream need not flow in a linear path, but may flow in a circular path or vortex (e.g., about a vertical or horizontal axis), or may flow along a random path. The gas flow may be laminar or turbulent, and the direction, flow rate and/or velocity of the gas flow may be constant or variable. For example, the gas flow may change from a downward vertical direction to a substantially horizontal direction. By placing the gas port 64 on the side 20 of the container 18, the gas vented through the channel 66 in this embodiment travels horizontally or substantially horizontally within the compartment 28 so that it may ride over and traverse the top surface 31. In some embodiments, the gas stream may be oxygenated sufficiently to independently oxygenate the culture to the extent needed for growth of cells or microorganisms, without any other form of oxygenation, such as bubbling. In other embodiments, gas vapor oxygenation may be used in conjunction with bubbling or other oxidation processes.

In one embodiment, gasOxygenation of the fluid stream enables mass transfer of oxygen using only air without the need to resort to bubbling with a kLa factor greater than 3 and more typically greater than 5 or 7. Gas flow oxygenation can also maintain a stable oxygen concentration set point within an active culture, without the need for separate sparging, that is within a range of 30% to 50% of air saturation. The above values can be obtained in stirred tank reactors with mixing by means of impellers and in other types of reactors. In one specific example, gas stream oxygenation using air only was able to oxygenate CHO cultures at a target value of 50% air saturation (868mbar ambient pressure) and to oxygenate CO₂Is peeled off to

Cell concentration of 3.5E +06 cells/mL at container volume. At this point, the culture is then fed to the full vessel volume. Notably, at such levels of culture density and vessel fill volume, oxygenation and CO provided by oxygenation of the gas stream₂Too high of peeling; it requires the addition of N mixed with air₂And CO₂To maintain target pH and dissolved O₂A target value.

During operation, compartment 28 of container 18 is filled with culture 29 such that top surface 31 is positioned adjacent to channel 66. In one embodiment, the distance D between the channel 66 and the top surface 31₁In the range of between about 0.75cm to about 15cm, with about 1cm to about 10cm or about 2cm to about 5cm being more common. Other distances may also be used. Furthermore, a distance D₁May vary based on factors such as the size of the vessel 18, the angle of protrusion of the gas (where flow perpendicular to the liquid surface is optimal), the flow rate of the gas, and the superficial velocity of the gas. When measuring the distance D₁When used, the top surface 31 may be the maximum liquid wave height of the culture 29 under agitation, or may be the top surface 31 without agitation. For scalable representation, the flow rate can be measured at the rate of the container volume per minute (VVM) of the maximum rated liquid working volume of the system. The flow rate of gas flowing out through passages 66 is typically in the range of between about 0.06VVM and about 0.2VVM, with about 0.08VVM to about 0.1 VVMVVM or about 0.16VVM to about 0.18VVM is more common. Other flow rates may also be used depending on the intended application. The velocity of the gas exiting the channel 66 or traveling across the top surface 31 within the compartment 28 is typically in a range between about 25 m/sec to about 275 m/sec, with about 25 m/sec to about 175 m/sec or about 30 m/sec to about 100 m/sec being more common. The speed may be greater than 25 m/sec, and more typically greater than 40 m/sec, 60 m/sec, 80 m/sec, or 100 m/sec. To achieve the desired gas velocity exiting passage 66, passage 66 may have a minimum flux exit area in terms of the volume of compartment 12 (i.e., the container volume (VV)). This minimum flux exit area may be about VV (liter)/80 (liter/mm)²) To about VV (liter)/7.8 (liter/mm)²) In a range of between, wherein about VV (liter)/40 (liter/mm)²) To about VV (liter)/30 (liter/mm)²) Or about VV (liter)/8.5 (liter/mm)²) To about VV (liter)/6.25 (liter/mm)²) More commonly. Other areas may be used.

If desired, the port 45 may be configured such that during operation, the rod 64 is angled such that gas expelled therethrough is directed slightly downwardly toward the top surface 31. For example, the rod 64 has a central longitudinal axis 88. The ports 45 may be formed such that, during use, the axis 88 of the rod 64 is inclined to the horizontal by an angle α in the range between 1 ° and about 10 °, such that gas exhausted therethrough flows slightly downwardly to the top surface 31. Other angles may be used.

As previously discussed, gas stream oxygenation is most effective for shallow depths of culture 29 within vessel 18. In one embodiment, the maximum distance D between top surface 31 and lower end wall 34 is measured as the diameter of vessel 18 (i.e., the Vessel Diameter (VD))₂(see fig. 11) may be over a wide range of distances, and at the maximum distance, the gas stream oxygenation may independently oxygenate the culture 29 to grow cells or microorganisms. For example, the maximum distance D₂May range between about vd (cm) 0.3 to about vd (cm) 0.4. In the case where vessel 18 does not have a circular cross-section, VD can be calculated on average diameter. In some embodiments, D depends on the diameter of the container₂And may range between about 5cm and 30cm or between 10cm and 100 cm. Other distances may also be used. At some depths, the system mayTo operate without the use of sparging or other oxygenation systems. In addition, for some depths, due to natural circulation caused by the aeration gas, the desired oxygenation can be achieved throughout the culture without the use of a separate mixer. However, as the depth increases, proper oxygenation of the culture requires both gas vapor oxygenation and a separate mixing system (such as thorough impeller or shaking) to ensure that all cultures are properly oxygenated.

As the depth of culture 29 increases, sensor 50 may detect that additional oxygenation is required even when mixing is achieved. Subsequently, the flow of sparging gas through spargers 52 and 54 can be regulated using an electrical or manual regulator for further controlling oxygenation and CO in culture 29₂And (4) content. Although bubbling with air or oxygen may not be required at shallow depths when using gas vapor oxygenation, bubbling with nitrogen (e.g., by bubbler 54) may be used at all depths to control oxygen in the culture, i.e., to strip off excess oxygen produced by gas vapor oxygenation. Although gas delivery system 60A is shown in fig. 11 as being located at or corresponding to distance D₂But two or more gas delivery systems 60A may be positioned at or near that same elevation and operated simultaneously.

Gas delivered to container 18 by gas delivery system 60A may be drawn through inlet port 41 so that container 18 does not over-inflate. Due to the relatively large amount of gas passing through vessel 18, the evaporation rate of the medium may be higher relative to conventional systems. As such, the reactor system 10 may operate with a condenser coupled to the inlet port 41. One example of a condenser that may be used with the reactor system 10 is disclosed in U.S. patent publication No. 2011/0207218a1, published 2011-8-25, which is specifically incorporated by reference herein in its entirety.

Culture 29 continues to grow at a level below that of channel 66 until a defined mass density or other desired value is determined in culture 29. Subsequently, valve 86 may be closed and media and other components added to culture 29 until the level of top surface 31 rises to within an operating distance from second gas delivery system 60B shown in fig. 11. Subsequently, gas delivery system 60B is activated to again pass the gas stream over top surface 31, and thereby continue gas stream oxygenation of culture 29. This process may then continue for a subsequent gas delivery system 60C. Likewise, any number of additional gas delivery systems may be vertically spaced along the side 20 of the vessel 18 for continuing gas flow oxygenation at other elevation angles.

Fig. 13 illustrates a fluid mixing system 1300, in accordance with various embodiments. In various embodiments, the mixing system 1300 may include a compartment 1302 having a drive shaft 1304 with an impeller 1306 and a sparger 1308 with a bubble path 1310 that illustrates the trajectory of bubbles rising from the sparger 1308.

In various embodiments, the compartment 1302 contains an aspect ratio of 1.5 and the bubblers 1308 are positioned relative to the single impeller 1306 in a location that is suboptimal for bubble re-entrainment. Various applications may require that the bubbles not be re-entrained, but more frequent re-entrainment increases the residence time of the bubbles in the mixing system 1300, which is preferred in most cases.

FIG. 14 illustrates a fluid mixing system 1400 according to various embodiments. In various embodiments, the mixing system 1400 can include a compartment 1402 having a drive shaft 1404 disposed therein; a second impeller 1406 and a first impeller 1408 attached to the drive shaft 1404; and a bubbler 1410 attached to the bottom of the compartment 1402 with a bubble path 1412 extending therefrom.

In various embodiments, the bubbler 1410 position relative to the drive shaft 1404 and the first and second impeller 1406, 1408 positions is suboptimal for bubble re-entrainment. Such a system is sometimes preferred when additional mixing is required and no increase in bubble residence time is required. However, in most applications, the relative positions of the bubbler 1410 and the impellers 1406, 1408 may be selected to alter the bubble path 1412 and produce a longer residence time for the bubbles.

FIG. 15 illustrates a fluid mixing system 1400 according to various embodiments. In various embodiments, the mixing system 1500 may include a compartment 1502; a drive shaft 1504 disposed within the compartment; a first impeller 1506 and a second impeller 1508 attached to the drive shaft 1404; a bubbler 1510; and a bubble path 1512.

In various embodiments, the relative positions of bubbler 1510 and first and second impellers 1506, 1508 are sub-optimal for bubble re-entrainment. There are some applications where a shorter bubble residence time is optimal, but in most cases this is not a desirable configuration.

Fig. 16 illustrates a fluid mixing system 1600 in accordance with various embodiments. In various embodiments, the mixing system 1600 can include a compartment 1602; a drive shaft 1604 disposed within the compartment 1402; a first impeller 1606 and a second impeller 1608 attached to the drive shaft 1404; a bubbler 1610; and a bubble path 1612 that depicts an ascending flow of bubbles from the bubbler 1410.

In various embodiments, the fluid mixing system 1600 of fig. 16 is optimized for re-entrainment. The relative positions of bubbler 1410 and first and second impellers 1606, 1608 are selected to affect bubble path 1612 to increase the residence time of the bubbles within mixing system 1600. In most applications, this is an ideal arrangement.

Fig. 17 shows a bubbler layout 1700 for experimental purposes. The bubbler layout 1700 includes an impeller tip sweep 1702, bubbler locations 1704, and compartments 1706.

Fig. 17 sets up an experiment in which a drive shaft (not shown) is offset toward the side walls of the compartment 1706 and various bubbler locations 1704 are operated at once to determine an optimal bubble dispersion pattern. In various embodiments, the corners of the compartment 1706 serve to increase bubble distribution by acting as baffles.

Fig. 18A to 18F show the bubble distribution pattern of each of the bubbler locations 1704 illustrated in fig. 17. The grayed bubbler location indicates the bubbler being operated. The other bubblers are turned off. For example, bubbler location 1 is in operation in fig. 18A, while the other bubbler locations are turned off.

Fig. 19 shows the performance of bubbler location 1704 as shown in fig. 17. The x-axis indicates bubbler position 1704 in use, while the y-axis shows elapsed time. The solid line shows the time (in seconds) it takes for the first released bubble to reach the surface, and the dashed line shows the time it takes for 95% of the bubbles to reach the surface after stopping the gas flow to the bubbler. In general, having the bubbles stay longer in the fluid means that more dissolved oxygen can enter the fluid and nourish the cells, which is preferred for most applications. When creating a scalable system, increasing or decreasing the bubble residence time to match kLa between two or more mixing systems will provide a better predictive model for commercial scale production. As previously discussed, it may be helpful to be able to alter the bubbler and impeller positions to change the re-entrainment properties.

Fig. 20 shows the performance of bubbler location 1704 as shown in fig. 17. The x-axis indicates the bubbler position 1704 in use, and the y-axis shows the normalized kLa values obtained. In general, higher kLa values are preferred. In a scalable system, the bubbler position may be selected to attempt to match kLa values between two or more mixing systems having different geometries and volumes.

Fig. 21 shows comparative data between the volume of a mixing system using the sparging method and system described in the present application and a prior art sparging system. kLa is depicted on the y-axis and flow volume per minute per container volume is depicted on the x-axis. It is clearly demonstrated that the prior art bubbling systems and methods do not produce similar kLa values between 50 liter to 2000 liter systems. However, the bubblers disclosed herein produce very similar values of kLa between systems having different volumes.

Using the methods described above, the aperture and count of the bubbler were scaled, and kLa was measured empirically by comparing the system with FRIT + open tube with the system with FRIT + DHS (bubbler system and method disclosed above).

This approach improves scaling performance across scale, reducing the maximum standard deviation of kLa from 20% to 8.6% across scale, and reducing the average standard deviation of kLa with open tubes or DHS from 16% to 6% across scale.

Fig. 22 shows the mixing uniformity across a volume at a given power input per volume. The hybrid system 200 used to generate the data seen in fig. 22 is the system depicted in fig. 2 and elsewhere in this document. The chart shown is from data taken from: 50 liter vessel and 3 impellers with an aspect ratio of 2.5, 500 liter vessel and 3 impellers with an aspect ratio of 2.5 and 5000 liter vessel and 3 impellers with an aspect ratio of 2.5. The impeller diameter is different on each scale, as is the ratio of impeller diameter to vessel width. As can be seen, at a given power per volume, the t95 mixing time remains constant across the scale.

Fig. 23 shows a table demonstrating constant or nearly constant impeller tip speed and power per volume across 50 liter, 100 liter, 250 liter, 500 liter, 1000 liter and 2000 liter systems. The diameter and number of impellers has been varied to accommodate systems of different sizes.

Fig. 24 illustrates a method 2400 of matching fluid mixing characteristics between bioreactors having different volumes, according to various embodiments. Block 2402 includes the step of selecting a first bioreactor having a set of operating parameters, the first bioreactor comprising: a first bioprocessing container having a first end, a second end, and a sidewall; and a first configurable mixing assembly suspended between the first end and the second end of the first bioprocessing container. Block 2404 includes the step of selecting a first impeller having a first diameter. Block 2406 includes the step of attaching the first impeller to the configurable mixing assembly, wherein the first diameter and attachment location are selected to conform to the operating parameters. Block 2408 includes the step of selecting a second bioreactor, the second bioreactor comprising: a second bioprocessing container having a first end, a second end, and a sidewall, wherein the second bioprocessing container is a different volume than the first bioprocessing container; and a second configurable mixing assembly suspended between the first end and the second end of the second biological treatment vessel. Block 2410 includes the step of selecting a second impeller having a second diameter that is different from the first diameter. Block 2412 includes the step of attaching a second impeller to the second configurable mixing assembly, wherein the second diameter and the second attachment position are selected to match a set of operational parameters to within five percent, wherein the set of operational parameters includes power per volume and impeller tip speed.

Fig. 25 illustrates a method 2500 of matching fluid mixing characteristics between bioreactors having different volumes, according to various embodiments. Block 2502 comprises the step of selecting a first bioreactor having operating parameters, the first bioreactor comprising: a first bioprocessing container having a first end, a second end, and a sidewall; and a first configurable mixing assembly suspended between the first end and the second end of the first bioprocessing container. Block 2504 includes the step of selecting a first bubbler having a first number of apertures, wherein the apertures have a first diameter, wherein the first number and the first diameter are selected to comply with operating parameters, wherein the first bubbler is attached to a first end. Block 2506 includes the step of selecting a second bioreactor comprising: a second bioprocessing container having a first end, a second end, and a sidewall, wherein an aspect ratio of the second bioprocessing container is different from an aspect ratio of the first bioprocessing container; and a second configurable mixing assembly suspended between the first end and the second end of the second biological treatment vessel. Block 2508 includes the step of selecting a second bubbler having a second number of wells, wherein the wells have a second diameter and attaching the second bubbler to the first end of the second biological treatment container, wherein the second number of wells is different from the first number of wells and the second diameter is different from the first diameter, wherein the second number of wells and the second diameter are selected to match an operating parameter to within five percent, wherein the operating parameter is kLa.

A series of predicted and actual data were collected and presented in figures 26, 27 and 28. The following is a way to predict the required pore size to ensure scaling across bioreactors with different volumes.

1. Using regression based on empirical data and literature, the following are predicted:

a. bubble size from pore size and flow rate (regression of internal empirical data)

b. Bubble terminal velocity (talaria, 2007, the terminal velocity of bubble rise in the liquid column, also verified by measuring the velocity of bubbles in the chamber).

c. Residence time based on liquid column height divided by bubble terminal velocity

kLa score is based on:

FIG. 26 shows the cross-scale predictive Kscore comparison data on a conventional bioreactor.

Fig. 27 shows actual kLa comparison data across scale on a conventional bioreactor.

FIG. 28 shows predicted Kscore comparison data across scales on some embodiments disclosed herein.

Using the systems and methods disclosed herein, operating parameters can be kept constant across mixing systems that accommodate different volumes. When moving from a test or bench scale to a commercial scale, the most important aspect is the ability to easily predict the growth conditions of the bioreactor. The scalable system herein allows a user to create such a system by adjusting various aspects of the bubbler design, drive system, and headspace gas flow system, where the components across the system may be completely different, but produce the same or similar results. It should be noted that all systems interact with each other and other aspects need to be considered when making a selection. For example, if increased bubble residence time is desired, the drive system can be altered to re-entrain the bubbles. Additional sparging and gas flow across the headspace may be added if needed to reduce CO2 build-up. To date, users need to determine more important metrics (shear force, power per volume, or kLa) and design scalable systems (vessel aspect ratio, impeller diameter, vessel width/diameter, etc.) that are the same in size. With the disclosed invention, various operating parameters can be kept constant with an understanding of how the various systems affect each other.

While the present teachings are described in conjunction with various embodiments, there is no intent to limit the present teachings to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

Further, in describing various embodiments, the specification may have presented the method and/or process as a particular sequence of steps. However, to the extent that a method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. Other orders of steps may be possible, as will be appreciated by one of ordinary skill in the art. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. Additionally, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art will readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.