Linearly scalable bioreactor system

Abstract

A scalable, single-use, bioreactor bag and system for use in carrying out a scalable biomanufacturing process is provided. The small-scale system models larger scale systems from a shear / scalability / performance basis. The bioreactor system disclosed provides for improved scalability by using the same or substantially same vessel geometry ratios and maximum process parameters for small and large-scale biomanufacturing.

Description

LINEARLY SCALABLE BIOREACTOR SYSTEMFIELD OF INVENTION

[0001] The present disclosure relates to bioreactor systems and methods for processing biological materials. In some embodiments, the disclosed bioreactor systems include disposable components.BACKGROUND

[0002] Cell culturing is an essential step in manufacturing biological products. Bacterial, microbial and mammalian cells are often cultured in order to produce therapeutic proteins. Such proteins may include, e.g., monoclonal antibodies (MAbs), erythropoietin (EPO), and interferons. Such cells are also cultured to produce nucleic acids, viruses for use in vaccines, and recombinant protein production for use in pharmaceuticals.

[0003] In scaling up cultures from the laboratory bench-top bioreactor to larger commercial production bioreactors, it is important to consider changes in culturing conditions as the volume of the culture increases. In the biotech / pharmaceutical manufacturing industry, “technical transfer,” also referred to as “technology transfer” (hereinafter, “tech transfer”) of a biopharmaceutical process, such as, e.g., cell culturing, from the research or development level, i.e., small-scale production, to large scale, commercial production is generally a complex, costly, and time-consuming process.

[0004] In connection with the above, in cell culture, oxygen is a key and often limiting substrate for growth, production, and maintenance activities. Cells obtain oxygen in from soluble oxygen in elemental, noncompound forms known as ‘dissolvedoxygen’ (DO). One of the most important functions of bioreactors is continuously providing dissolved oxygen to cells through aeration. Aeration in the bioreactor typically occurs through two gas supply modes; a) when oxygen diffuses from an overlay gas supplied over the liquid for solubilization at the cell culture medium-gas interface and b) when oxygen is supplied below the liquid level sometimes into sparge tubes or sparging elements. Sparging elements are intended to provide the gas in the form of bubbles to increase surface area-to-volume contact and residence time of the gas in the liquid to encourage solubilization. Gas supplied below the liquid level can be solubilized further into the cell culture or solution media with the help of agitation. Agitation disperses the oxygen bubbles, improves retention time of the bubbles below the liquid surface, and promotes mass transfer of the gas into the liquid. The rate of oxygen transfer (OTR) from gas mass of oxygen into a soluble form of oxygen mass is a function of physicochemical properties of the cell culture medium, the geometric and operational parameters of the bioreactor, and presence of cells. Monitoring of dissolved oxygen (DO) under various operating conditions of agitation, gas flow rates, media or media simulant solution is performed to assess OTR in the absence of cells. The OTR is correlated by the oxygen mass transfer coefficient, ki.a (the volumetric mass-transfer coefficient that describes the efficiency with which oxygen can be delivered to a bioreactor for a given set of operating conditions), and the oxygen concentration gradient in the liquid.

[0005] Due to its low solubility in liquid phase and increasing metabolic consumption by the cells with time, oxygen is supplied continuously to the cell culture.Oxygen supply is carefully controlled for optimal cell growth by manipulating gas supply, agitation, or media additions in response to DO readings from immersedsensors. During batch cell culture, oxygen utilization / uptake rate (OUR) is initially low during the lag phase, where cells divert energy to sustaining internal cell functions and there is cell doubling or little gain in overall culture cell density. Cell density increases during the next phase, exponential phase, during which OUR increases until OTR (Oxygen Transfer Rate) becomes limiting under those conditions. Therefore, the OTR, through its correlation to ki.a, defines a theoretical maximum cell density that could be achieved in cell culture under the OTR test conditions before the supply of oxygen becomes a limiting control parameter. Because of this association with cell density, ki.a values obtained across multiple gas supply and agitation conditions are particularly useful in evaluating various bioreactor design features and evaluating scalability across possible bioreactor system sizes within a bioreactor platform. Any change to process, engineering parameters, or geometry of the system tank and component design has an impact on ki.a and thus is a good measure of the scalability of a bioreactor design for scaling into representative volumes across a bioreactor platform.

[0006] Mixing is used to maintain a homogenous state and eliminate gradients of concentration (cell, gas, pH, medium, and nutrient), temperature, and other properties. Gas bubble size and residence time are highly dependent upon an array of mixing conditions around the agitator design including impeller type, speed, and location(s) as well as sparge design, porosity, and performance with gas flow rate, ki.a values generally increase as agitation speed increases. However, high agitation speeds can lead to increased shear forces at the blade tip resulting in cell death.

[0007] Additionally, higher oxygen supply concentrations and higher gas flow rate improves oxygen availability which drive increases in OTR. Increasing oxygen supply to a bioreactor drives this availability by increasing the concentration gradient(correlating to a potential higher saturation value depending on the fluid composition) and can be controlled by modifying the oxygen concentration in the gas (air vs O2 enrichment) and volumetric flow. Although high ki.a values are desirable, it is important to consider the actual operating conditions and implications to cell viability and associated process costs. For example, high air flow rates can cause cell damage due to shear forces. Excessive foam might also be generated, requiring a high concentration of antifoam that can inhibit cell growth and may cause batch-to-batch variability in downstream process performance. Additionally, higher air flow rates require a larger exhaust filter area to limit bioreactor over pressurization, thus driving consumable cost increases.

[0008] Traditional bioreactors, therefore, are designed with different impeller types, spargers, combinations, and locations to achieve target ki.a values while minimizing the detrimental shear forces. But these modifications often make it difficult to scale between bioreactors within a given platform because the critical design parameters are not constant.

[0009] Thus, there is an on-going need in biopharmaceutical development and manufacturing for easily scalable systems and process transfer techniques that provide an easy, reliable, and repeatable tech transfer among bioreactor sizes for scale-up, scale-down, and scale-out. Scaling requires robustness and consistent performance between bioreactor scale with reduced process assessment, changes to process operations, or need for validation testing regardless of the purpose of process scale for process development, clinical production, or cGMP production.BRIEF SUMMARY OF THE DISCLOSURE

[0010] The present invention provides a scalable, single-use bioreactor systemand method for carrying out a scalable biomanufacturing process that solves many of the recognized problems scaling cell culture processes for commercial production or product / process evaluation at smaller scale. Use of the disclosed system and method minimizes risks and helps ensure that the robustness, purity, and potency of the scaled process are comparable to that of the original process scale. Tech transfer and scaling using the disclosed system can provide optimal results, and a safe and cGMP compliant scaled process for manufacturing, while eliminating or reducing the need for extensive, costly, and time-consuming process and parameter evaluation. In summary, the small- scale (e.g., 50L) bioreactor system disclosed provides for “linear scalability” by using the same, or substantially simulating the same, vessel and bag geometry, gas sparging system, impeller shape and type, and process control systems in the larger bioreactors (e.g., 200 liter through 2000 liter systems). The invention, inter alia, includes the following, alone or in combination.

[0011] In one aspect, the present invention relates to a scalable bioreactor system for use in carrying out a scalable biomanufacturing process, the system comprising: a bioprocessing bag having a flexible wall; a working volume turn down ratio (maximum working volume: minimum working volume) of about 5: 1; a liquid height to diameter ratio at maximum working volume of between about 1 : 1 to 2: 1, preferably about 1.7: 1; an impeller mounted on an impeller plate attached to an inside bottom surface of the flexible wall; and a ratio of the diameter of the impeller, Di, to the diameter of the installed bioprocessing bag, Dt, equal to a value of between about 0.3 to 0.5, preferably about 0.4.

[0012] The bioprocessing bag further includes at least one sparger attached to an inside bottom surface of the flexible wall for introducing gas into the bioprocessingbag. In 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 between 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 between about 0.008 to 0.024, preferably about 0.02. The gasses introduced through the first and second spargers are controlled such that the maximum gas exit velocity are under control. For the first sparger, the target linear velocity is between about 12 to 60 m / s, preferably about 24 m / s, corresponding to maximum gas flows between about 0.15 to 0.55 vvm (gas volumetric flow rate per vessel working liquid volume), preferably 0.2 vvm. For the second sparge channel, the maximum gas exit velocity for the second sparger is between about 36 to 60 m / s, preferably 36 m / s, and allowing maximum gas flow between about 0.05 to 0.18 vvm, preferably 0.1 vvm. Additionally, the overlay maximum gas flow can be maintained constant and may be set to about 0.05 vvm.

[0013] Throughout the entire text, “sparging surface area” is defined as the overall surface area of the sparger through which gases are introduced into the bioprocessing bag. Additionally, “cross-sectional area of the bag” is defined as the maximum cross-sectional area of the liquid surface in the bioreactor when the bioprocessing bag is inflated in place within the outer vessel. In other words, “cross- sectional area of the bag” is approximately the same as the cross-sectional area of the inner volume of the outer vessel that the bioprocessing bag sits in during use.

[0014] Further, the term “about” with respect to all values provided herein include values that are + / -15% the stated value, including the boundary values of any and all ranges.

[0015] According to embodiments, the first sparger is used for dissolved oxygen control of the culture media, and in order to do so may deliver a mixture of air and oxygen. The second sparger may be used for carbon dioxide stripping (removal) and may deliver nitrogen or air. Keeping the sparger surface area ratio the same across bioreactor scales helps to ensure an equal degree of gas dispersion, and thus better scalability.

[0016] Further, the distance between the bottom of the impeller blades and the gas producing surface of the sparging surface is between about 8 to 24mm, preferably about 16mm. This distance may be preserved to ensure scalable performance among the bioreactor sizes and may not be a scalable or proportional distance dependent on bioreactor liquid height or impeller height. This distance may be fixed to ensure similar bubble break-up of gas by the impeller between scales.

[0017] The bioreactor system further includes a vessel for housing the bioprocessing bag. The vessel has a liquid height to diameter ratio of between about 1 : 1 to 2:1, preferably about 1.7: 1 for the jacketed section of the system aligning with the maximum operating liquid volume. The vessel may include a plurality of baffles, the baffles having a height ratio (baffle height / vessel diameter) of between about 1 / 10 to 1 / 12, preferably about 1 / 11, noting that baffler height is the distance the baffle penetrates from the vessel perimeter toward the vessel center.

[0018] The impeller power number (NP) is dependent on the impeller shape, vessel geometry (baffle size, distance between impeller blades and vessel surfaces), and liquid (density, viscosity, and thus gasing conditions). The impeller power number in down pumping (e.g., clockwise impeller rotation from top view) is between about 0.8 to 3.9, preferably about 2.6, and in up pumping (e.g., counterclockwise rotation fromtop view) is between about 0.8 to 2.5, preferably about 1.7.

[0019] In another aspect, the present invention relates to a linearly scalable bioreactor system for use in carrying out scalable biomanufacturing processes, the system comprising:

[0020] a first bioprocessing bag having a first volume, a first flexible wall, and a liquid aspect ratio of the first bag height at the first bag maximum working volume to first bag diameter equal to a first H / D; a first impeller mounted on a first impeller plate attached to a first inside surface of the first flexible wall, the ratio of the first impeller, Di, to the diameter of the first bioprocessing bag, Dt, equal to Di / Dt; a first sparger attached to an inside bottom surface of the flexible wall, the ratio of the sparging surface area of the first sparger Asl, to the cross-sectional area of the first bioprocessing bag (in an inflated state), Ab, equal to Asl / Ab;

[0021] a second bioprocessing bag having a second volume that is larger than the first volume of the first bioprocessing bag, a second flexible wall, and an aspect ratio of second bag height at the second bag maximum working volume to second bag diameter equal to a second H / D, wherein the first and the second H / D are substantially equal to one another; a second impeller mounted on a second impeller plate attached to a second inside surface of the second flexible wall, the ratio of the diameter of the second impeller, D2i, to the diameter of the second bioprocessing bag, D2t, equal to D2i / D2t, wherein Di / Dt = D2i / D2t; a second sparger attached to an inside bottom surface of the second flexible wall, the ratio of the sparging surface area of the second sparger A2sl, to the cross-sectional area of the second bioprocessing bag (in an inflated state), A2b, equal to A2sl / A2b, wherein Asl / Ab = A2sl / A2b (or approximately equal to), wherein the first and the second impeller are of the same type, and are eachpositioned over a sparging surface of respective spargers, the distance between the bottom of the impeller blades and the sparging surface of each of the first and second bioprocessing bag being about same, and are each configured to operate within the first and second bag, respectively, such that, in a bioprocessing operation utilizing the same fluid within the first and the second bioprocessing bags, under similar conditions, a consistently similar maximum oxygen kia is achieved through control of agitation speed and gas flow rates while staying within acceptable shear rate and Kolmogorov Eddy length.

[0022] According to certain embodiments, H / D = between about 1-2, preferably about 1.7, Di / Dt = D2i / D2t = between about 0.3 to 0.5, preferably about 0.4, Asl / Ab = A2sl / A2b = between about 0.023 to 0.068, preferably about 0.04.

[0023] In embodiments, the first bioprocessing bag and second bioprocessing bag each include an additional sparger attached to an inside bottom surface of the first and second flexible wall, respectively. The ratio of the sparging surface area of the additional sparger, As2, to the cross-sectional area of the first bioprocessing bag (in an inflated state), Ab, equal to As2 / Ab. The ratio of the sparging surface area of the additional sparger, A2s2, to the cross-sectional area of the second bioprocessing bag (in an inflated state), A2b, equal to A2s2 / A2b, wherein As2 / Ab = A2s2 / A2b (or approximately equal to).

[0024] According to certain embodiments, As2 / Ab = A2s2 / A2b = between about 0.008 to 0.024, preferably 0.02.

[0025] Further, the distance between the bottom of the impeller blades and the sparging surface for the first and second bioprocessing bag is between about 8 to24mm, preferably 16mm.

[0026] Further, the gasses introduced through the first and second spargers of the first and second bioprocessing bags are controlled such that the maximum gas exit velocity and maximum gas flow rate relative to the nominal bioreactor volume are the same as each other. Similarly, the gasses introduced through the additional spargers of the first and second bioprocessing bags are controlled such that the maximum gas exit velocity and maximum gas flow are the same as each other, but different from the first and second spargers.

[0027] In certain embodiments, the maximum gas exit velocity and corresponding maximum gas flow for the first and second sparger are between about 12 to 60 m / s, preferably 24 m / s and between about 0.15 to 0.55 vvm, preferably about 0.2 vvm, and the maximum gas exit velocity and corresponding maximum gas flow for the additional spargers are between about 36 to 60 m / s, preferably 36 m / s and between about 0.05 to 0.18 vvm, preferably 0.1 vvm.

[0028] According to embodiments, the first and second spargers provide dissolved oxygen into the culture media, and in order to do so may deliver a mixture of air and oxygen. The additional spargers may be used for carbon dioxide stripping (removal) and may deliver nitrogen or air. Keeping the sparger surface area ratios the same across scales helps to ensure an equal degree of gas dispersion, and thus better scalability (e.g., control over scalable performance).

[0029] Still further, the power number for the first impeller and the second impeller are approximately the same. In certain embodiments, the power number for the first and second impeller in down pumping (e.g., clockwise impeller rotation) isbetween 0.8 to 3.6, preferably about 2.6 and in up pumping (e.g., counterclockwise rotation) is between about 0.8 to 2.5, preferably 1.7.

[0030] The first and second bioprocessing bag may each be disposed within a vessel. The vessel housing each of the first and second bioprocessing bags has a liquid height to diameter ratio of about H / D. The value of H / D being the same for both vessels. In particular embodiments, H / D for the vessel is also between about 1 : 1 to 2: 1, preferably 1.7: 1. Each vessel may include a plurality of baffles (e.g., 1-4), the baffles having a height ratio (baffle height / vessel diameter) Hb / D, noting that vessel height is the distance the baffle penetrates from the vessel perimeter toward the vessel center. The value of Hb / D being the same for both vessels.

[0031] In certain embodiments, Hb / D = between about 1 / 10 to 1 / 12, preferably about 1 / 11.

[0032] According to a further aspect of the invention, a method of scaling a biomanufacturing process is described. The method of scaling a biomanufacturing process, comprises providing a first bioprocessing bag having a first volume and a first flexible wall, the first bioprocessing bag including a first impeller mounted on a first impeller plate attached to a first inside surface of the first flexible wall and a first sparger attached to an inside bottom surface of the flexible wall, wherein when the first bioprocessing bag is in an inflated state, a ratio of a sparging surface area of the first sparger Asl, to the cross-sectional area of the first bioprocessing bag, Ab, is equal to Asl / Ab; performing a first biomanufacturing process in the first bioprocessing bag; providing a second single-use bioprocessing bag having a second volume, that is larger than the first volume, and a second flexible wall, the second bioprocessing bag including a second impeller mounted on a second impeller plate attached to a secondinside surface of the second flexible wall, wherein when the second bioprocessing bag is in an inflated state, a ratio of a sparging area of the second sparger A2sl, to the cross- sectional area of the second bioprocessing bag, A2b, is equal to A2sl / A2b; and scaling the first biomanufacturing process to a larger volume by performing a second biomanufacturing process in the second bioprocessing bag; wherein Asl / Ab is approximately equal to or equal to A2sl / A2b.

[0033] In embodiments, Asl / Ab = A2sl / A2b (or approximately equal to) = between about 0.023 to 0.068, and in one preferred embodiment, Asl / Ab is about 0.04.

[0034] In embodiments, the first bioprocessing bag and second bioprocessing bag each include an additional sparger attached to an inside bottom surface of the first and second flexible wall, wherein a ratio of a sparging surface area of the additional sparger, As2, to the cross-sectional area of the first bioprocessing bag, Ab, is equal to As2 / Ab and a ratio of the sparging surface area of the additional sparger, A2s2, to the cross-sectional area of the second bioprocessing bag, A2b, is equal to A2s2 / A2b, wherein As2 / Ab = A2s2 / A2b (or approximately equal to).

[0035] In embodiments, As2 / Ab = A2s2 / A2b = between about 0.008 to 0.024, and in one preferred embodiment As2 / Ab is about 0.02.

[0036] The method may further include operating the first and second bioprocessing bag in the same (or similar) way as described with regard to the other aspects of the invention.

[0037] In any and all of the aspects and embodiments of the present invention, the linearly scalable bioreactor system, and associated method, for use in carrying out a scalable biomanufacturing process may include a process measuring system; a systemof field actuating devices or field actuators; and a local processing unit and associated software logic configured to convert a measuring system input and pass the measuring system input to a supervisory controller or human machine input device for data manipulation and storage and to transform the measuring system input from the supervisory controller or human machine input device to a manipulation of the field actuating device or the field actuator.

[0038] The scalable bioprocessing system may have a human machine interface and associated software programming comprising at least one of a computer mouse, a keyboard and a touch-screen; and a supervisory controller and software programming capable of commanding system devices to respond to specific configurable parameters using data from a process inputs, a sensor measurement, a device state, a setpoint, a deviation from setpoint, an alarm condition, and combinations thereof.

[0039] The design of the disclosed scalable, bioreactor system, which may be a single-use system, is such that the operating range and geometry of each bioreactor in the system provide the ability to achieve consistent and similar maximum oxygen mass transfer while staying within acceptable volume fractions containing excessive shear rates and Kolmogorov Eddy lengths for targeted cell types.

[0040] In any of the aspects and embodiments of the present invention, the single-use bioprocessing bag is disposed within a vessel, also referred to herein as a “tank.” The vessel may be comprised of a rigid or slightly rigid material and serves to support the bag disposed therein. “Linear scalability,” as the term is used herein, refers to the relatively constant value of one or more geometric and process parameters of the vessel or of the bag disposed therein, and the ability to achieve and maintain consistent and similar oxygen mass transfer across each of the bioreactors in the system for eachbioreactor in the system, e.g., from 50L to about 2000L. A geometric similarity for the vessel or the bag disposed therein, and consistent and similar maximum oxygen mass transfer are maintained for each bioreactor in the system, across a wide range of working volumes. This linear scalability provides a means to model the performance of a large bioreactor in a smaller bioreactor (scale-down), or conversely for a development process that is carried out in a relatively small bioreactor to be easily scaled up for use in a larger commercial scale process with minimal changes in protocol.

[0041] The design of the disclosed scalable, single-use, bioreactor system is such that its gas sparging / aeration system is substantially the same across scales, such that the smaller bioreactor can simulate the performance of the larger systems aeration and gassing performance. This additional feature of linear scalability provides a means to model the performance of a large bioreactor in a smaller bioreactor, or conversely for a development process that is carried out in a relatively small bioreactor to be easily scaled up for use in a larger commercial scale process with minimal changes in protocol. Specifically, by keeping, for example, the Di / Dt, power number, and sparger surface area ratios constant between the different volumes, agitation speed and sparging rates can be varied within acceptable shear rate and Kolmogorov Eddy length in all bioreactor sizes while maintaining substantially the same maximum ki.a.

[0042] In summary, the bioreactor system disclosed provides for “linear scalability” by using the same or substantially simulating the same bag and vessel geometry, gas sparging system, impeller shape and type, and process control system as used in larger systems. The disclosed bioreactor system is well suited for mammalian cell, microbial, bacterial, plant, insect, protozoan, organ, and / or fungal cell culture.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Fig l is a schematic side elevational, cutaway view of a 50L bioreactor vessel with a single-use, flexible bag disposed therein, an impeller, magnetic impeller hub, impeller plate, and external magnetic drive according to an embodiment if the invention.

[0044] Fig 2 is a top-down view of a bioreactor vessel with the impeller plate and spargers illustrated, according to embodiments of the invention.

[0045] Fig 3 is a top-down view of a bioreactor vessel with the impeller plate, spargers, and impellers illustrated, according to embodiments of the invention.

[0046] Fig. 4 is a contour plot illustrating empirical dissolved oxygen ki.a for a 50L and 200L bioreactor, according to embodiments of the invention.DETAILED DESCRIPTION

[0047] A description of preferred embodiments of the invention follows. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. At the outset, the invention is described in its broadest overall aspects, with a more detailed description following. The features and other details of the compositions and methods of the invention will be further pointed out in the claims.

[0048] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of these words mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the context requires otherwise.

[0049] A scalable, single use, bioreactor system according to an embodiment of the invention is a small-scale bioreactor that provides linear scalability from small scale to large scale, e.g., from a 50L single-use bioreactor bag to a 2000L single-use, bioreactor bag in the same single-use platform within a family of bioreactors of varying sizes.

[0050] FIG. l is a schematic side elevational, cutaway view of a disclosed bioreactor system 100 comprising a 50L bioreactor vessel or support structure 20 with a single-use, flexible bag 30 disposed therein; an impeller 22 attached to a magnetic hub 24 at the bottom center of the inside of the bag 30; an impeller plate 26 positioned on the inside bottom of the bag and upon which the magnetic puck 24 rotates; and an external magnetic drive 23.

[0051] The bioreactor system 100 has been designed to achieve high cell density culture conditions. In particular, the small-scale (e.g., 50L) bioreactor system has been designed to include sparger(s) having a sparging surface area sufficient to provide enough oxygen transfer to not negatively impact oxygen ki.a. Specifically, because sparging surface area independently effects oxygen ki.a (i.e., independent of agitation and flow conditions), the small-scale bioreactor system is designed such that oxygen transfer into the cell culture is maintainable at sufficient levels to obtain high cell density culture conditions within maximum agitation and gas flow rate constraints. The inventors have advantageously found that by maintaining the same (or substantially the same) sparging surface area ratio (sparging surface area / bag cross- sectional area in an inflated state within the vessel) between scales, as well as other geometric parameters as described in greater detail below, scalability is improved. Saidanother way, by determining a sparging surface area at small scale that provides sufficient oxygen transfer and maintaining a sparging surface area ratio based on that value between scales, culture conditions at each scale can be similar without exceeding maximum agitation rate and gas flow rates.

[0052] In addition to sparging surface area, keeping additional geometric parameters constant (or nearly constant) between scales further improves scalability by making the geometry of the different scales the same (or substantially the same). For example, by fixing the aspect ratio H / D, the ratio of Di / Dt (where Di is the impeller diameter and Dt is the vessel diameter), the impeller blade - sparger distance, and / or the baffle height ratio (baffle height / Vessel diameter) between scales, an even greater similarity of culture conditions at each scale is achieved.

[0053] Still further, capping certain operational parameters between scales further improves scalability by avoiding operating the bioreactor system in a regime that would be detrimental to cell growth. For example, by capping maximum gas flow rate (and thus gas flow per minute) and mixing contribution from agitation rate (i.e., power input to volume ratio (P / V)), cell culturing conditions between all scales is kept within desirable limits (e.g., maintaining acceptable shear rates, dissolved oxygen and carbon dioxide concentrations, and Kolmogorov Eddy).

[0054] While the invention is directed to keeping certain geometric and process parameters fixed, a range of values for each of these parameters, as described below, is within the scope of the invention. Specifically, any value within a given range can provide the advantages described herein, so long as it is fixed (or substantially the same) across scales. Additionally, the ranges are based upon mathematical calculations given overall design constraints of the present bioreactor system. It is noted, however,that the specific values recited as preferred values described herein are meant to denote optimal values for a scalable bioreactor system. And the invention is explicitly not limited to those values, and rather encompasses all of the ranges described herein.

[0055] In embodiments, the aspect ratio H / D of height of liquid working volume H to diameter D of the bag 30 is between about 1-2, preferably 1.7. The ratio of Di / Dt (where Di is the impeller diameter and Dt is the vessel diameter) is between about 0.3 to 0.5, preferably about 0.4.

[0056] As best illustrated by Fig. 2, there is also at least one sparger attached to a bottom of the inside of bag 30. The at least one sparger can take the form of an array of sparging discs 27, 28 located on the impeller plate 26. In embodiments, the at least one sparger comprises two spargers. The first sparger 27, which in the embodiment illustrated in Fig. 2 includes three sparging discs, has a ratio of the sparging surface area to bag cross-sectional area (in an inflated state within the vessel) of between about 0.023 to 0.068, preferably about 0.04. The second sparger 28, which in the embodiment illustrated in Fig. 2 includes one sparging disc, has a ratio of the sparging surface area to bag cross-sectional area (in an inflated state within the vessel) of between about 0.008 to 0.024, preferably about 0.02. The gasses introduced through the first and second spargers 27, 28 are controlled such that the maximum gas exit velocity for the first sparger 27 is about 12 - 60 m / s, preferably 24 m / s corresponding to a maximum gas flow of between about 0.15 to 0.55 vvm (vessel volume per minute), preferably 0.2 vvm, while the maximum gas exit velocity for the second sparger 28 is between about 36 to 60 m / s, preferably 36 m / s corresponding to a maximum gas flow of between about 0.05 to 0.18 vvm, preferably 0.1 vvm. The overlay maximum gas flow is about 0.05 vvm. Additionally, maximum agitation rate (i.e., power input to volume ratio(P / V)) is capped at about 150W / m3.

[0057] A reusable bioreactor vessel according to the disclosed system may include a reusable polymeric or metal vessel stand, support, or holder 20 with integral, external motor / agitation assembly 23, and a measurement and control system (not shown). The vessel stand, support, or holder may be comprised of a mixed material such as a polymer with metals. The polymeric or metal vessel can be any shape or size as long as it is capable of supporting the disclosed stirred tank single-use flexible bioreactor bag 30 design. For example, according to one embodiment of the invention a polymeric or metal stand, support, or holder 20 is capable of accepting and supporting a 50L flexible or collapsible bioprocess bag assembly 30.

[0058] The measurement and control system is built on a configurable software platform. The measurement and control system operates in real-time; accepts process inputs (measurements) such as temperature, RPM, pH, DO, weight, pressure, etc.; and controls field devices such as mass flow controllers, pumps, solenoid valves, heating elements, and the like. According to one embodiment, human interaction with the system is through a touch-screen based interface that may be on a desk-top, laptop, or notebook computer, via remote computer operating over the internet, or a remote control device. The interface allows users to enter set-points, manage gas-sparging, calibrate field devices, manage alarms, configure operator identity and access levels, and view process values graphically, over time. The measurement and control system may further include a server for storage of current and historical data related to the culture process.

[0059] Scalability, as described above, is a very important factor especially in the biotech / pharmaceutical manufacturing industry, because it allows easy and simpletech transfer from the research level (small-scale) to the large-scale production. The geometrical similarity, e.g., the aspect ratio (H / D), impeller geometry, sparger geometry, and impeller power number, as well as process similarities, e.g., sparger gas entrance velocity and gas flow rate - are key factors to control and minimize changes in the performance of the bioreactor process during scale up.

[0060] In addition to the above, shear rate and Kolmogorov Eddy length are parameters that must be taken into consideration. Specifically, the shear rate and Kolmogorov eddy length come into play as limits to avoid operating the bioreactor in a regime that would be detrimental to cell growth for cell types suitable for growing in suspension cell culture. The process parameters above have been designed such that the bioreactor does not exceed 2000 s'1for the shear rate and the Kolmogorov eddies being larger than 62 micrometers from a static analysis of the system at maximum agitation by power density (W / m3) and preferred targeted gas flow rates through both sparge assemblies. Both of these limits being satisfied for no less than 95% of the volume of the vessel, i.e., small, localized regions beyond the limits are acceptable.

[0061] Tables 1 and 2 below provide a summary of geometric and operational parameter ranges, within the scope of the invention. Each bioreactor has the same or approximately the same H / D aspect ratio, impeller geometry, sparger geometry, and impeller power number, as well as the same or similar operational limit similarities, e.g., sparger gas entrance velocity, for all reactors. All ranges provided are approximations (i.e., each boundary can be adjusted by up to 15%).

[0062] Tables 3 and 4 below provide a summary of preferred geometric and operational parameter values and ranges that are within the scope of the invention. Specifically, each bioreactor has the same or approximately the H / D aspect ratio, impeller geometry, sparger geometry, and impeller power number, as well as operational limit similarities, e.g., sparger gas entrance velocity, for all reactors. All values are approximations (i.e., each boundary can be adjusted by up to 15%).*Baffle height is the distance the baffle penetrates into the vessel interior

[0063] Having these values constant (or approximately the same), within the recited ranges, among the scales helps to achieve linear scalability and highly predictable scale-up condition modeling, while also maintaining substantially consistent oxygen mass transfer (i.e., oxygen ki.a (+ / -30%)). Throughout the text, the 50L, 200L, 500L, lOOOL and 2000L single-use bioreactor bags disclosed herein are each designated as the X-50, X-200, X-500, X-1000 and X-2000, respectively.

[0064] The data in the above Table 1 through Table 4 show some linear scaling factors for the disclosed single-use bioreactors. Assuming a substantially cylindrical shape throughout the working volume of the single-use bioreactor bag, according to an embodiment of the invention, the volume turndown ratio, which is the ratio of the maximum working volume to the minimum working volume, is about 5: 1 for allbioreactor sizes. “H” is the height of the working volume in the single-use bag and “D” is the diameter of the tank or the single use bag, which is disposed within the supporting tank. For a series of maximum working volumes, the H / D ratio of either the tank or the bag disposed within is substantially constant, having a value of about 1 : 1 to 2:1, preferable about 1.7. The impeller diameter, “Di,” varies (i.e., increases as vessel size increases), but the ratio of the impeller diameter Di to the vessel diameter Dt remains constant, at a value of about 0.3-0.5, preferably about 0.4. In some embodiments, the impeller type of the invention is a 6-blade combination Rushton 50- degree pitch blade and is the same type in each system. The number of blades on the impeller may vary.

[0065] The impeller, in one embodiment, is mounted on the impeller plate and is magnetically driven by an external motor.

[0066] The impeller power number, “NP”, which is calculated from the formulawhere Po is the power input into the medium at unareoted conditions, N (s-1) is the stirrer speed, D; (ml is the impeller diameter and p (Kg nr3) is the medium density [3],(for both up-pumping and down-pumping) is similarly constant among the bioreactor sizes. Specifically, as shown the up-pumping power number is about 0.8- 2.5, preferably about 1.7 and the down-pumping power number is about 0.8-3.6, preferably 2.6

[0067] The impeller blade - sparging distance, which is the vertical distance between the top surface of the sparger and the bottom surface of the impeller blades, is also constant, with a value of about 8-24mm, preferably 16 mm.

[0068] The bioreactor bags of the present invention may include two types ofsparges 27, 28, e.g., in the form of sparging discs, located underneath the impeller (see, e.g., Figs. 2 and 3). Sparger 1 (as provided in Tables 1 and 3), corresponding to sparger 27 of Fig. 2, is used for dissolved oxygen (DO) control and is configured to deliver a mixture of nitrogen and oxygen. Sparger 2 (as provided in Tables 1 and 3), corresponding to sparger 28, is used for carbon dioxide (CO2) stripping (removal) and is configured to deliver nitrogen and / or air. The total surface area of the sparging surface of each sparger divided by the vessel / bag cross-sectional area, as shown in Table 1 and 3 is also kept constant (or approximately the same). Specifically, Sparger 1 sparging surface area ratio is between about 0.023 to 0.068, and in a preferred embodiment 0.04, and Sparger 2 sparging surface area ratio is between about 0.008 to 0.024, and in a preferred embodiment about 0.02. These values are constant (or approximately the same) among the scales.

[0069] One significant advantage of the present invention is providing the same sparging area ratios among all scales of bioreactors. By making this ratio constant, an equivalent amount of gas dispersion is achieved, which greatly helps control the scaling process as related to oxygen ki.a (and thus oxygen mass transfer) as well as dissolved CO2 stripping. More specifically, because sparging surface area independently effects oxygen ki.a (i.e., independent of agitation and flow conditions), a constant surface area ratio for the spargers that does not limit desired oxygen ki.a is established, thereby providing higher maximum ki.a with a fixed geometry among scales.

[0070] In addition to geometric scaling parameters, it has been found to be advantageous to set operational limits at all bioreactor scales. As provided in Tables 2 and 4, the same maximum gas entrance velocities and maximum gas flow rates for the spargers is constant for all bioreactor scales. Sparger 1 has a maximum gas entrancevelocity of about 12 - 60 m / s, preferably about 24 m / s and a maximum total gas flow of about 0.15 - 0.55 vvm, preferably about 0.2 vvm, while Sparger 2 has a maximum gas entrance velocity of about 36 - 60 m / s, preferably about 36 m / s and a maximum total gas flow of about 0.05 - 0.18 vvm, preferably about 0.1 vvm. Additionally, a maximum input of power per unit volume (max P / V), which is the amount of power transferred to a working volume of culture through the rotation of the impeller, and is maintained constant among bioreactor scales for the nominal bioreactor volume, having a value of about 150 W / m3. Ensuring that these operational parameters have a constant maximum among the scales aides in controlling the scaling process as related to oxygen ki.a (and thus oxygen mass transfer) and CO2 stripping, as well as ensuring that the bioreactors all perform within the prescribed shear rate and Kolmogorov eddy limits. Said another way, having these operational limits the same among scales provides a design space that provides the same or similar performance at each scale. Further, this design space allows for the alteration of oxygen concentration in the feed stream and utilizing sparge two in an effort to match gas flow rates (in vvm) across scales enabling similarity in pCO2profiles, while delivering similar oxygen ki.a at fixed P / V.

[0071] It may also be advantageous to control the maximum overlay gas flow rate, which is the rate of gas flow that is provided into the headspace of the bioreactor bag 30. As Tables 2 and 4 provide, this maximum value is constant among all scales, and set to 0.05 vvm.

[0072] In embodiments, the bag or vessel uses the same disposable or singleuse materials of construction and rigid plastics product contact components as larger scale bioreactors in the same system. The use of the same materials of construction or use of the same class of polymers for construction eliminates the regulatory disconnectbetween small scale optimization and large-scale implementation with respect to extractable and leachable components, biocompatibility conformance, and regulatory testing. As a result, the 50L system models larger scale systems not just from a scalability / performance basis, but also from a regulatory compliance basis.

[0073] Some of the features of a stirred tank bioreactor in the disclosed system include: a bottom mounted or a side wall-mounted impeller positioned close above substantially the same sparging surface, as described above; USP Class VI materials; full integration with a controller; biocompatible materials in construction; and linear scalability over a wide range of working tank or bag volumes, as described above, for example from 50L to 2000L in the same single use bioreactor system.Single Use Bioreactor Scale-Up Factors

[0074] There are many parameters that effect cell growth, and deviations from acceptable ranges is detrimental to health and growth. Accordingly, it is critical, and often difficult when scaling, to keep these parameters within acceptable limits. As discussed above, the present invention provides bioreactors that are highly scalable. By fixing certain geometric attributes and establishing the same operational limits among scales, many of the fluid response parameters are kept within acceptable ranges, and in some instances can be kept constant, which greatly aids in scaling between bioreactor sizes. This can be achieved, for example, by choosing a scale-up basis, such as agitation power density (P / V), to be constant. With this value constant, identical OTR can be delivered by virtue of similar system ki.a across scales that can be fine-tuned (e.g., by adjusting inlet oxygen concentration and matching overall gas flows on a vvm basis through the supplemental second sparger which results in similar pCO2 profiles).

[0075] It is important to ensure that there is adequate oxygen mass transfer into the cell culture and CO2 stripping, otherwise unnecessary cell death occurs, and cell proliferation is reduced or halted. Complementarily, mixing time also has a large effect on cell health and proliferation, as slow mixing times prevent cells from obtaining the required nutrients in a timely manner or can expose them to harmful concentration gradients.

[0076] Because each scale-up parameter is dependent on another parameter, all the parameters cannot be kept constant during scale-up. For example, oxygen ki.a, agitation speed, and gas flow rate cannot all be held constant. Rather, two of these parameters can be held constant, while the third varies in order to maintain the other two.

[0077] We have now discovered a system comprising two or more single-use bioreactor bags, each having one or more of the aforementioned geometric parameters and operational limits fixed. By fixing these, the other scale-up parameters can all be maintained within desirable ranges even though they cannot all be held constant.

[0078] Fig. 4 provides contour plots illustrating empirical dissolved oxygen ki.a for a 50L and 200L bioreactor, according to embodiments of the invention. As illustrated, and with the above design constraints, at the maximum P / V (150W / m3) and a sparger 1 vvm of 0.19, a maximum oxygen ki.a (h-1) for the 50 L bioreactor is approximately 45. Based upon characterization data, each of the other scales (e.g., 100- 2000 L) can achieve, or exceed, the same oxygen ki.a while still being within the aforementioned operational limits. For example, at a P / V of 108 W / m3and sparge 1 gas flow rate of 0.15 vvm, a maximum oxygen mass transfer coefficient (kr a) for the 200 L bioreactor at nominal volume is approximately 45 h’1. In this way, it becomes evidentthat a sufficiently high oxygen ki.a (e.g., these ki.a values can achieve high cell densities (e.g., in excess of lOOxlO6cells / mL)) can be achieved among all scales within the same operational limits, which aids in scale up (or scale down).

[0079] Successful scale-up (SU) and / or scale-down (SD) of biologies production requires that performance equivalence be achieved between scales of operation. The SU and SD platform design of the disclosed scalable, single-use bioreactor design provide a unique system for accomplishing this. As Table 3 shows, maximum Oxygen ki.a and dissolved CO2 stripping is constant among the scales, which greatly helps to ensure that the cell culture maintains adequate oxygen mass transfer and dissolved CO2 removal regardless of scale. Specifically, the present design provides a SU and SD platform that provides consistent maximum ki.a within maximum tolerance limits (e.g., P / V max, shear, eddy)at all scales. In this way, each bioreactor can consistently provide enough dissolved oxygen and CO2 stripping, by adjusting agitation speed and / or sparging rate, without causing toxic conditions within the cell culture (e.g., unacceptably high shear rates).

[0080] Fittings are added to the bag to enable functionality required in a bioreactor such as penetrations and filters to allow for fluid and gas transfer, a mixing interface, sensors and a sparging surface to control bubble size. Multiple sparging surfaces and options can be utilized in various embodiments of the invention. A sparging surface can be attached to the bioprocessing bag as an adapter or a bulkhead fitting, designed to connect a hose or tubing such as an oxygen supply tubing to the bag. The porosity and the area of a sparge surface can vary (within the aforementioned limits). In one embodiment, a sparging surface may be simply a single hole or a plurality of holes configured to add oxygen gas or air to the bioreactor bag. At leastsome of the sparging surface may be positioned under the impeller (e.g., the sparger for DO control) to allow for efficient circulation of gas through the media in the bag and its position and type is substantially the same as the position and type in the larger 50L- 5000L systems.

[0081] The linearly scalable bioreactor system according to an embodiment of the invention may include a temperature controller and at least one sensor and / or a probe (not shown). To eliminate utilities required for temperature control through a heat exchanger, heating can be provided by means of closed loop water jacket heated and / or cooled by control system mounted on the bioreactor system or by an electric heating blanket(s), or a Peltier heater. The heat blanket may include a thermocouple(s) for sensing a temperature of the contents of the bioprocessing bag, working in conjunction with the temperature controller to control a set temperature of the contents of the bioprocessing bag. A temperature conducting material may be embedded in the surface of the bioprocessing bag to counteract the insulating effect of plastic if necessary.

[0082] Cooling may also be provided by a closed loop water jacket heated and / or cooled by control system mounted on the bioprocessing bag or by standard heat exchange through a cover or jacket on the tank supporting the bioprocessing bag. Cooling may also be provided by means of Peltier coolers. For example, a Peltier cooler may be applied to an exhaust line (e.g., to a chamber similar to a small bag, with a large volume to decelerate air and a large surface area situated in a heat exchanger) to condense gas in the exhaust air to help prevent an exhaust filter from wetting out. Alternatively, the exhaust filter may be heated by a filter heater to help prevent condensation of moisture from the exhaust line. This heat source could also be thewaste heat from active cooling side of the Peltier unit by directing the hot gas flow through a conduit and holder for the exhaust filter.

[0083] The bioprocessing bag, including all attachments, penetrations, sensors, etc,) may be sterilized prior to use ( e.g., gamma-irradiation). After sterilization, the inside of the bag, tubing and components may be considered sterile, providing a "sterile envelope" protecting the contents of the vessel from airborne contaminants outside.

[0084] It should also be appreciated that there are at least three basic modes of operation used by stirred tank bioreactors, and that the disclosed bioreactor system comprises bioreactors that can be easily modified to operate in any of the three modes. The three modes are as follows.

[0085] Semi -continuous, Continuous or Perfusion Mode: In a semi- continuous, continuous or perfusion mode, nutrients and supporting process fluids are continuously added to the system; waste products are continuously removed either continuously or periodically (“drain and fill”); and product is harvested intermittently or throughout the culture period. With the continuous mode, the on-going difficulty in obtaining sufficiently high product titers is well recognized. In addition to the low titers, there is a need to concentrate product of the continuous mode.

[0086] Batch Mode: In batch mode, all nutrients are added at the beginning and products are not removed until the end of the batch. Waste products accumulate during the run, and nutrients are used up, making the batch process inefficient for many applications. The simplicity of operating in this mode makes it a desirable choice for seed train expansion.

[0087] Fed-Batch Mode: A fed-batch mode is similar to the batch mode in thatproducts are removed only at the end of the run, but differs in that nutrients are added at multiple intervals during the process. Many biotherapeutics and most virus-producing, microcarrier cultures are carried out, post infection, in a fed-batch process.

[0088] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims and abstract), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims and abstract), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

What is claimed is:

1. A bioreactor system for use in carrying out a scalable biomanufacturing process, the system comprising: a bioprocessing bag having a flexible wall; an impeller mounted on an impeller plate attached to an inside surface of the flexible wall; and a first sparger attached to an inside surface of the flexible wall, wherein when the bioprocessing bag is in an inflated state, a ratio of a sparging surface area of the first sparger to a cross-sectional area of the bag is between about 0.023 to 0.068.

2. 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 bag is about 0.04.

3. The bioreactor system of claim 1 or 2, further comprising a second sparger attached to the inside surface of the flexible wall, wherein when the bioprocessing bag is in the inflated state, a ratio of the sparging surface area of the second sparger to a cross- sectional area of the bag is between about 0.008 to 0.024.

4. The bioreactor system of claim 3, wherein the ratio of the sparging surface area of the second sparger to a cross-sectional area of the bag is about 0.02.

5. The bioreactor system of any one of claims 1-4, wherein a ratio of the diameter ofthe impeller, Di, to the diameter of the bioprocessing bag, Dt, is equal to between about 0.3 to 0.5.

6. The bioreactor system of claim 5, wherein the ratio of the diameter of the impeller, Di, to the diameter of the bioprocessing bag, Dt, is equal to about 0.4.

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

8. The bioreactor system of any of claims 1-7, wherein a distance between impeller blades of the impeller and a sparging surface of the first sparger is between about 8 to 24mm, preferably about 16mm.

9. A bioreactor system for use in carrying out a scalable biomanufacturing process, the system comprising: a first bioprocessing bag having a first volume and a first flexible wall; a first impeller mounted on a first impeller plate attached to a first inside surface of the first flexible wall; a first sparger attached to an inside surface of the flexible wall, wherein when the first bioprocessing bag is in an inflated state, a ratio of a sparging surface area of thefirst sparger Asl, to the cross-sectional area of the first bioprocessing bag, Ab, is equal to Asl / Ab; a second single-use bioprocessing bag having a second volume, that is larger than the first volume, and a second flexible wall; a second impeller mounted on a second impeller plate attached to a second inside surface of the second flexible wall, wherein when the second bioprocessing bag is in an inflated state, a ratio of a sparging surface area of the second sparger D2sl, to the cross- sectional area of the second bioprocessing bag, A2b, equal to A2sl / A2b; wherein Asl / Ab is approximately equal to or equal to A2sl / A2b.

10. The bioreactor system of claim 9, wherein Asl / Ab is between about 0.023 to 0.068.

11. The bioreactor system of claim 9 or 10, wherein Asl / Ab is about 0.04.

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

13. The bioreactor system of claim 12, wherein As2 / Ab is between about 0.008 to0.024.

14. The bioreactor system of claim 13, wherein As2 / Ab is about 0.02.

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

16. A method of scaling a biomanufacturing process, comprising: providing a first bioprocessing bag having a first volume and a first flexible wall, the first bioprocessing bag including a first impeller mounted on a first impeller plate attached to a first inside surface of the first flexible wall and a first sparger attached to an inside surface of the flexible wall, wherein when the first bioprocessing bag is in an inflated state, a ratio of a sparging surface area of the first sparger Asl, to the cross-sectional area of the first bioprocessing bag, Ab, is equal to Asl / Ab; performing a first biomanufacturing process in the first bioprocessing bag; providing a second single-use bioprocessing bag having a second volume, that is larger than the first volume, and a second flexible wall, the second bioprocessing bag including a second impeller mounted on a second impeller plate attached to a second inside surface of the second flexible wall, wherein when the second bioprocessing bagis in an inflated state, a ratio of a sparging area of the second sparger A2sl, to the cross- sectional area of the second bioprocessing bag, A2b, is equal to A2sl / A2b; scaling the first biomanufacturing process to a larger volume by performing a second biomanufacturing process in the second bioprocessing bag; wherein Asl / Ab is approximately equal to or equal to A2sl / A2b.

17. The method of claim 16, wherein Asl / Ab is between about 0.023 to 0.068.

18. The method of claim 17, wherein Asl / Ab is about 0.04.

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

20. The method of claim 19, wherein As2 / Ab is between about 0.008 to 0.024.

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

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