Linear Scalable Bioreactor System

Abstract

Scalable single-use bioreactor bags and systems are provided for use in implementing scalable biomanufacturing processes. The small-scale systems model larger-scale systems in terms of shear force, scalability, and performance. The bioreactor systems of the present disclosure allow for improved scalability by using identical or substantially identical vessel geometric ratios and maximum process parameters for small-scale and large-scale biomanufacturing.

Description

[Technical Field]

[0001] FIELD OF THE DISCLOSURE The present disclosure relates to bioreactor systems and methods for processing biological materials. In some embodiments, the bioreactor systems of the present disclosure include disposable components. [Background technology]

[0002] Cell culture is an essential step in the manufacturing of biological products. Bacterial, microbial, and mammalian cells are often cultured for the production of therapeutic proteins. Such proteins can include, for example, monoclonal antibodies (MAbs), erythropoietin (EPO), and interferons. In addition, such cells are cultured for the production of nucleic acids, viruses for vaccine applications, and recombinant protein products for pharmaceutical applications.

[0003] When scaling up a culture from a benchtop bioreactor in the laboratory to a larger commercial production bioreactor, it is important to consider changes in culture conditions due to the increased culture volume.In the biotechnology / pharmaceutical manufacturing industry, the "technical transfer" also known as "technology transfer" of a biopharmaceutical process, such as cell culture, from the research or development level, i.e., small-scale production, to large-scale commercial production is typically a complex, costly, and time-consuming process.

[0004] Related to the above, oxygen is an important and often limiting substrate for growth, production, and maintenance activities in cell culture. Cells obtain oxygen from soluble oxygen, an elemental, non-compound form known as "dissolved oxygen" (DO). One of the most important functions of a bioreactor is to continuously supply dissolved oxygen to cells through aeration. Aeration in bioreactors is typically achieved through two gas supply modes: a) oxygen from an overlay gas supplied over the liquid diffuses and is solubilized at the interface between the cell culture medium and the gas; and b) oxygen is supplied below the liquid level, sometimes into a sparge tube or sparging element. Sparging elements aim to increase the surface area-to-volume contact and residence time of the gas in the liquid by supplying the gas in the form of bubbles, thereby promoting solubilization. The gas supplied below the liquid level can be further solubilized into the cell culture medium or solution medium with the aid of agitation. Agitation disperses oxygen bubbles, improves bubble retention time below the liquid surface, and promotes mass transfer of the gas into the liquid. The oxygen transfer rate (OTR) from the gaseous oxygen to the soluble form of the oxygen substance is determined by the physicochemical properties of the cell culture medium, the geometric and operational parameters of the bioreactor, and the presence of cells. Monitoring of dissolved oxygen (DO) under various operating conditions of agitation, gas flow rate, and medium or medium-simulant solution is performed to assess the OTR in the absence of cells. This OTR is calculated using the oxygen mass transfer coefficient k L There is a correlation between a (volumetric mass transfer coefficient, which indicates the efficiency with which oxygen can be supplied to a bioreactor under given operating conditions) and the oxygen concentration gradient in the liquid.

[0005] Oxygen is continuously supplied to cell cultures because it has low solubility in the liquid phase and increases over time as metabolic consumption by cells increases. Oxygen supply is carefully controlled to optimize cell growth by manipulating gas supply, agitation, or medium addition in response to DO measurements from an immersion sensor. In batch cell cultures, the oxygen utilization / uptake rate (OUR) is low during the early lag phase, when cells divert energy to maintaining internal cellular functions and the cells double or there is virtually no increase in overall culture cell density. As cell density increases during the subsequent log phase, the OUR increases during this period, and the OTR (oxygen transfer rate) increases until it becomes a limiting factor under those conditions. Thus, the OTR is expressed as k L The correlation with a defines the theoretical maximum cell density that can be achieved in a cell culture under OTR test conditions before oxygen supply becomes the limiting control parameter. This correlation with cell density allows the k obtained from multiple gas supply and agitation conditions to be calculated. L The a value is particularly useful in evaluating various bioreactor design features and assessing scalability across possible bioreactor system sizes within a bioreactor platform. Any changes to the process, engineering parameters, or geometry of the system tank and component design will affect the k L a, and therefore k L a provides a good indication of the scalability of the bioreactor design in scaling to a representative volume of the entire bioreactor platform.

[0006] Mixing is used to maintain homogeneity and eliminate gradients in concentration (cells, gases, pH, medium, and nutrients), temperature, and other properties. Gas bubble size and residence time are highly dependent on a set of mixing conditions related to agitator design, including impeller type, speed, and position, and sparge design, porosity, and performance relative to gas flow rate. L The a-value generally increases with increasing agitation speed, but high agitation speeds can increase shear forces at the blade tips, potentially resulting in cell death.

[0007] Furthermore, higher oxygen supply concentrations and gas flow rates increase the amount of available oxygen, thereby increasing the OTR. Increasing the oxygen supply to the bioreactor increases this available oxygen by increasing the concentration gradient (correlated with an increased potential saturation value depending on the fluid composition), and this increase in oxygen supply can be controlled by changing the oxygen concentration in the gas (air vs. O2 enriched) and volumetric flow rate. L While a high a value is desirable, it is important to consider the actual operating conditions, its relevance to cell viability, and the associated process costs. For example, high airflow rates can cause cell damage due to shear forces. They can also result in excessive foaming, which can require high concentrations of antifoam agents, which can inhibit cell growth and lead to batch-to-batch variability in downstream process performance. Furthermore, higher airflow rates require larger exhaust filter areas to prevent bioreactor overpressure, thus increasing consumable costs.

[0008] Therefore, conventional bioreactors are designed to achieve a target k L Various types of impellers, spargers, combinations, and configurations are designed to achieve a value of a while minimizing detrimental shear forces, but these modifications often make scaling between bioreactors within a given platform difficult due to variations in critical design parameters. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there is a continuing need in biopharmaceutical development and manufacturing for scalable systems and process transfer technologies that allow technology transfer between bioreactor sizes for scale-up, scale-down, and scale-out in an easy, reliable, and repeatable manner. Scaling practices, whether for process development, clinical production, or cGMP production, require robustness and consistency of performance between bioreactor scales, along with reduced need for process evaluation, process operation changes, or validation studies. [Means for solving the problem]

[0010] The present invention provides a scalable single-use bioreactor system and method for implementing scalable biomanufacturing processes that solves many of the recognized problems associated with scaling cell culture processes for small-scale commercial production or product / process evaluation. Use of the disclosed system and method minimizes risk and helps ensure that the robustness, purity, and potency of the scaled process are comparable to the original process scale. Technology transfer and scaling using the disclosed system can eliminate or reduce the need for costly and time-consuming process and parameter evaluation on a large scale while achieving optimal results and a safe, cGMP-compliant scaled process. In summary, the disclosed small-scale (e.g., 50 L) bioreactor system achieves "linear scalability" by using or substantially replicating the same vessel and bag geometries, gas sparging systems, impeller shapes and types, and process control systems as those found in larger bioreactors (e.g., 200- to 2000-liter systems). Among other features, the present invention includes the following, either alone or in combination:

[0011] In one aspect, the invention relates to a scalable bioreactor system for use in conducting a scalable biomanufacturing process, comprising a bioprocess bag having flexible walls, a working volume turndown ratio (maximum working volume to minimum working volume) of about 5:1, a liquid level to diameter ratio at the maximum working volume of about 1:1 to 2:1, preferably about 1.7:1, an impeller mounted on an impeller plate attached to the interior bottom surface of the flexible walls, and a ratio of the impeller diameter Di to the installed bioprocess bag diameter Dt of about 0.3 to 0.5, preferably about 0.4.

[0012] The bioprocess bag further comprises at least one sparger attached to the bottom surface of the interior of the flexible wall for introducing gas into the bioprocess bag. In some embodiments, the at least one sparger comprises a first sparger and a second sparger. The first sparger has a sparging surface area ratio (sparging surface area / bag cross-sectional area) of about 0.023 to 0.068, preferably about 0.04. The second sparger has a sparging surface area ratio (sparging surface area / bag cross-sectional area) of about 0.008 to 0.024, preferably about 0.02. Gas introduced through the first and second spargers is controlled to maintain a maximum gas exit velocity. For the first sparger, the target linear velocity is about 12-60 m / s, preferably about 24 m / s, which corresponds to a maximum gas flow rate (gas volumetric flow rate per vessel working liquid volume) of about 0.15-0.55 vvm, preferably 0.2 vvm. For the second sparger channel, the maximum gas exit velocity of the second sparger is about 36-60 m / s, preferably 36 m / s, which allows for a maximum gas flow rate of about 0.05-0.18 vvm, preferably 0.1 vvm. Furthermore, the overlay maximum gas flow rate can be maintained constant and may be set to about 0.05 vvm.

[0013] Throughout this text, "sparging surface area" is defined as the total surface area of ​​the sparger for introducing gas into the bioprocess bag. Additionally, "cross-sectional area of ​​the bag" is defined as the maximum cross-sectional area of ​​the liquid surface within the bioreactor when the bioprocess bag is expanded in place within the outer vessel. In other words, the "cross-sectional area of ​​the bag" is approximately the same as the cross-sectional area of ​​the interior volume of the outer vessel in which the bioprocess bag rests during use.

[0014] Furthermore, the term "about" in reference to all numerical values ​​provided herein includes a numerical value of + / -15% of the stated numerical value, which includes the boundaries of any range.

[0015] According to some embodiments, the first sparger is used for dissolved oxygen control of the culture medium and may deliver a mixture of air and oxygen to accomplish this. The second sparger may be used for carbon dioxide stripping and may deliver nitrogen or air. Maintaining a constant surface area ratio of the sparger across bioreactor scales helps ensure comparable gas dispersion and therefore better scalability.

[0016] Additionally, the distance between the bottom of the impeller blades and the gas-generating surface of the sparging surface is about 8-24 mm, preferably about 16 mm. This distance may be maintained to ensure scalable performance across bioreactor sizes and may not be a scalable or proportional distance depending on the liquid level or impeller height of the bioreactor. This distance may be constant across scales to ensure comparable gas bubble breakdown by the impeller.

[0017] The bioreactor system further includes a vessel for housing the bioprocess bag. The vessel has a height-to-diameter ratio of about 1:1 to 2:1, preferably about 1.7:1, for the jacketed section of the system corresponding to the maximum working liquid volume. The vessel may include a plurality of baffles, the baffles having a height ratio (baffle height / vessel diameter) of about 1 / 10 to 1 / 12, preferably about 1 / 11. Note that the baffle height is the distance the baffle protrudes from the periphery of the vessel toward the center of the vessel.

[0018] Impeller power number (N p ) depends on the impeller shape, the vessel geometry (baffle size, distance between the impeller blades and the vessel surface), and the liquid (density, viscosity, and therefore gas generation conditions). The impeller power number for down-pumping (e.g., impeller rotation in a clockwise direction when viewed in a plan view) is about 0.8 to 3.9, preferably about 2.6, and the impeller power number for up-pumping (e.g., impeller rotation in a counterclockwise direction when viewed in a plan view) is about 0.8 to 2.5, preferably about 1.7.

[0019] In another aspect, the present invention relates to a linear scalable bioreactor system for use in conducting a scalable biomanufacturing process, the system comprising: a first bioprocess bag having a first volume, a first flexible wall, and a liquid aspect ratio of the height of the first bag at the first bag's maximum working volume to the diameter of the first bag that is a first H / D; a first impeller mounted on a first impeller plate mounted against a first interior surface of the first flexible wall, wherein the ratio of the first impeller diameter Di to the first bioprocess bag diameter Dt is Di / Dt; and a first sparger mounted against an interior bottom surface of the flexible wall, wherein the ratio of the sparging surface area As1 of the first sparger to the cross-sectional area Ab of the first bioprocess bag (in an expanded state) is As1 / Ab; a second bioprocess bag having a second volume greater than the first volume of the first bioprocess bag, a second flexible wall, and an aspect ratio of a second bag height to a diameter of the second bag at the second bag's maximum working volume that is a second H / D, wherein the first H / D and the second H / D are substantially equal; a second impeller mounted on a second impeller plate mounted against a second interior surface of the second flexible wall, wherein a ratio of a diameter D2i of the second impeller to a diameter D2t of the second bioprocess bag is D2i / D2t, where D2i / Dt=D2i / D2t; and a second sparger mounted against an interior bottom surface of the second flexible wall, wherein a sparging surface area A2s1 of the second sparger and a diameter D2t of the second bioprocess bag (in an expanded state) are equal to each other. the ratio of the cross-sectional area A2b of the first impeller and the cross-sectional area A2s1 is A2s1 / A2b, where A2s1 / Ab=A2s1 / A2b (or approximately equal thereto), the first impeller and the second impeller are of the same type, each of the impellers is positioned above the sparging surface of a respective sparger, the distance between the bottom of the impeller blade and the sparging surface of each of the first bioprocess bag and the second bioprocess bag is approximately the same, and each of the first impeller and the second impeller is configured to operate within the first bag and the second bag, respectively, so that, under similar conditions, in a bioprocessing operation using the same fluid in the first bioprocess bag and the second bioprocess bag, the ratio of the cross-sectional area A2b of the first impeller and the cross-sectional area A2s1 is A2s1 / A2b, where A2s1 / Ab=A2s1 / A2b (or approximately equal thereto), the ratio of the cross-sectional area A2s1 / Ab=A2s1 / A2b L a is achieved by controlling the agitation speed and gas flow rate, and a second sparger Equipped with.

[0020] According to a particular embodiment, H / D=about 1 to 2, preferably about 1.7, Di / Dt=D2i / D2t=about 0.3 to 0.5, preferably about 0.4, and As1 / Ab=A2s1 / A2b=about 0.023 to 0.068, preferably about 0.04.

[0021] In some embodiments, the first bioprocess bag and the second bioprocess bag each include an additional sparger attached to the interior bottom surface of the first flexible wall and the second flexible wall, respectively. The ratio of the sparging surface area As2 of the additional sparger to the cross-sectional area Ab of the first bioprocess bag (in the expanded state) is As2 / Ab. The ratio of the sparging surface area A2s2 of the additional sparger to the cross-sectional area A2b of the second bioprocess bag (in the expanded state) is A2s2 / A2b, where As2 / Ab = A2s2 / A2b.

[0022] According to a particular embodiment, As2 / Ab=A2s2 / A2b=about 0.008 to 0.024, preferably 0.02.

[0023] Additionally, the distance between the bottom of the impeller blades and the sparging surfaces of the first and second bioprocess bags is approximately 8-24 mm, preferably 16 mm.

[0024] Additionally, the gas introduced through the first and second spargers of the first and second bioprocess bags is controlled to have the same maximum gas exit velocity and maximum gas flow rate relative to the nominal bioreactor volume. Similarly, the gas introduced through the additional spargers of the first and second bioprocess bags is controlled to have the same maximum gas exit velocity and maximum gas flow rate relative to the nominal bioreactor volume, but different from the speed and flow rate of the first and second spargers.

[0025] In certain embodiments, the maximum gas exit velocity and corresponding maximum gas flow rate of the first and second spargers is about 12-60 m / s, preferably 24 m / s, and about 0.15-0.55 vvm, preferably about 0.2 vvm, and the maximum gas exit velocity and corresponding maximum gas flow rate of the additional spargers is about 36-60 m / s, preferably 36 m / s, and about 0.05-0.18 vvm, preferably 0.1 vvm.

[0026] According to some embodiments, the first and second spargers provide dissolved oxygen in the culture medium and may provide a mixture of air and oxygen for this purpose. Additional spargers may be used for carbon dioxide stripping and may deliver nitrogen or air. Maintaining a consistent surface area ratio of the spargers across scale helps ensure comparable gas distribution and therefore better scalability (e.g., scalable performance control).

[0027] Furthermore, the power numbers of the first and second impellers are approximately the same. In certain embodiments, the power numbers of the first and second impellers for down-pumping (e.g., clockwise impeller rotation) are between 0.8 and 3.6, preferably about 2.6, and the power numbers of the first and second impellers for up-pumping (e.g., counterclockwise impeller rotation) are between 0.8 and 2.5, preferably about 1.7.

[0028] Each of the first and second bioprocess bags may be disposed within a container. The containers housing the first and second bioprocess bags each have a height-to-diameter ratio of approximately H / D. The H / D value is the same for both of these containers. In certain embodiments, the H / D ratio of the containers is also approximately 1:1 to 2:1, preferably 1.7:1. Each container may include multiple baffles (e.g., 1 to 4), each of which has a height ratio (baffle height / container diameter) of Hb / D. Note that the height of the container is the distance the baffle protrudes from the periphery of the container toward the center of the container. The Hb / D value is the same for both of these containers.

[0029] In a specific embodiment, Hb / D=about 1 / 10 to 1 / 12, preferably about 1 / 11.

[0030] According to a further aspect of the present invention, a method of scaling a biomanufacturing process is described. The method of scaling a biomanufacturing process includes the steps of providing a first bioprocess bag having a first volume and a first flexible wall, the first bioprocess bag comprising a first impeller mounted on a first impeller plate mounted against a first interior surface of the first flexible wall and a first sparger mounted against an interior bottom surface of the flexible wall, wherein when the first bioprocess bag is in an expanded state, a ratio of a sparging surface area As1 of the first sparger to a cross-sectional area Ab of the first bioprocess bag is As1 / Ab; performing a first biomanufacturing process in the first bioprocess bag; and expanding the first bioprocess bag to a second volume and a second flexible wall, the second volume and the second flexible wall being larger than the first volume. 1. The method of claim 1, further comprising: providing a second single-use bioprocess bag having a flexible wall, the second bioprocess bag comprising a second impeller mounted on a second impeller plate attached to a second interior surface of the second flexible wall, wherein when the second bioprocess bag is in an expanded state, a ratio of a sparging area A2s1 of the second sparger to a cross-sectional area A2b of the second bioprocess bag is A2s1 / A2b; and scaling a first biomanufacturing process to a larger volume by performing a second bioprocess bag in the second bioprocess bag, wherein A2s1 / Ab is approximately equal to or equal to A2s1 / A2b.

[0031] In some embodiments, As1 / Ab=A2s1 / A2b (or approximately equal thereto)=about 0.023 to 0.068, and in one preferred embodiment, As1 / Ab is about 0.04.

[0032] In some embodiments, the first bioprocess bag and the second bioprocess bag each include an additional sparger attached to the interior bottom surface of the first flexible wall and the second flexible wall, wherein the ratio of the sparging surface area As2 of the additional sparger to the cross-sectional area Ab of the first bioprocess bag is As2 / Ab, and the ratio of the sparging surface area A2s2 of the additional sparger to the cross-sectional area A2b of the second bioprocess bag is A2s2 / A2b, where As2 / Ab = A2s2 / A2b (or approximately equal).

[0033] In some embodiments, As2 / Ab=A2s2 / A2b=about 0.008 to 0.024, and in one preferred embodiment, As2 / Ab is about 0.02.

[0034] The method may further include manipulating the first bioprocess bag and the second bioprocess bag in the same (or similar) manner as described with respect to other aspects of the invention.

[0035] In any of the aspects and embodiments of the invention, a linear scalable bioreactor system and associated methods for use in implementing a scalable biomanufacturing process may comprise a process measurement system, a system of field operating devices or field actuators, and a local processing unit and associated software logic configured to translate the measurement system inputs and send them to a supervisory controller or human-machine input device for data manipulation and storage, and to translate the measurement system inputs from the supervisory controller or human-machine input device into operations of the field operating devices or field actuators.

[0036] The scalable bioprocessing system may have a human-machine interface and associated software programming comprising at least one of a computer mouse, keyboard, and touch screen, and a supervisory controller and software programming capable of using data from process inputs, sensor measurements, device states, set points, deviations from set points, alarm conditions, and combinations thereof to command system devices to respond to specific configurable parameters.

[0037] The design of the disclosed scalable bioreactor system, which can be a single-use system, is such that the operating range and geometry of each bioreactor in the system allows for consistent, approximately equivalent maximum oxygen mass transfer while remaining within an acceptable range of volume fractions, including excess shear rates and Kolmogorov vortex lengths, for the target cell type.

[0038] In any aspect and embodiment of the present invention, the single-use bioprocess bag is disposed within a vessel, also referred to herein as a "tank." This vessel may be constructed of a rigid or semi-rigid material and serves to support the bag disposed therein. As used herein, the term "linear scalability" refers to the relative constancy of one or more geometric and process parameter values ​​of the vessel or the bags disposed therein, and the ability to achieve and maintain consistent, approximately equivalent oxygen mass transfer for each bioreactor in the system, e.g., from 50 L to about 2000 L. The geometric equivalence of the vessel or the bags disposed therein, and consistent and approximately equivalent maximum oxygen mass transfer, are maintained over a wide range of working volumes for each bioreactor in the system. This linear scalability provides a means for modeling (scaling down) the performance of large-scale bioreactors in smaller-scale bioreactors, or, conversely, for easily scaling up development processes performed in relatively small-scale bioreactors for use in larger, commercial-scale processes with minimal protocol changes.

[0039] The scalable single-use bioreactor system of the present disclosure is designed such that its gas sparging / aeration system is substantially identical between scales, allowing the smaller bioreactor to simulate the aeration and gassing performance of a larger system. This additional feature of linear scalability provides a means to model the performance of a larger bioreactor in a smaller bioreactor, or conversely, to easily scale up a development process performed in a relatively small bioreactor for use in a larger commercial-scale process with minimal protocol changes. Specifically, for example, by maintaining constant Di / Dt, power number, and sparger surface area ratio across different volumes, substantially identical maximum k LWhile maintaining a, it is possible to vary the agitation and sparge rates while still keeping the shear rate and Kolmogorov vortex length within acceptable ranges for all bioreactor sizes.

[0040] In summary, the bioreactor system of the present disclosure achieves "linear scalability" by using or substantially replicating the same bag and vessel geometries, gas sparging systems, impeller shapes and types, and process control systems used in larger systems. The bioreactor system of the present disclosure is well suited for the cultivation of mammalian cells, microbial, bacterial, plant, insect, protozoan, organ, and / or fungal cells. [Brief explanation of the drawings]

[0041] [Figure 1] FIG. 1 is a schematic side elevation cutaway view of a 50 L bioreactor vessel having a single-use flexible bag, impeller, magnetic impeller hub, impeller plate, and external magnetic drive disposed therein, according to one embodiment of the present invention. [Figure 2] FIG. 1 is a plan view of a bioreactor vessel illustrating an impeller plate and spargers, according to one embodiment of the present invention. [Figure 3] FIG. 1 is a plan view of a bioreactor vessel illustrating an impeller plate, spargers, and impeller according to one embodiment of the present invention. [Figure 4] FIG. 10 is a contour plot showing experimental dissolved oxygen kLa for a 50 L bioreactor and a 200 L bioreactor, according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0042] Preferred embodiments of the present invention are described below. It will be understood that the specific embodiments of the present invention are presented by way of example and not as limitations of the present invention. The present invention will first be described in its broadest overall aspect, followed by a more detailed description. Features and other details of the compositions and methods of the present invention will be further pointed out in the claims.

[0043] Throughout the description and claims of this specification, the terms "comprise" and "contain," as well as variations thereof, mean "including but not limited to," and are not intended to (and do not) exclude other parts, additions, components, wholes, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood as contemplating the plural as well as the singular, unless the context requires otherwise.

[0044] A scalable single-use bioreactor system according to one embodiment of the present invention is a small-scale bioreactor that provides linear scalability from small to large scale, for example, from a 50 L single-use bioreactor bag to a 2000 L single-use bioreactor bag within the same single-use platform within a family of bioreactors of different sizes.

[0045] FIG. 1 is a schematic side elevation cutaway view of a bioreactor system 100 of the present disclosure, comprising a 50 L bioreactor vessel or support structure 20 having a single-use flexible bag 30 disposed therein, an impeller 22 mounted to a magnetic hub 24 centrally located at the bottom inside bag 30, an impeller plate 26 located on the inside bottom of the bag and on which magnetic puck 24 rotates, and an external magnetic drive 23.

[0046] The bioreactor system 100 is designed to achieve high cell density culture conditions. In particular, small scale (e.g., 50 L) bioreactor systems are designed to achieve high oxygen k L The system is designed with a sparger having sufficient sparging surface area to achieve sufficient oxygen transfer without adversely affecting the oxygen k L Because oxygen transfer independently affects a (i.e., regardless of agitation and flow conditions), this small-scale bioreactor system is designed to maintain oxygen transfer to the cell culture at a level sufficient to achieve high-cell-density culture conditions within the constraints of maximum agitation and gas flow rates. The inventors have advantageously discovered that maintaining the same (or substantially the same) sparging surface area ratio (sparging surface area in expanded state within vessel / bag cross-sectional area) between scales, as well as maintaining other geometric parameters, which will be described in more detail below, improves scalability. In other words, by determining the sparging surface area that achieves sufficient oxygen transfer at the small scale and maintaining the sparging surface area ratio between scales based on that value, culture conditions at each scale can be made approximately equivalent without exceeding the maximum agitation rate and gas flow rate.

[0047] In addition to sparging surface area, scalability is further improved by keeping additional geometric parameters constant (or nearly constant) between scales, thereby making the geometries of different scales identical (or substantially identical). For example, by keeping the aspect ratio H / D, the Di / Dt ratio (where Di is the impeller diameter and Dt is the vessel diameter), the impeller blade-to-sparger distance, and / or the baffle height ratio (baffle height / vessel diameter) constant between scales, even greater comparability of culture conditions is achieved at each scale.

[0048] Additionally, by placing upper limits on certain operational parameters across scales, scalability is further improved by avoiding operating the bioreactor system in certain situations that may be detrimental to cell growth. For example, by placing upper limits on the maximum gas flow rate (and therefore gas flow rate per minute) and the mixing contribution from the agitation rate (i.e., power input to volume ratio (P / V)), cell culture conditions are maintained within desirable ranges across all scales (e.g., acceptable shear rates, dissolved oxygen and carbon dioxide concentrations, and Kolmogorov vortices are maintained).

[0049] Although the present invention aims to hold certain geometric and process parameters constant, the numerical ranges for each of these parameters, as described below, are included within the scope of the present invention. Specifically, any numerical value within a given range can provide the benefits described herein, so long as that value remains constant (or is substantially the same) across scales. Furthermore, these ranges are based on mathematical calculations subject to the overall design constraints of the bioreactor system. However, it should be noted that the specific numerical values ​​cited as preferred values ​​described herein are intended to represent optimal values ​​for a scalable bioreactor system. Furthermore, the present invention is not expressly limited to these numerical values, but rather encompasses all ranges described herein.

[0050] In some embodiments, the aspect ratio H / D of the working liquid volume height H to the diameter D of the bag 30 is about 1-2, preferably 1.7. The ratio Di / Dt (where Di is the impeller diameter and Dt is the vessel diameter) is about 0.3-0.5, preferably about 0.4.

[0051] As best shown in FIG. 2, there is also at least one sparger mounted to the interior bottom of bag 30. This at least one sparger can take the form of an array of sparging disks 27, 28 disposed on impeller plate 26. In some embodiments, this at least one sparger consists of two spargers. In the embodiment shown in FIG. 2, the first sparger 27 has three sparging disks and has a ratio of sparging surface area to bag cross-sectional area (in the expanded state within the vessel) of about 0.023 to 0.068, preferably about 0.04. In the embodiment shown in FIG. 2, the second sparger 28 has one sparging disk and has a ratio of sparging surface area to bag cross-sectional area (in the expanded state within the vessel) of about 0.008 to 0.024, preferably about 0.02. The gas introduced through the first sparger 27 and the second sparger 28 is controlled so that the maximum gas exit velocity of the first sparger 27 is about 12-60 m / s, preferably 24 m / s, corresponding to a maximum gas flow rate of about 0.15-0.55 vvm (vessel volumes per minute), preferably 0.2 vvm, while the maximum gas exit velocity of the second sparger 28 is about 36-60 m / s, preferably 36 m / s, corresponding to a maximum gas flow rate of about 0.05-0.18 vvm, preferably 0.1 vvm. The overlay maximum gas flow rate is about 0.05 vvm. Furthermore, the maximum agitation speed (i.e., input power-to-volume ratio (P / V)) is about 150 W / m. 3 An upper limit can be set.

[0052] A reusable bioreactor vessel according to the disclosed system may include a reusable polymeric or metallic vessel stand, vessel support, or vessel holder 20 with an integrated external motor / agitation assembly 23 and a measurement and control system (not shown). The vessel stand, vessel support, or vessel holder may be constructed of a composite material, such as a polymer mixed with a metal. The polymeric or metallic vessel can be of any shape or size as long as it is capable of supporting the stirred tank single-use flexible bioreactor bag 30 design of the disclosed system. For example, according to one embodiment of the present invention, the polymeric or metallic stand, support, or holder 20 is capable of receiving and supporting a 50 L flexible or compressible bioprocess bag assembly 30.

[0053] The measurement and control system is built on a configurable software platform. The measurement and control system operates in real time, receiving process inputs (measurements) such as temperature, RPM, pH, DO, weight, and pressure, and controlling field devices such as mass flow controllers, pumps, solenoid valves, and heating elements. According to one embodiment, human interaction with the system occurs through a touchscreen-based interface that may be located on a desktop, laptop, or notebook computer, through a remote computer operating over the Internet, or through a remote control device. The interface allows a user to enter setpoints, manage gas sparging, calibrate field devices, manage alarms, set operator IDs and access levels, and view graphical representations of process values ​​over time. The measurement and control system may further include a server for storing current and historical data related to the fermentation process.

[0054] As mentioned above, scalability is a very important factor, especially in the biotechnology / pharmaceutical manufacturing industry, because it allows for easy and simple technology transfer from research level (small scale) to large scale production. Geometric equivalences, such as aspect ratio (H / D), impeller shape, sparger shape, and impeller power number, as well as process equivalences, such as sparger gas inlet velocity and gas flow rate, are important factors for controlling and minimizing changes in bioreactor process performance during scale-up.

[0055] In addition to the above, shear rate and Kolmogorov vortex length are also parameters that need to be considered. Specifically, shear rate and Kolmogorov vortex length act as limiting values ​​to avoid operating the bioreactor in a condition detrimental to cell growth for cell species suitable for growth in suspension cell culture. The above process parameters are related to power density (W / m 3 ) and the desired target gas flow rate through both sparge assemblies. Static analysis of the system at maximum agitation achieved by the desired target gas flow rate through both sparge assemblies revealed that the shear rate of the bioreactor was 2000 s -1 and Kolmogorov vortices are designed to be larger than 62 micrometers. Both of these limits need only be met for more than 95% of the vessel volume; small localized areas exceeding these limits are acceptable.

[0056] Tables 1 and 2 below provide an overview of the ranges of geometric and operational parameters within the scope of the present invention. Each bioreactor has the same or nearly the same H / D aspect ratio, impeller geometry, sparger geometry, and impeller power number, as well as the same or nearly equivalent operational limits, such as sparger gas inlet velocity, across all reactors. All ranges provided are approximate (i.e., each boundary can be adjusted by up to 15%).

[0057] [Table 1]

[0058] [Table 2]

[0059] Tables 3 and 4 below provide an overview of preferred geometric and operational parameter values ​​and ranges within the scope of the present invention. Specifically, each bioreactor has the same or nearly the same H / D aspect ratio, impeller geometry, sparger geometry, and impeller power number, as well as equivalence in operational limits, such as sparger gas inlet velocity, across all reactors. All values ​​are approximate (i.e., each boundary can be adjusted by up to 15%).

[0060] [Table 3]

[0061] [Table 4]

[0062] Keeping these values ​​constant (or nearly identical) across scales within the ranges provided herein helps achieve linear scalability and highly predictable modeling of scale-up conditions, while also providing substantially consistent oxygen mass transfer (i.e., oxygen k L a (+ / - 30%) is maintained. Throughout this text, the 50 L, 200 L, 500 L, 1000 L, and 2000 L single-use bioreactor bags disclosed herein are designated as X-50, X-200, X-500, X-1000, and X-2000, respectively.

[0063] The data in Tables 1 through 4 above illustrate several linear scaling factors for the single-use bioreactors of the present disclosure. According to one embodiment of the present invention, the volume turndown ratio, which is the ratio of the maximum working volume to the minimum working volume, is approximately 5:1 for all bioreactor sizes, assuming the entire working volume of the single-use bioreactor bag is substantially cylindrical. "H" is the height of the working volume within the single-use bag, and "D" is the diameter of the tank or single-use bag disposed within the support tank. For a range of maximum working volumes, the H / D ratio for either the tank or the bag disposed within the tank remains substantially constant, ranging from approximately 1:1 to 2:1, and preferably approximately 1.7. While the impeller diameter "Di" varies (i.e., increases with increasing vessel size), the ratio of the impeller diameter Di to the vessel diameter Dt remains constant, ranging from approximately 0.3 to 0.5, and preferably approximately 0.4. In some embodiments, the impeller type of the present invention is a six-blade Rushton 50 degree pitch blade, and is the same type for each system. The number of blades in the impeller may vary.

[0064] In one embodiment, the impeller is mounted on an impeller plate and is magnetically driven by an external motor.

[0065] Impeller power number "N p " is calculated using the following formula:

[0066]

number

[0067] where P0 is the input power to the medium under non-aerated conditions, and N(s -1 ) is the stirring speed, and D i (m) is the impeller diameter, and ρ (kg m -3 ) is the medium density [3]. p" (for both up-pumping and down-pumping) remains similarly constant across bioreactor sizes. Specifically, as shown, the up-pumping power number is about 0.8 to 2.5, preferably about 1.7, and the down-pumping power number is about 0.8 to 3.6, preferably 2.6.

[0068] The impeller blade-to-sparging distance, which is the vertical distance between the upper surface of the sparger and the lower surface of the impeller blade, is also constant and is about 8 to 24 mm, preferably 16 mm.

[0069] The bioreactor bag of the present invention may include two types of spargers 27, 28 in the form of, for example, a sparging disk positioned below the impeller (see, e.g., Figures 2 and 3). Sparger 1 (as presented in Tables 1 and 3), corresponding to sparger 27 in Figure 2, is used for dissolved oxygen (DO) control and is configured to deliver a mixture of nitrogen and oxygen. Sparger 2 (as presented in Tables 1 and 3), corresponding to sparger 28, is used for carbon dioxide (CO2) stripping and is configured to deliver nitrogen and / or air. As shown in Tables 1 and 3, the total surface area of ​​the sparging surface of each sparger divided by the cross-sectional area of ​​the vessel / bag is also kept constant (or approximately the same). Specifically, the sparging surface area ratio of Sparger 1 is about 0.023 to 0.068, and in a preferred embodiment is 0.04, and the sparging surface area ratio of Sparger 2 is about 0.008 to 0.024, and in a preferred embodiment is about 0.02. These values ​​are constant (or nearly identical) between scales.

[0070] One important advantage of the present invention is that it achieves the same sparging area ratio between bioreactors of all scales. Maintaining this ratio constant ensures uniform gas distribution, which is achieved by reducing the oxygen k La (and therefore oxygen mass transfer) as well as scaling processes related to dissolved CO stripping. More specifically, the sparging surface area contributes to the oxygen k L a (i.e., independent of stirring and flow conditions), the desired oxygen k L A constant surface area ratio of the sparger that does not limit a is established, thereby allowing for a higher maximum k at a constant geometry across scales. L a is realized.

[0071] In addition to the geometric scaling parameters, it has been found advantageous to establish operational limits for every bioreactor scale. As presented in Tables 2 and 4, the maximum sparger gas inlet velocity and maximum gas flow rate are constant for all bioreactor scales. Sparger 1 has a maximum gas inlet velocity of about 12-60 m / s, preferably about 24 m / s, and a maximum total gas flow rate of about 0.15-0.55 vvm, preferably about 0.2 vvm. Meanwhile, Sparger 2 has a maximum gas inlet velocity of about 36-60 m / s, preferably about 36 m / s, and a maximum total gas flow rate of about 0.05-0.18 vvm, preferably about 0.1 vvm. Furthermore, the maximum power input per unit volume (max P / V), which is the amount of power transferred to the working volume of the medium via impeller rotation, remains constant across bioreactor scales for the nominal volume of the bioreactor, at approximately 150 W / m 3 By ensuring that the maximum values ​​of these operating parameters are constant across scales, the oxygen k LThis helps control the scaling process not only with respect to a (and therefore oxygen mass transfer) and CO2 stripping, but also ensures that all of the bioreactors function within the limits of the given shear rate and Kolmogorov vortex. In other words, by equating these operating limits across scales, a design space is created that provides identical or similar performance at each scale. Furthermore, this design space allows for variation of oxygen concentration in the feed stream and the use of sparge2, which allows for matching gas flow rates (in vvm) across scales to achieve comparable pCO2 profiles, while also providing equivalent oxygen k at a constant P / V. L a is delivered.

[0072] Additionally, it may also be advantageous to control the maximum overlay gas flow rate, which is the gas flow rate delivered into the headspace of the bioreactor bag 30. As presented in Tables 2 and 4, this maximum value is constant across all scales and is set at 0.05 vvm.

[0073] In some embodiments, the bag or container uses the same disposable or single-use construction materials and rigid plastic product-contact components as the larger-scale bioreactor in the same system. Using the same construction materials or the same class of polymer for construction eliminates regulatory conflicts between small-scale optimization and large-scale implementation with respect to extractable and leachable components, biocompatibility compliance, and regulatory testing. As a result, the 50 L system models larger systems not only in terms of scalability / performance, but also in terms of regulatory compliance standards.

[0074] Some of the features of the stirred tank bioreactor in the disclosed system include a bottom-mounted or sidewall-mounted impeller located near the top of substantially the same sparging surface as described above, USP Class VI materials, full integration with the controller, construction from biocompatible materials, and linear scalability over a wide range of working tank or bag volumes, e.g., from 50 L to 2000 L in the same single-use bioreactor system as described above.

[0075] Scale-up factors for single-use bioreactors There are many parameters that affect cell growth, and deviations from acceptable ranges result in harm to health and growth. Therefore, maintaining these parameters within acceptable ranges is very important and often difficult when scaling. As discussed above, the present invention provides a highly scalable bioreactor. By fixing certain geometric characteristics and setting the same operational limits across scales, many of the fluid response parameters can be maintained within acceptable ranges, and in some cases, even constant, which greatly aids in scaling between bioreactor sizes. This can be achieved, for example, by selecting a scale-up criterion, such as the agitation power density (P / V), to be constant. This constant value allows for the k of the system to be maintained across scales. L The equality of a and the ability to fine-tune this (e.g., by adjusting the inlet oxygen concentration and matching the overall gas flow rate on a vvm basis via an auxiliary second sparger, resulting in an identical pCO2 profile) allows for a constant OTR to be achieved.

[0076] It is important to ensure adequate oxygen mass transfer and CO stripping to the cell culture; otherwise, unwanted cell death will occur and cell growth will be reduced or halted. Additionally, mixing time also has a significant impact on cell health and growth, as long mixing times can prevent cells from receiving necessary nutrients in a timely manner or expose them to harmful concentration gradients.

[0077] Because each scale-up parameter depends on another, it is not possible to keep all parameters constant during scale-up. For example, oxygen k L It is not possible to maintain a constant a, agitation speed, or gas flow rate. Rather, two of these parameters can be held constant while the third parameter is varied to maintain the other two.

[0078] We have now discovered a system comprising two or more single-use bioreactor bags in which one or more of the aforementioned geometric parameters and operational limits are constant, allowing any of the other scale-up parameters to be kept within a desired range, although not all of them can be kept constant.

[0079] FIG. 4 shows experimental dissolved oxygen k for 50 L and 200 L bioreactors according to some embodiments of the present invention. L As shown and based on the design constraints above, the maximum P / V (150 W / m 3 ) and a gas flow rate of 0.19 vvm for Sparger 1, the maximum oxygen k for the 50 L bioreactor L a(h -1 ) is approximately 45. Based on the characterization data, each of the other scales (e.g., 100 L to 2000 L) can achieve the same oxygen k while still remaining within the aforementioned operating limits. L For example, a P / V of 108 W / m 3and the maximum oxygen mass transfer coefficient (k) of the 200 L bioreactor at nominal volume when the gas flow rate of Sparger 1 is 0.15 vvm. L a) is about 45 hours -1 By doing this, the oxygen k L a (e.g., these k L The a value is higher at high cell densities (e.g., 100 × 10 6 It has been shown that it is possible to achieve high cell counts (exceeding 1000 cells / mL) within the same operating limits at all scales, which is useful for scaling up (or down).

[0080] Successful scale-up (SU) and / or scale-down (SD) of biologics manufacturing requires comparable performance between operational scales. The disclosed SU and SD platform design of the scalable single-use bioreactor design provides a unique system to achieve this. As shown in Table 3, the maximum oxygen k L a and dissolved CO2 stripping are constant across these scales, which goes a long way to ensuring that cell cultures maintain adequate oxygen mass transfer and dissolved CO2 removal regardless of scale. Specifically, the design achieves a constant maximum k within maximum allowable limits (e.g., maximum P / V, shear, vortex, etc.) at all scales. L In this way, each bioreactor can be adjusted by adjusting the agitation and / or sparging rates to consistently provide sufficient dissolved oxygen and CO stripping without causing toxic conditions within the cell culture (e.g., unacceptably high shear rates).

[0081] To achieve the necessary functions of the bioreactor, fittings are added to the bag, such as perforations and filters to allow fluid and gas transfer, mixing interfaces, sensors, and sparging surfaces to control bubble size. Multiple sparging surfaces and options can be used in various embodiments of the invention. Sparging surfaces can be attached to the bioprocess bag as adapters or bulkhead fittings designed to connect hoses or tubing, such as oxygen supply tubing, to the bag. The porosity and area of ​​the sparging surface can vary (within the limits described above). In one embodiment, the sparging surface can simply be a single hole or multiple holes configured to add oxygen gas or air to the bioreactor bag. While at least some of the sparging surfaces can be located below the impeller (e.g., spargers for DO control) to provide efficient circulation of gas through the medium in the bag, the location and type of sparging surfaces are substantially identical to the location and type in larger 50 L to 5000 L systems.

[0082] A linear scalable bioreactor system according to one embodiment of the present invention may include a temperature controller and at least one sensor and / or probe (not shown). To eliminate the need for utilities required for temperature control via a heat exchanger, heating can be achieved by a closed-loop water jacket that is heated and / or cooled by a control system mounted on the bioreactor system or by an electric heating blanket or Peltier heater. The heating blanket may include a thermocouple for sensing the temperature of the contents of the bioprocess bag, which works in conjunction with the temperature controller to control the set point temperature of the contents of the bioprocess bag. Optionally, a thermally conductive material may be embedded in the surface of the bioprocess bag to offset the insulating effect of the plastic.

[0083] Additionally, cooling may be achieved by a closed-loop water jacket heated and / or cooled by a control system mounted on the bioprocess bag, or by standard heat exchange through a cover or jacket on the tank supporting the bioprocess bag. Additionally, cooling may be achieved by a Peltier cooler. For example, a Peltier cooler may be applied to the exhaust line (e.g., to a small bag-like chamber with a large capacity for decelerating the air and a large surface area disposed within the heat exchanger) to condense gases in the exhaust air and help prevent wetting of the exhaust filter. Alternatively, the exhaust filter may be heated by a filter heater to help prevent condensation of moisture from the exhaust line. Additionally, the heat source can be waste heat from the actively cooled side of the Peltier unit by sending a hot gas stream through the exhaust filter conduit and holder.

[0084] The bioprocess bag, including any attachments, penetrations, sensors, etc., may be sterilized (e.g., by gamma irradiation) prior to use. After sterilization, the interior of the bag, tubing, and components can be considered sterile, thereby achieving a "sterile envelope" that protects the contents of the container from external airborne contaminants.

[0085] It should further be understood that there are at least three basic modes of operation used by stirred tank bioreactors, and the bioreactor system of the present disclosure consists of a bioreactor that can be easily modified to operate in any of these three modes. These three modes are as follows:

[0086] Semi-continuous, continuous, or perfusion mode: In semi-continuous, continuous, or perfusion modes, nutrients and supporting process fluids are continuously added to the system, waste is removed either continuously or periodically ("drain and fill"), and product is harvested intermittently or throughout the entire culture period. It is well known that in continuous mode it is always difficult to obtain a sufficiently high product titer. In addition to this low titer, continuous mode requires product concentration.

[0087] Batch Mode: In batch mode, all nutrients are added at the beginning and product is not removed until the end of the batch. Waste accumulates during the run and nutrients are used up, making the batch process inefficient for many applications. However, the simplicity of operating a bioreactor in this mode makes it a desirable option for seed train expansion.

[0088] Fed-batch mode: Fed-batch mode is similar to batch mode in that product is removed only at the end of the run, but differs in that nutrients are added at multiple intervals during the process. Many biotherapeutic and most virus production microcarrier cultures are performed in a fed-batch process after infection.

[0089] It is to be understood that any feature, integer, property, compound, chemical moiety, or chemical group described in connection with a particular aspect, embodiment, or example of the invention is also applicable to any other aspect, embodiment, or example described herein, unless inconsistent. All features disclosed in this specification (including the appended claims and abstract) and / or all steps of any method or process similarly disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to every novel feature or every novel combination of features disclosed in this specification (including the appended claims and abstract), or to every novel feature or every novel combination of any method or process similarly disclosed. [Explanation of symbols]

[0090] 20 50L bioreactor vessel or support structure, reusable polymeric or metallic vessel stand, vessel support, or vessel holder 22 impeller 23 External magnetic drive, integrated external motor / agitator assembly 24 Magnetic Hubs, Magnetic Packs 26 Impeller plate 27 First sparger, sparging disc 28 Second sparger, sparging disc 30 Single-Use Flexible Bioreactor Bags, 50L Flexible or Shrinkable Bioprocess Bag Assembly 100 Bioreactor Systems

Claims

1. 1. A bioreactor system for use in conducting a scalable biomanufacturing process, comprising: a bioprocess bag having flexible walls; an impeller mounted on an impeller plate mounted against an interior surface of the flexible wall; a first sparger mounted against the interior surface of the flexible wall, wherein the ratio of the sparging surface area of ​​the first sparger to the cross-sectional area of ​​the bioprocess bag when the bioprocess bag is in an expanded state is between about 0.023 and 0.068; A bioreactor system comprising:

2. 10. The bioreactor system of claim 1, wherein the ratio of the sparging surface area of ​​the first sparger to the cross-sectional area of ​​the bioprocess bag is about 0.

04.

3. 3. The bioreactor system of claim 1 or 2, further comprising a second sparger mounted against the interior surface of the flexible wall, wherein the ratio of the sparging surface area of ​​the second sparger to the cross-sectional area of ​​the bioprocess bag when the bioprocess bag is in the expanded state is between about 0.008 and 0.

024.

4. 4. The bioreactor system of claim 3, wherein the ratio of the sparging surface area of ​​the second sparger to the cross-sectional area of ​​the bioprocess bag is about 0.

02.

5. 5. The bioreactor system of claim 1, wherein the ratio of the impeller diameter Di to the bioprocess bag diameter Dt is between about 0.3 and 0.

5.

6. 6. The bioreactor system of claim 5, wherein the ratio of the diameter Di of the impeller to the diameter Dt of the bioprocess bag is about 0.

4.

7. 7. The bioreactor system of any one of claims 3 to 6, wherein the first sparger is configured to introduce oxygen and / or air into the bioprocess bag and the second sparger is configured to introduce nitrogen and / or air into the bioprocess bag.

8. 8. The bioreactor system of claim 1, wherein the distance between the impeller blades of the impeller and the sparging surface of the first sparger is about 8 to 24 mm, preferably about 16 mm.

9. 1. A bioreactor system for use in conducting a scalable biomanufacturing process, comprising: a first bioprocess bag having a first volume and a first flexible wall; a first impeller mounted on a first impeller plate mounted against a first interior surface of the first flexible wall; a first sparger mounted against the interior surface of the flexible wall, wherein when the first bioprocess bag is in an expanded state, the ratio of the sparging surface area A of the first sparger to the cross-sectional area A of the first bioprocess bag is A / A; a second single-use bioprocess bag having a second volume larger than the first volume and a second flexible wall; a second impeller mounted on a second impeller plate attached to a second interior surface of the second flexible wall, wherein when the second bioprocess bag is in an expanded state, a ratio of a sparging surface area A2s1 of the second sparger to a cross-sectional area A2b of the second bioprocess bag is A2s1 / A2b; and Equipped with A bioreactor system in which As1 / Ab is approximately equal to or equal to A2s1 / A2b.

10. 10. The bioreactor system of claim 9, wherein As1 / Ab is about 0.023 to 0.

068.

11. 11. The bioreactor system of claim 9 or 10, wherein As1 / Ab is about 0.

04.

12. 12. The bioreactor system of any one of claims 9 to 11, wherein each of the first bioprocess bag and the second bioprocess bag comprises an additional sparger attached to an interior surface of the first flexible wall and the second flexible wall, respectively, wherein a ratio of a sparging surface area As2 of the additional sparger to a cross-sectional area Ab of the first bioprocess bag is As2 / Ab, and a ratio of a sparging surface area A2s2 of the additional sparger to the cross-sectional area A2b of the second bioprocess bag is A2s2 / A2b, and wherein As2 / Ab is approximately equal to or equal to A2s2 / A2b.

13. 13. The bioreactor system of claim 12, wherein As2 / Ab is about 0.008 to 0.

024.

14. 14. The bioreactor system of claim 13, wherein As2 / Ab is about 0.

02.

15. 10. The bioreactor system of claim 9, wherein a ratio of a diameter Di of the first impeller to a diameter Dt of the first bioprocess bag is Di / Dt, a ratio of a diameter D2i of the second impeller to a diameter D2t of the second bioprocess bag is D2i / D2t, and Di / Dt is equal to or approximately equal to D2i / D2t.

16. 1. A method for scaling a biomanufacturing process, comprising: providing a first bioprocess bag having a first volume and a first flexible wall, the first bioprocess bag comprising a first impeller mounted on a first impeller plate mounted against a first interior surface of the first flexible wall, and a first sparger mounted against the interior surface of the flexible wall, wherein when the first bioprocess bag is in an expanded state, a ratio of a sparging surface area As1 of the first sparger to a cross-sectional area Ab of the first bioprocess bag is As1 / Ab; conducting a first biomanufacturing process in the first bioprocess bag; providing a second single-use bioprocess bag having a second volume larger than the first volume and a second flexible wall, the second bioprocess bag comprising a second impeller mounted on a second impeller plate mounted against a second interior surface of the second flexible wall, wherein when the second bioprocess bag is in an expanded state, a ratio of a sparging area A2s1 of the second sparger to a cross-sectional area A2b of the second bioprocess bag is A2s1 / A2b; Scaling the first biomanufacturing process to a larger volume by conducting a second biomanufacturing process in the second bioprocess bag. Including, A method wherein As1 / Ab is approximately equal to or equal to A2s1 / A2b.

17. 17. The method of claim 16, wherein As1 / Ab is about 0.023 to 0.

068.

18. 18. The method of claim 17, wherein As1 / Ab is about 0.

04.

19. 19. The method of any one of claims 16 to 18, wherein each of the first bioprocess bag and the second bioprocess bag comprises an additional sparger attached to an interior surface of the first flexible wall and the second flexible wall, wherein a ratio of a sparging surface area As2 of the additional sparger to a cross-sectional area Ab of the first bioprocess bag is As2 / Ab, and a ratio of a sparging surface area A2s2 of the additional sparger to the cross-sectional area A2b of the second bioprocess bag is A2s2 / A2b, and wherein As2 / Ab is approximately equal to or equal to A2s2 / A2b.

20. 20. The method of claim 19, wherein As2 / Ab is about 0.008 to 0.

024.

21. 21. The method of claim 20, wherein As2 / Ab is about 0.02.

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